Jatropha curcas is a tropical and subtropical shrub and its growth is affected by one of major abiotic stress, chilling. Therefore, we adopt the phosphoproteomic analysis, physiological measurement and ultrastructure observation to illustrate the responsive mechanism of J. curcas seedling under chilling (4 °C) stress. After chilling for 6 h, 308 significantly changed phosphoproteins were detected. Prolonged the chilling treatment for 24 h, obvious physiological injury can be observed and a total of 332 phosphoproteins were examined to be significantly changed. After recovery (28 °C) for 24 h, 291 phosphoproteins were varied at the phosphorylation level. GO analysis showed that significantly changed phosphoproteins were mainly responsible for cellular protein modification process, transport, cellular component organization and signal transduction at the chilling and recovery periods. On the basis of protein-protein interaction network analysis, phosphorylation of several protein kinases, such as SnRK2, MEKK1, EDR1, CDPK, EIN2, EIN4, PI4K and 14-3-3 were possibly responsible for cross-talk between ABA, Ca2+, ethylene and phosphoinositide mediated signaling pathways. We also highlighted the phosphorylation of HOS1, APX and PIP2 might be associated with response to chilling stress in J. curcas seedling. These results will be valuable for further study from the molecular breeding perspective.As a promising energy plant for biodiesel,  Oryza sativa, Zea mays and Lycopersicon esculentum, are sensitive to chilling stress and easily damaged by chilling temperature [As one of the most critical limiting factors, low temperature affects the plant growth and development broadly as well as yield, quality, postharvest life and geographic distribution . Chillinperature . The celperature ,6,7. Comperature . Low temperature ,9,10. Itperature ,12,13. Aperature  and SnRKperature . ABI5 anperature ,17,18,19perature , as wellperature  and appeArabidopsis thaliana, Brachypodium distachyon, Triticum aestivum, Broussonetia papyrifera after hormonal and dehydration treatment, salt and chilling stress [As the major and reversible post-translational modification, protein phosphorylation was crucial for providing the basis for complex signaling networks and the regulation of diverse cellular functions in plants . Phosphog stress ,28,29,30Jatropha curcas L., which originated from the tropics or subtropics, is a woody oil plant that belongs to the Euphorbiaceae family [J. curcas has been considered as the ideal material for biodiesel in the world [J. curcas has been achieved with the application of genomic [J. curcas was calculated as about 410 Mb and approximately 286 Mb consisting of 120,586 contigs and 29,831 singlets have been sequenced. These databases are valuable for further studies focusing on all aspects of molecular mechanisms. As an arisen economic woody species, J. curcas has been paid worldwide attentions. However, low temperature is still the major factor limited its distribution and affects the production of J. curcas seeds consequently. In consideration of the research and application, it is valuable to explore the metabolic network and regulated signal pathway of J. curcas under low temperature stress. Nevertheless, only few studies focused on molecular mechanism of chilling response in J. curcas. A previous study had employed transcriptome to analyze J. curcas under cold stress (12 °C), however, the result was sweeping without accurate mapping the regulated network basing on the experimental data, besides, the treatment temperature was also too moderate [J. curcas seedling under cold stress (4 °C) instead of high-throughput identification [J. curcas seedling at phosphorylation level. We aim to understand how sensitive is J. curcas to chilling stress and further unveil the specific phosphorylated proteins involved in potential pathways in J. curcas under chilling stress. The results will be benefit for providing clue to screen the chilling resistant J. curcas species.e family . Due to he world . Taking  genomic ,34, tran genomic ,36 and p genomic ,39,40,41moderate  to evalufication . In thisJ. curcas seedling drooped and pseudostem tilted after 24 h of chilling treatment (4 °C) and mostly recovered after being returned to 28 °C for 24 h (2 concentration (Ci) showed the opposite change tendency, the C6 h and C24 h of Ci increased to 160% and 408% respectively and almost returned to C0 h level after 24 h recovery. Conductance to H2O (Cond) and transpiration rate (Trmmol) shared the similar change curve, only the R24 h of Cond and Trmmol showed the significant changes compared with C0 h (O. sativa and Musa paradisiaca under chilling stress (4 °C), the species also originated in the tropics [The leaves of for 24 h A. To evaith C0 h B. These  tropics ,45.J. curcas seedling under chilling treatment, the fourth-leaves at each treatment  were prepared for ultrastructural observation. In the leaf cell from C0 h, the morphological structures were intact chloroplasts with numerous embedded starch granules, which mainly distributed closely to the cell membrane. Besides, intact vacuole and mitochorina could also be observed clearly  via the PRIDE partner repository [In total, 3101 phosphopeptides with 3101 phosphorylated sites corresponding to 1810 phosphoproteins were identified  and the pository  with theJ. curcas seedling (C0 h), 308, 332 and 291 phosphorylation sites corresponding to 279, 313 and 270 phosphoproteins were differentially changed after chilling for 6, 24 h  and recovery for 24 h (R24 h), respectively  were used for GO annotation. The distribution pie charts for biological process, cellular component and molecular function are shown in O. sativa and A. thaliana were compared against J. curcas to determine the degree of conservation of phosphorylation sites among different plant species. The thresholds were set as score ≥ 80, E-value < 1 × 10−10 and identity ≥ 30%. In all, 561 (69.7%) of the 805 phosphoproteins had phosphorylated orthologs in the two species, 163 (20.2%) had phosphorylated orthologs in only one species (The sequences of the 805 significant changed phosphoproteins were used as queries to blast phosphoprotein databases that were constructed using data sets in the Plant Protein Phosphorylation DataBase (P3DB)  and PhoP species , 81 phos species .The kinase related phosphorylation motifs of the significantly changed phosphopeptides were identified by employing WebLogo and motif-X. Briefly, the significant changed phosphopeptides were centered at the phosphorylated amino acid residues of each experimental group  and thenhttp://string-db.org, version 9.1). A total of 514 KOGs representing 610 phosphoproteins  were observed to be significantly up-regulated phosphorylated in the chilling treatment process and return to normal condition at the recovery process, which might indicate that the JcSRK2a phosphorylation was activated by osmotic stress caused by chilling treatment. Multiple sequence alignments between JcSRK2a and 10 subfamily of SnRK2 from A. thaliana showed the JcSRK2a shared the highest similarity (86.44%) with AtSnRK2.4 which could be activated by osmotic stress [TaSnRK2.4 in A. thaliana enhanced the plant tolerance to drought, salinity and low temperature [J. curcas. In other hand, as the member of the SnRK1 subfamily [J. curcas seedling.When plants were exposed to chilling stress, the osmotic stress mediated by ABA accompanied and the corresponding signal pathways were triggered . Accordic stress . Furtherperature . The JcSubfamily , KIN10 iubfamily ,60. We oJ. curcas seedling under chilling stress. Coincidently, as a negative regulator, JcKEG (id 2703) showed down regulation trend at phosphorylation level, which possibly indicated that ABA had induced KEG degradation. This assumption was supported by previous studies [O. sativa [ABI5 was highly inducible by either ABA treatment or stress . In the  studies ,62. In r. sativa ,63. The . sativa . It was 2+ transiently increased as the result from chilling stress [2+ signal transduction [J. curcas. MAPK subsequently interacted with ICE1, which was degraded by an E3 ligase HOS1 [The neighbors of SnRKs were chilling stress related protein kinases, which participated in multiple biological processes. Cytosolic Cag stress . CDPK plsduction . The phosduction . In thissduction . Two prosduction . Althougsduction ,70,71,72sduction . In thisase HOS1 . The pho2+ into the cytoplasm [JcPLC2 and BpPLC2 were constitutive even under low temperature stress [J. curcas seedling, PLC2s  were both differential phosphorylated at recovery stage instead of the chilling stage. Their phosphorylation sites were Ser312 and Ser278 respectively. The phosphorylation site Ser312 from PLC2  was located in the catalytic Y domain  was significantly and continuously up-regulated at the phosphorylation level under chilling stress and returned to normal level at the recovery stage  and recovery period. Under chilling stress, many proteins were misfolded and these proteins could not be assembled and function as normal. There were two approaches to handle these misfolded proteins. One was refolding in the assistance of molecular chaperones. In this study, CPN60B2 (id 1375) and CHSP70 (id 3984) were both phosphorylated changed and might result in the activation of RuBisCo assembly ,89,90. NJ. curcas seedling within 24 h chilling treatment  dephosphorylation matched very well with those of PSII recovery. These results might suggest that PSII reactivation from low temperature photoinhibition was closely related to CP29 dephosphorylation. Similar result had been illuminated at O. sativa during recovery from chilling induced photoinhibition [In response to the chilling stress, changes could be observed at the physiological and cellular level in reatment , which wecreased B and theecreased , which ienhanced  and the enhanced  and phosenhanced . In thiscavenged . Coincidhibition .A. thaliana [pmi1 exhibited severely attenuated chloroplast movements under low and high light intensities, indicating that PMI1 was necessary for both chloroplast accumulation and avoidance movements [J. curcas seedling, subsequently, all of them inducible regulated at the phosphorylation level in the recovery stage. To date, there were relatively few physiological and molecular evidences for explaining chloroplast movement through phosphorylation. Therefore, we speculated that the activities of CHUP1 and PMI1 might be regulated by other post-translational modification, such as glycosylation or sumoylation. Chloroplast movement was an efficient strategy to alleviate PSII damage and maintained maximal photosynthetic output . In planthaliana . In thisthaliana . CHUP1 movements . NeverthJ. curcas seeds with same provenance were collected from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Yunnan Province. The mature J. curcas seeds were sowed and grown in a mixed soil of peat and vermiculite (1:1) in a growth chamber under 28/25 °C (day/night) temperature regime, a photon flux density of 150 μmol m−2·s−1 throughout a 14-h photoperiod and a relative humidity of 80%. Four-leaf stage seedlings were used in this experiment. Low temperature treatments were started at 11:00 AM on the first day by setting the temperature to 4 °C, which was reached about 50 min later. The temperature was returned to 28 °C after 24 h of chilling treatment. The fourth true leaves were detached from the top of J. curcas seedling at each time point , which indicated as C0 h, C6 h, C24 h and R24 h respectively for each biological replicate. one part of the collected leaves were used for tissue preparation of transmission electron microscopy and other part of the collected leaves from the five plants were frozen in liquid nitrogen and stored at −80 °C until use. However, in consideration of the functional maturity, the first true leaves were chosen for measurements of photosynthesis, stomatal conductance and transpiration. The measurement was carried out by using a portable gas analysis system, LI-COR 6400 with a light-emitting diode light source . The measurement conditions were set as follows: block temperature 20 °C, photon flux density 1200 μmol m−2·s−1, humidity 80% and the CO2 concentration was stabilized by 5 L surge flask. The uniform mature g for 10 min at 4 °C. The supernatant was transferred to a fresh tube and precipitated by adding 10% cold trichloroacetic acid on ice for over 30 min. The mixture was centrifuged at 15,000 g for 10 min at 4 °C and the supernatant was discarded. After washed three times with acetone containing 1% DTT, the pellet was collected by centrifugation, air-dried and then suspended in SDT buffer containing 4% SDS, 1 mM DTT, 100 mM Tris-HCl (pH 7.5), sonicated for 2 min (with cooling on ice) and boiled for 15 min. The protein mixtures were harvested via centrifugation at 15,000× g for 20 min at 4 °C to remove insoluble material. Protein concentrations of experimental samples were quantified according to Bradford method [The fourth true leaves from C0 h, C6 h, C24 h and R24 h were fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.0) for 4 h at room temperature. Then the samples were handled as the previous operation . Leaf tod method . Albumin2 = 1:1, w/w) of TiO2 beads  for 40 min. The mixture was centrifuged at 10, 000× g for 3 min at 4 °C and the supernatant was discarded. After washing with 800 μL wash buffer (65% ACN/0.1% TFA) for 40 min, then centrifugation as above, then the phosphopeptides were eluted twice with 800 μL elution buffer (500 mM NH4OH/60% ACN), centrifugation and the eluate was dried and reconstituted in 0.1% formic acid/H2O for MS analysis.The same amount of extracted protein mixture in each sample was directly reduced with DTT, alkylated with iodoacetamide and subsequently digested with endoproteinase Lys-C and trypsin as previously described . The enrm/z) with the lock mass option enabled. A lock mass function was used to obtain high mass accuracy. The 12 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 37%. The threshold for precursor ion selection was 500 and the mass window for precursor ion selection was 2.0 Da. The dynamic exclusion duration was 120 s, the repeat count was 1 and the repeat duration was 30 s. Three biological replicates were performed independently from sample collection to the phosphopeptide identification using LC-MS/MS.The enriched phosphopeptides were separated on a self-packed C18 reverse-phase column  that directly connected to the nanospray ion source to an Q Exactive mass spectrometer  running in the positive ion mode. The pump flow was split to achieve a flow rate of 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% formic acid (A) and 0.1% formic acid and 90% ACN (B). A four-step linear gradient of 2% to 5% B in 5 min, 5% to 22% B in 55 min, 22% to 90% B in 5 min and keeping 90% B for 10 min. The spray voltage was set at 2.0 kV and the temperature of the heated capillary was 270 °C. For data acquisition, each MS scan was acquired at a resolution of 60,000 (at 400 p ≥ 0.75) and the PTM score (≥5) was considered as the reliable. An FDR of 0.01 was used for phosphorylation site identification.The raw files were processed with MaxQuant (version 1.3.0.5) and searched against the NCBI Jatropha protein database  concatenated with a decoy consisting of reversed sequences. The following parameters were used for database searches: trypsin/P was chosen as enzyme specificity, Carbamidomethyl (C) was selected as a fixed modification, Oxidation (M), Acetyl (Protein N-term) and Phospho (STY) were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm and the fragment ion mass tolerances for the MS/MS spectra were 20 ppm. The false discovery rate (FDR) was set to ≤0.01 for peptide and protein. The minimum peptide length was set to 6. The localization of the phosphorylation site was based on the PTM scores that assigned probabilities for each of the possible sites according to their site-determining ions. The software MaxQuant was used to calculate the PTM scores and PTM localization probabilities. Only when the phosphorylation site of localization probabilities  phosphopeptides detected in all three biological replicates; (2) phosphopeptides with credible ANOVA analysis or Student’s-test analysis (FDR<0.05); (3) phosphorylation localization probability ≥ 0.75 and (4) phosphorylation site score difference ≥ 5.http://www.blast2go.com/b2ghome), significantly enriched phosphorylation motifs were extracted from phosphopeptides with confidently identified phosphorylation residues using the Motif-X algorithm (http://motif-x.med.harvard.edu/). The phosphopeptides were centered at the phosphorylated amino acid residues and aligned and seven positions upstream and downstream of the phosphorylation sites were included. Because the upload restriction of Motif-X is 10 Mb, a FASTA format data set (nearly 10 Mb) containing the protein sequences from the J. curcas protein database was used as the background database to normalize the scores against the random distributions of amino acids. The occurrences threshold was set to 5% of the input data, set at a minimum of 20 peptides and the probability threshold was set to p < 10−6. The phosphoproteins blasted by the National Center for Biotechnology Information (NCBI) were used to obtain the KOG numbers of those proteins by eggNOG (http://eggnogdb.embl.de/). A data set containing all the KOG numbers was then used for PPI by using the Search Tool for the Retrieval of Interaction Genes/Proteins (STRING) database (http://string-db.org/) and the PPI network was displayed by Cytoscape software (Version 3.6.3).Protein function was annotated through Blast2GO software (J. curcas seedling was sensitive to chilling stress. It was studied by employing a comparative phosphoproteomic analysis, physiological measurement, ultrastructure observation under chilling stress and recovery. For the 805 significantly changed phosphopeptides, 9 phosphorylation motifs were extracted, which were mainly regulated by CDK, SnRK2, MAPK and CDPK. A complex PPI subnetwork were constructed and indicated the crucial roles of SnRK, 14-3-3 and ADP-ribosylation factor in the responsive network. Our results showed the phosphorylation was an essential event and responsible for chilling response and defense in J. curcas seedling. Consequently, Ca2+, ABA, ethylene, phosphoinositide and 14-3-3 mediated signal pathways cross linked for chilling response. In response to chilling stress, the phosphorylation of SnRK2a, ABI5 and TRAB1 were possibly related to ABA mediated signal pathway. Additionally, the phosphorylation level of transport, photoinhibition and chloroplast movement related proteins were also significantly regulated. We also highlighted that the phosphorylation of JcHOS1, JcKEG, JcAPX, JcPIP2 and JcPI4K might activate the potential functions to defense against chilling stress. Finally, we depicted a schematic presentation including signal transduction, metabolism and ion transport on the basis of changes at phosphorylation level, which is valuable for us to understand the chilling response and defense network in J. curcas seedling. In summary, the woody oil plant species,


Cyclina sinensis (CSH). CSH was separated into four different molecular weight (MW) fractions by ultrafiltration. Fraction CSH-I showed the strongest ACE inhibitory activity. A peptide was purified by fast protein liquid chromatography (FPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) and its sequence was determined to be Trp-Pro-Met-Gly-Phe . The Lineweaver-Burk plot showed that WPMGF was a competitive inhibitor of ACE. WPMGF showed a significant degree of stability at varying temperatures, pH, and simulated gastrointestinal environment conditions. We investigated the interaction between this pentapeptide and ACE by means of a flexible molecular docking tool. The results revealed that effective interaction between WPMGF and ACE occurred mainly through hydrogen bonding, hydrophobic interactions, and coordination bonds between WPMGF and Zn(II). In conclusion, our study indicates that a purified extract derived from Cyclina sinensis or the WPMGF peptide could potentially be incorporated in antihypertensive functional foods or dietary supplements.Marine-derived angiotensin-I converting enzyme (ACE) inhibitory peptides have shown potent ACE inhibitory activity with no side effects. In this study, we reported the discovery of a novel ACE-inhibitory peptide derived from trypsin hydrolysates of  Hypertension is the most common chronic disease and a critical factor in the development of cardiovascular pathologies. The number of yearly deaths due to hypertension complications is approximately 9.4 million worldwide , accountAngiotensin-I converting enzyme  belongs to the zinc-binding protease family , and plaSynthetic drugs such as captopril, enalapril, benazepril, and other ACE inhibitors  have beeSynodus macrops , and simulated intestinal fluid, SIF[sp]. The results showed that the ACE inhibitory activities of peptide WPMGF after incubation with SGF[sp] and SIF[sp] were 38.16 ± 0.52% and 49.40 ± 0.30%, respectively, whilst that of the control was 51.52 ± 0.72%  prosthetic group and with a binding energy value of −29.97 kJ/mol . The tissue homogenate was incubated 3 times with 0.1 M NaOH, and each incubation lasted 2 h, then filtered with a gauze. The filter was then washed with water until the pH reached neutrality, and the homogenate was dehydrated and stored in sealed bags at −20 °C until the next separation and purification steps.l-histidyl-l-leucine (HHL) were purchased from Sigma Chemicals Co. . Other chemicals and reagents used were of analytical grade, except acetonitrile and trifluoroacetic acid (TFA), which were HPLC grade .Trypsin  and pepsin  were purchased from YTHX Biotechnology Co., Ltd. . ACE (from rabbit lung), and the ACE synthetic substrate hippuryl-Cyclina sinensis tissue homogenate was subjected to hydrolysis with trypsin under the conditions reported by Luo et al. Any ACE inhibitory effects from other proteins can be excluded by testing a purified synthetic peptide, which can also be used in subsequent experiments. Mimotopes Biotechnology Co., Ltd.  synthesized the synthetic peptide using the Fmoc-solid phase method based on the sequence identified measured in the previous step. The identity of the peptide was verified by mass analysis using ESI-MS, and the purity of the synthetic peptide was detected above 95% by HPLC.−1 of HHL against the rate of formation V−1 of HA. The rate of HA generation was calculated based on the following equation:HAC is the concentration of HA (mmol/L).We followed the method described by Bita Forghani  to deterThe method described by Kittiphattanabawon et al.  was usedWe followed the methods described in the Pharmacopoeia of the People’s Republic of China 2015 Edition  (Volume [sp]): Concentrated hydrochloric acid (37%) was diluted to a 9.5% to 10.5% solution. Then, 800 mL of water and 10 g of pepsin were added to 16.4 mL of this diluted hydrochloric acid solution, mixed well, and the volume completed to 1000 mL with water.Simulated gastric fluid (SGF[sp]): 6.8 g of KH2PO4 was dissolved in 500 mL of water, and the pH was adjusted to 6.8 with 0.1 M NaOH. Then ten grams of trypsin were dissolved in an appropriate amount of water, added to the solution described above, and diluted to 1000 mL with water.Simulated intestinal fluid (SIF[sp] or SIF[sp] (0.5 mg/mL) and incubated for 2 h at 37.5 °C. In addition, the peptide solution was first incubated with SGF[sp] at 37.5 °C for 2 h, the solvent was removed by means of a centrifugal filter concentrator, and was then incubated with SIF[sp] for 2 h. The reaction mixtures were then boiled to inactivate the enzyme. After cooling to room temperature, samples were centrifuged at −4 °C  and the supernatant was adjusted to pH = 7 for the determination of the ACE inhibitory activity.The in vitro stability of the ACE inhibitory peptide in the simulated gastrointestinal environments was determined according to the method described by Yu et al . The moshttp://www.rcsb.org). Prior to docking analysis, water molecules and the Lisinopril inhibitor were removed using the Chimera 1.13 software  [Gbinding was computed according to the following equation:GvdW+hb+desolv is the sum of the van der Waals, hydrogen bond, and desolvation energies. ΔGelec is the electrostatic energy, ΔGintern is the total internal energy, ΔGtor is the torsional free energy, and ΔGunb is the unbound system’s energy.The crystal structure of human ACE (PDB: 1O86) was obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank  , whereasCA, USA)  with freAll experimental data were expressed as the mean ± standard deviation . The purified pentapeptide exhibited potent ACE inhibitory activity with an IC50 value of 0.789 mM, and its inhibitory pattern was shown to be of a competitive nature. WPMGF exhibited sustained thermal stability at 100 °C, maintained high inhibitory activity after being subjected to a wide range of pH values (2–8), and showed satisfactory residual activity after in vitro simulation of gastrointestinal digestive conditions. The peptide (WPMGF) has hydrophobic amino acid residues at both the C-terminus and N-terminus, a feature commonly found in peptides with good ACE inhibitory activity. Molecular docking simulation indicates that the peptide binds well to the active site of ACE and the conformation of the pentapeptide-ACE complex is stable. Therefore, this peptide derived from trypsinized hydrolysates of Cyclina sinensis, together with a certain proportion of food matrix components, could be considered in the future for the industrial production of functional foods and dietary supplements.This study reports, for the first time, that trypsinized hydrolysates of


Greenhouse‐gas‐induced warming in the Arctic has caused declines in sea ice extent and changed its composition, raising concerns by all circumpolar nations for polar bear conservation.Negative impacts have been observed in three well‐studied polar bear subpopulations. Most subpopulations, however, receive little or no direct monitoring, hence, resource selection functions (RSF) may provide a useful proxy of polar bear distributions. However, the efficacy of RSFs constructed from past data, that is, reference RSFs, may be degraded under contemporary conditions, especially in a rapidly changing environment.We assessed published Arctic‐wide reference RSFs using tracking data from adult female polar bears captured in the Beaufort Sea. We compared telemetry‐derived seasonal distributions of polar bears to RSF‐defined optimal sea ice habitat during the period of RSF model development, 1985–1995, and two subsequent periods with diminished sea ice: 1996–2006 and 2007–2016. From these comparisons, we assessed the applicability of the reference RSFs for contemporary polar bear conservation.In the two decades following the 1985–1995 reference period, use and availability of optimal habitat by polar bears declined during the ice melt, ice minimum, and ice growth seasons. During the ice maximum season , polar bears used the best habitat available, which changed relatively little across the three decades of study. During the ice melt, ice minimum, and ice growth seasons, optimal habitat in areas used by polar bears decreased and was displaced north and east of the Alaska Beaufort Sea coast. As optimal habitat diminished in these seasons, polar bears expanded their range and occupied greater areas of suboptimal habitat.Synthesis and applications: Sea ice declines due to climate change continue to challenge polar bears and their conservation. The distribution of Southern Beaufort Sea polar bears remained similar during the ice maximum season, so the reference RSFs developed from data collected >20 years ago continue to accurately model their winter distribution. In contrast, reference RSFs for the ice transitional and minimum seasons showed diminished predictive efficacy but were useful in revealing that contemporary polar bears have been increasingly forced to use suboptimal habitats during those seasons. Concurrently, average global air temperature rose > 1°C above preindustrial levels for the first time in 2015 . Across the Arctic, the presence of seasonal sea ice decreased 5 days decade−1 between 1979 and 2013 . Since the early 2000s, synergistic interactions between decreasing ice thickness, increased mobility and fracturing, and reduced surface albedo have rendered the ice pack more vulnerable to melting  and advance (5.3 days decade−1 later) has also occurred in the adjacent region that includes the Chukchi Sea polar bear subpopulation  model the relationship between animal locations and environmental covariates with the goal of estimating the relative probability of a resource being used , remained fairly consistent across those two decades despite declining ice trends. The assessment by Durner et al.  launched a suite of comprehensive studies to assess the long‐term impact of a changing Arctic on polar bears. One component of that work involved an international collaboration to assess observed and predicted changes in polar bear sea ice habitat throughout the Arctic Ocean and adjoining peripheral seas ], where β = 15). We assigned β = 15 because it performed well for our specific tracking data across seasons and regions. We excluded locations of bears that were on land or in maternal dens, and from PTTs that had become detached as evidenced by invariant activity sensor data, temperature sensor data emulating ambient conditions, or location data either persistently stationary or persistently following the prevailing ice drift.From 1985 to 2016, we captured adult female polar bears in the southern Beaufort Sea and equipped them with either collar, ear tag, or glue‐on platform transmitter terminals (PTT) that provided Doppler‐derived or GPS locations. We restricted our analysis to data from adult female polar bears because adult male and subadult polar bears cannot wear radio collars, and the vast majority of tracking data has been of adult females. We retained data from a PTT if it was deployed on a bear captured between 123° and 157° west longitude Figure . DAF ret2.2For the reference RSFs, we used the four seasonal RSFs , the four environmental covariates , and the original coefficients Table . Durner We obtained ocean depth from the International Bathymetric Chart of the Arctic Ocean (IBCAO) Version 3.0  encompassing all polar bear locations, 1985–2016. One location per day per bear was selected for the MCP analysis based on the best location quality ; ties were decided randomly. A single MCP was derived from the daily polar bear locations using the mcp function in the R package adehabitatHR Calenge, . The MCP2.4http://nsidc.org/arcticseaicenews/2018/09/arctic-sea-ice-extent-arrives-at-its-minimum/, accessed 26 February 2019).Identical to Durner et al. , we used2.5We assigned monthly SIC grids to one of four temporally dynamic seasons  using the methods from Durner et al. . Season 2.6Monthly RSF grids were derived using the appropriate seasonal model Table  with the2.7We calculated the percentage of polar bear locations within each equal area RSF zone using only higher‐accuracy locations  that were no less than 72 hr apart . Locations that occurred outside of RSF zones were excluded. For each decadal period, the seasonal mean percentage of bear locations within each equal area interval and 95% confidence intervals were calculated by bootstrapping. For each of 25 bootstrap iterations, we set the sample size to the number of bears within the respective period and season.2.86 km2). We emulated that method but calculated it for our study area (2.3 × 106 km2). We averaged the lower RSF threshold‐value for the 17th equal area bin  across all months for each season and every year during 1985–1995. We then calculated a single average for each season to establish the lower threshold of optimal polar bear habitat for the reference period and applied those thresholds to the respective seasonal RSF grids of subsequent periods.Durner et al.  chose thα < 0.05.To assess optimal habitat within an area overlapping the spatial distribution of polar bears, we developed kernel utilization distributions (UD) of polar bear locations for each decadal period and season. We defined an overall area of polar bear occupancy as the region that encompassed 95% of the UD, and a “core” area that included 50% of the UD. We used the same higher‐quality, temporally restricted subset of polar bear locations described above to generate period‐season UDs with function kernelUD in the R package adehabitatHR . A smaller proportion of polar bear locations in 1996–2006 and 2007–2016, relative to 1985–1995, often occurred in the highest RSF equal area zones during the ice melt season . The ability of the RSF to predict the distribution of polar bears during the ice minimum season after the reference period of 1985–1995 was often diminished . Like the ice melt and ice minimum seasons, there was often diminished ability of the RSF to predict the distribution of polar bears during the ice growth season after the reference decade of 1985–1995 . The ice maximum season had the best RSF performance and the most consistent performance in decades after the reference period. During ice maximum seasons, the proportion of polar bear locations occurring within equal area bins was similar across periods and CIs often overlapped , as there was less optimal habitat in 2007–2016 (10.1%) than in 1985–1995 (19.4%) and in 1996–2006 . During the maximum ice season, the percent of optimal habitat within the 50% UD was greater in 1996–2006 (50.1%) than in 1985–1995 (45.9%) and greater in 2007–2016 (48.6%) than in 1985–1995  as all periods were significantly different from the others , with, again, all periods were significantly different from the others , however, this was a result of a greater percentage of optimal habitat in 1985–1995 (35.1%) than in 1996–2006 (27.8%) and in 2007–2016 (25.0%; Figure The decrease in the average percent of monthly optimal habitat between periods was even greater within the 95% UDs Figure . OptimalAcross periods, optimal habitat centroids generally moved northward and eastward in the ice transitional and minimum seasons but were relatively unchanged during the ice maximum season Figure . The gre4Our assessment of Arctic‐wide seasonal RSFs from 1985–1995 when applied to environmental data in the Beaufort and Chukchi seas between 1996 and 2016 indicates that the efficacy of the reference RSF models to predict adult female polar distribution has diminished. In 1996–2006 and 2007–2016, and during seasons of ice melt, ice minimum, and ice growth, the distribution of polar bears and the distribution of optimal habitat showed relatively little overlap. During those seasons, as optimal habitat decreased and shifted north, polar bears expanded their range and used more sub‐optimal habitat. Utilization of suboptimal habitats can be maladaptive for wildlife (Hollander, Dyck, Martin, & Titeux, Across all seasons and Arctic‐wide, Durner et al.  reportedDuring spring ice melt, summer ice minimum, and autumn ice growth seasons, there was a diminishing amount of optimal habitat in our study area and within the kernel UDs of polar bear distribution Figure . Also, wA changing Arctic has diminished the abundance of optimal habitat for SB polar bears during the ice melt, minimum, and growth seasons. The loss of optimal habitat in these seasons, coupled with increasing duration (Stern & Laidre, Our findings elevate concerns for the future status of SB polar bears as the transitional seasons of sea ice lengthen and the extent of optimal polar bear habitat during those seasons declines. Indeed, the first conservation criterion of the U.S. Marine Mammal Protection Act is that the health and stability of marine ecosystems be maintained at a level such that polar bears may persist as “significant functioning elements” within those systems USFWS, . ConsequThe authors claim that they have no conflict of interest.G.M.D. and D.C.D. conceptualized the study and developed the problem formulation. G.M.D. conducted all analyses. G.M.D., D.C.D., and T.C.A. reviewed and interpreted the results of all analyses. G.M.D. led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.


Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1–PI3K–Akt–mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions.This article has an associated First Person interview with the first author of the paper. Summary: The authors present a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of sphingolipids undermines basic cell function, with potential implications for a variety of genetic sphingolipidoses and adult neurodegenerative conditions. Glycosphingolipids (GSLs) are key components of neural membranes, and defects in their metabolism have negative consequences on neuronal and glial survival . Severalin vitro and in vivo , resulting in its activation. After release from the membrane, Akt regulates a myriad of downstream targets in the cytoplasm, including GSK3β and mTOR complex 1 (mTORC1)  , 2009. L in vivo ; however(mTORC1) . In thisHere, we show that Akt inhibition by psychosine specifically takes place within LR, where psychosine prevents the recruitment of the key upstream kinases PI3K and mTORC2 to these domains downstream of IGF-1R activation. This inhibition in Akt phosphorylation can be overcome by sustained stimulation with IGF-1 but only during a time window before psychosine accumulation reaches a threshold level. We also show a previously unknown aspect of mTORC2 activation that involves its recruitment to and phosphorylation in LRs, which may be relevant in other contexts such as neurodegenerative diseases and cancer.in vitro model for psychosine toxicity studies .The IGF-1–Akt pathway is known to be downregulated by psychosine , but theFig. S1D, compare lanes 1 and 2). Therefore, GF-stimulated Akt phosphorylation is sensitive to inhibition by psychosine in NSC34 cells within the timeframe tested.We observed that switching to growth media containing fetal bovine serum (FBS) following psychosine treatment rescued cell morphology  and p-Akt levels B. A simiFig. S1B, lanes 1 and 2). IGF-1 leads to the phosphorylation of its receptor, followed by downstream phosphorylation of its known effectors: p-Akt (S473), p-GSK3β (S9) and p-p70S6K1 (T389). As control, p-PKCα/βII (Thr638/641), a GF-independent phosphorylation event, is not stimulated upon IGF-1 treatment in NSC34 cells. IGF-1 stimulates the phosphorylation of Akt at the expense of ERK, however , cumulative over time  and independent of receptor phosphorylation . We noticed that IGF-1 stimulation also triggered morphological changes in cells, such as inducing neurite-like formations. Similarly to as shown in Fig. S1F). Akt phosphorylation is fully recovered if psychosine is subsequently removed from the media during IGF-1 treatment, except when using higher doses of psychosine . Therefore, psychosine levels and the duration of exposure appear to be important factors in determining whether pathway inhibition can be overcome by GFs in vitro. Overall, these results suggest that damage to neuronal survival pathways begins shortly after cellular exposure to psychosine, accumulates over time, and is independent of cellular proliferation state. Most importantly, there may only be a brief time window during which inhibitory effects are reversible before psychosine accumulates to levels at which it causes irreversible neuronal damage.We next investigated whether pre-treatment with psychosine prior to IGF-1 stimulation would inhibit initial pathway activation. Cells were serum deprived and treated with 10 µM psychosine for 1 h, followed by IGF-1 (100 ng/ml) for 15 min E. Pre-trFig. S1B). Cells were serum deprived for 24 h, pre-treated for 1 h with 10 µM psychosine with or without 250 nM okadaic acid (OA), followed by IGF-1 (100 ng/ml) stimulation for 10 min . Additionally, specific inhibition of protein phosphatases PP1 and PP2A , or the inhibition of lipid phosphatases PTEN and Ship2 , all had similar effects in rescuing p-Akt, p-p70S6K1 and p-ERK . Protein/lipid phosphatases are the main mechanism employed by cells to quench and fine-tune signal intensity following pathway activation, and the above phosphatases have all been implicated as negative regulators of the IGF-1R pathway. It is perhaps not surprising that inhibition of any of these upstream negative regulators would lead to an increase in pathway activity and hence rescue Akt phosphorylation. Therefore, we conclude that, even though a psychosine-mediated increase in phosphatase activity may be contributing to reduced Akt phosphorylation, it does not appear to be the sole mechanism of inhibition by psychosine.Psychosine causes an upregulation in the activities of cellular protein phosphatases PP1 and PP2A, leading to a loss in the phosphorylation of GSK3β and neurofilaments , 2012. Tr 10 min F. Althour 10 min F, it hadFig. S1B, compare lanes 2 and 6), and can be antagonized by elevating PIP3 levels indirectly by inhibiting the lipid phosphatases PTEN and Ship2 . Therefore, we hypothesized that there would be synergy between the inhibition of PI3K activity, and hence levels of PIP3 generated, and the inhibition of p-Akt by psychosine, especially when combined at suboptimal doses. Cells were serum deprived and pre-treated for 30 min with psychosine , PI3K/p110α inhibitor wortmannin , or a combination of both at each dose. Cells were subsequently stimulated with IGF-1 (100 ng/ml) under the same respective pre-treatment conditions and the levels of p-Akt and its downstream target p-GSK3β were assessed in whole-cell lysates using western blotting. As expected, either 10 μM psychosine or robust inhibition of PI3K activity by 1 µM wortmannin significantly but not completely blocks Akt phosphorylation downstream of receptor activation . IGF-1R is unique in that it is embedded in the plasma membrane as an inactive hetero-tetramer, and this autoinhibition is released upon ligand binding as opposed to the activation of receptors such as EGFR and insulin receptor, for which dimerization through ligand binding is required for activation. Additionally, plasma membrane rigidity does not affect ligand binding to IGF-1R . Similarly, IGF-1 stimulates, and 1 µM wortmannin significantly inhibits, p70S6K1 phosphorylation (i.e. mTORC1 activity) and its downstream target, p-Rictor (T1135) (i.e. p70S6K1 activity) . We therefore conclude that both are activated by the IGF-1R–PI3K axis in NSC34 cells. Treating cells with 10 µM psychosine in addition to 1 µM wortmannin eliminates p70S6K1 phosphorylation , which can be rescued by elevating PIP3 levels through PTEN/Ship2 inhibition . p-Rictor (T1135) follows the same pattern , suggesting distinct regulation of Akt targets based on the compartment in which Akt is activated  domain, and rendering the protein in a conformation amenable for phosphorylation by upstream kinases in the absence of GF stimulation . Therefoctivated . As expectivated B. This rctivated A, pointsFig. S3A). Cells were serum-deprived for 6 h followed by IGF-1 (100 ng/ml) stimulation for 15 min, and were subjected to fractionation of subcellular compartments. IGF-1 leads to IGF1-Rβ and Akt phosphorylation at the membrane, with subsequent translocation of p-Akt to the cytoplasm. p70S6K1, its target Rictor, as well as mTORC2 are also phosphorylated at the membrane in response to IGF-1 pathway activation, but not ERK1/2 or PKCα. Membrane-associated p70S6K1 likely catalyzes Rictor phosphorylation within that compartment, since an exclusively cytoplasmic target of p70S6K1, p-IRS-1 (S636/639), is phosphorylated only within the cytoplasmic fraction . Results from our studies using wortmannin and SC-79 suggest that a membrane-associated step other than receptor phosphorylation in the Akt activation cascade may be compromised by psychosine. To further understand the mechanism of this inhibition, we compared the dynamics of IGF-1-mediated pathway activation in the presence or absence of psychosine within distinct subcellular compartments. Since this has not been studied in NSC34 cells before, we first determined the subcellular localization of IGF-1R pathway components and their phosphorylation in response to IGF-1 stimulation . As expected, psychosine treatment caused a significant inhibition of Akt activation in whole lysates without inhibiting receptor phosphorylation. This Akt inhibition is also reflected separately within the membrane, cytoplasmic and nuclear fractions. To our surprise, psychosine also caused an inhibition of mTORC2 phosphorylation, but not that of mTORC1, specifically at the membrane, an effect likely missed in total lysates due to dilution by the large volume of the cytoplasmic fraction. Based on this observation, we predict that the decrease in p-Akt levels in the cytoplasm and nucleus are collateral to an initial absence of Akt phosphorylation at the plasma membrane due to failed mTORC2 activation. Furthermore, our data suggest that Akt phosphorylation within the cytoplasmic and nuclear compartments can be rescued by IGF-1 stimulation at the plasma membrane when psychosine is present  stimulation with continued presence of psychosine. At the end of stimulation, we subjected cells to subcellular fractionation and compared phosphorylation levels in each fraction with total lysates  at least within the timeframe that we have tested. Stimulation of Rictor phosphorylation is mediated by mTORC1/p70S6K1 downstream of Akt, as the mTOR inhibitor rapamycin inhibits SC-79-stimulated Rictor phosphorylation . We should also note that there is appreciable Rictor phosphorylation in the absence of p-Akt . This suggests that Rictor is under the regulation of different kinases under different conditions, with p70S6K1 being the main kinase under IGF-1 stimulation. We have not investigated the contribution of p-Rictor to overall mTORC2 activity in detail; however, we expect that the decrease in p-Rictor levels might additionally contribute to mTORC2 inhibition by psychosine, especially since Rictor translocation out of the nucleus is regulated by phosphorylation  stimulation for 15 min with continued presence of 10 µM psychosine. Cells were homogenized (H) and post-nuclear plasma membrane (PM) fractions were isolated using 30% Percoll. Purified PM was sonicated and subjected to fractionation on a 5-35% sucrose gradient overnight using a detergent-free method. Fractions 1-10 (low to high density) were collected, and proteins in each fraction were precipitated and resuspended in an equal volume of loading buffer. An equal volume of resuspended protein from each fraction was loaded in individual lanes and subjected to immunoblotting. Under basal conditions, there is minimal Akt protein at the PM A, row 1.We next asked whether we would see a similar defect in LR recruitment in cells that have first been stimulated with IGF-1 followed by psychosine treatment, as opposed to pre-treatment with psychosine followed by IGF-1. Although the outcome is analogous in terms of decreased Akt phosphorylation overall compare B. LR fraFigs S1B, S2A). Similar to conditions in In addition, the level of IGF-1R is not affected, consistent with our previous observation that inhibition is downstream of receptor activation . In this scenario, we can fully rescue the levels of Akt phosphorylation judging from whole-cell lysates ; however, the pattern of p-Akt distribution on the plasma membrane is altered . There is ectopic recruitment and activation of Akt outside of LRs in cells treated with psychosine prior to IGF-1 stimulation, but only when psychosine is removed from media during IGF-1 incubation. This appears to result from an increased recruitment of PI3K components p110α/p85, followed by PDK1 and mTORC2, to non-raft regions , even though there is no change in upstream IGF-1R activity.Akt distribution was not significantly affected upon psychosine treatment especially when we did not detect any Akt phosphorylation under this condition (data not shown). This is in contrast to our observation in This suggests that structural perturbations in LRs persist even after psychosine is removed, resulting in altered patterns of Akt activation. In summary, the deregulation in Akt phosphorylation is a result of a rapid redistribution of its upstream kinases on the plasma membrane due to disruption of the LR structure by psychosine . Hence, in vivo. Since effects in NCS34 cells are visible soon after psychosine exposure, we hypothesized that a dysregulation in signaling would start at a much earlier time point at the cellular level, before symptoms presented themselves visibly at the peak of myelination after postnatal day 20 (P20). For this, levels of Akt and mTOR phosphorylation were determined in protein extracts from brains of wild-type and pre-symptomatic twitcher mice at P15  on how psychosine could inhibit mTORC2 activity based on our observations from the two conditions that we have tested: in cells treated with psychosine prior to IGF-1 stimulation versus treated with psychosine after IGF-1 stimulation. Although these conditions correspond to the inhibition of initial activation (A lack of mTORC2 activation in LRs is the immediate step that leads to Akt inhibition. PI3K is necessary for receptor tyrosine kinase (RTK)-mediated mTORC2 activation, and the binding of PIP3 directly to one of the mTORC2 subunits relieves its auto-inhibition, stimulating its kinase activity towards Akt . In our tivation F and dowtivation G, respecFig. S1G); (3) mimicking increased cytoplasmic PIP3 levels using SC-79 is sufficient to bypass inhibition at the plasma membrane and stimulate mTORC2 activity towards Akt  inards Akt ; and (5)ards Akt A. The fiards Akt . These oards Akt B, are fuFig. S4).In the dispersion model G, in whiFinally, mTORC1 and mTORC2 have diverse functions within the cell that are tightly regulated by nutrients and GFs . Unlike In conclusion, our work predicts that pathway activities that rely mainly on LR integrity in any given cellular context would be affected by psychosine treatment, especially GF-mediated RTKs and chemokine-mediated G protein-coupled receptors. Examples include IGF-1-mediated Akt activation in muscle cells, TGFβ-mediated ERK activation in epithelial cells, or LR-mediated signaling in neuroinflammation. We hypothesize that, depending on which pathways are active in any given cell type at any given time, the ones that depend significantly on LRs for activation and/or continued activity will be sensitive to psychosine exposure.The fact that psychosine exposure does not completely shut down Akt phosphorylation, and that IGF-1R pathway components are intact and inhibition is reversible under certain conditions, offers a window of opportunity for therapeutic interventions. An attractive idea for therapy would be to treat patients with IGF-1 and/or other GFs to improve intracellular signaling and therefore cellular resilience to psychosine. We believe that this treatment should be done in conjunction with hematopoietic stem cell transplantation or gene therapy, and continued for a specified period of time after genetic correction. Persistence of psychosine in rafts is a limiting factor for therapeutic effectiveness in mouse models of KD , suggestTable S2), and imply that neurogenesis, and possibly other developmental processes, could be affected during this period in utero. There have been cases of some infants already born with symptoms, and by this point the damage is irreversible and these infants are no longer candidates for transplantation. Therefore, it may be essential for combination therapies to be initiated at the earliest time point possible.Additionally, we believe that treatment should begin very early to be effective. We see negative effects on cellular signaling soon after the cells are exposed to psychosine A, and thIn conclusion, surmounting evidence suggests that psychosine exerts its effects mainly through structural disruption of LRs rather than specific psychosine-protein interactions, with its demonstrated interaction with α-synuclein being an exception . We hypoThe use of twitcher mice and the experimental studies were approved by the Animal Care and Use Committee of the University of Illinois at Chicago (protocol number 15–101). Breeder twitcher heterozygous mice  were originally purchased from Jackson Laboratory, maintained and treated under standard housing conditions using the Animal Care and Use Committee protocols of our institution. Male and female twitcher mice were used indistinctly. Mice were anesthetized by isofluorane, and killed by cervical dislocation.’s protocol. Neurospheres (passage 8) were dissociated, plated on Matrigel-coated 6-well plates (0.5×106 cells/well), and differentiated into mixed glial cultures for 2 days in vitro (DIV) using Neurocult media containing differentiation supplement (cat. no. 05704). Whole-cell lysates were prepared in RIPA buffer with protease and phosphatase inhibitors. An equal amount of protein was analyzed from wild-type and twitcher cultures on a western blot.Neural progenitors were isolated from wild-type and twitcher embryonic telencephalons at E12 according to previously published protocols . Neural Neuronal progenitors from wild-type and twitcher mice were grown under differentiation conditions for 7 DIV, and cultured in the presence or absence of IGF-1 (100 ng/ml) for 24 h. Cells were fixed in 4% paraformaldehyde/PBS and then blocked in 5% BSA/PBS. Cells were incubated with 1:100 dilution of cholera-toxin–Alexa-Fluor-488 (Molecular Probes) and 1:100 dilution of rabbit anti-PI3K  at 4°C overnight. After incubation with anti-rabbit IgG-Alexa-Fluor-546 , samples were confocally imaged in an upright Leica TCS DM5500Q confocal microscope .4 NSC34 cells were plated on Matrigel-coated 6-well plates (day 0) and grown in 5% FBS/DMEM for 3 days until 60% confluent (day 3). If conditions did not require serum deprivation, cells were washed with warm 1× PBS on day 3 and treated with psychosine working solution with the indicated concentrations  at 37°C for the indicated times . If conditions required serum deprivation, cells were washed with warm 1× PBS on day 3, and serum-free DMEM was added for 24 h. Each treatment condition was performed in triplicate on day 4 (i.e. the same condition was used in three separate wells on a 6-well plate). After treatment, cells were washed in cold 1× PBS and 80 μl of ice-cold RIPA lysis buffer  containing protease/phosphatase inhibitors  were added in each well and incubated on ice for 15 min. Lysates were transferred to an Eppendorf tube and centrifuged at 16,000 g for 30 min, and supernatant was transferred to a fresh tube. Protein concentrations were determined using the Pierce MicroBCA Protein Assay kit (cat. no. 23235). To analyze phosphorylation levels, an equal amount of protein (5-7.5 μg) from each triplicate per condition was pooled and run in a single lane on a 4-12% NuPAGE Bis-Tris protein gel (Invitrogen). This was repeated at least three times for each condition, representing the average of nine data points per condition. See Table S1 for a complete list of inhibitors and reagents, and the concentrations that were used.Details of the NSC34 cells used in this study were previously published elsewhere . NSC34 cg for 2-3 min. For whole-cell lysates, cell pellet from the INPUT fraction was processed with RIPA buffer containing protein and phosphatase inhibitors, and vortexed periodically to lyse the cells while being kept on ice. Cells were then centrifuged at 16,000 g for 10 min and supernatant was saved as INPUT whole-cell extract (WCE). To obtain the cytoplasmic fraction, cold cytosol extraction buffer (CEB) with protease and phosphatase inhibitors was added to the remaining pellet (∼20 μl packed volume) and incubated for 10 min at 4°C while rotating. Cells were centrifuged at 500 g for 5 min at 4°C and supernatant was collected as the cytoplasmic extract (CE). The CE was centrifuged once again at 16,000 g for 10 min at 4°C to further clear the fraction. Cold membrane extraction buffer (MEB) (+inhibitors) was added to the pellet, vortexed for 5 s and incubated at 4°C for 15 min while rotating. Lysate was centrifuged at 3000 g for 5 min at 4°C and supernatant was collected as the membrane extract (ME). Cold nuclear extraction buffer (NEB) (+inhibitors) was added to the pellet, vortexed for 15 s and incubated for 30 min at 4°C while rotating. Lysate was centrifuged at 5000 g for 5 min at 4°C and supernatant was collected as the soluble nuclear extract (SNE). Protein concentration in each fraction was determined with the Pierce MicroBCA Protein Assay kit (cat. no. 23235) and 10 μg protein from each fraction for each condition was run on a western blot for comparison.Subcellular fractionation of NSC34 cells was performed using the Thermo Fisher Scientific Subcellular Protein Fractionation Kit for Cultured Cells (cat. no. 78840) according to the manufacturer's protocol. A total of 750,000 NSC34 cells were transferred to a T75 flask and grown in complete media (5% FBS/DMEM) for 3 days until 50-60% confluent, and serum deprived in DMEM for 24 h. Following treatment, cells were dissociated from the flask using Cellstripper solution , 5 ml cold DMEM (no FBS) was added and cell suspension was transferred to a 15 ml Falcon tube. Cells were centrifuged at 4°C, 1000 rpm for 5 min, and the supernatant was discarded. The pellet was resuspended in 1 ml cold PBS; 100 μl was transferred to an Eppendorf tube and saved for INPUT (to obtain whole-cell lysate); the remaining cell suspension (900 μl) was transferred to another Eppendorf and centrifuged at 4°C, 500 Table S1. For antibody validation profiles, please refer to the manufacturer’s website.Samples were run on a 4-12% NuPAGE Bis-Tris protein gel (Invitrogen) using Invitrogen NuPAGE™ MOPS SDS Running Buffer (cat. no. NP000102) for 2.5 h at 120 V, and proteins were transferred onto PVDF membrane in a wet transfer tank using Invitrogen NuPAGE™ Transfer Buffer (cat. no. NP00061) (without methanol) for 2 h at 350 mA. After the wet transfer, PVDF membranes were transferred to a container and covered in 100% methanol, and left to dry overnight at room temperature. In our experience, this helped with the binding of proteins to the membrane surface and gave better chemiluminescent signals. PVDF membranes were then cut along molecular mass markers into multiple pieces containing distinct proteins of interest . When ready for blotting, PVDF sections were re-activated briefly in methanol, and blocked in 5% milk at 4°C overnight. For comparing phosphorylated versus total protein , membranes were first incubated with the phospho-antibody, stripped in stripping buffer and re-probed with an antibody for the non-phospho protein. Band intensities were quantified using the freely available NIH ImageJ software. For a complete list of antibodies and reagents used for the study, please see 6 NSC34 cells were seeded in a T175 flask and grown in complete media for 3 days until 50-60% confluent, washed twice with warm 1× PBS and serum deprived for 24 h. Following treatment, cells were immediately placed on ice and scraped in detergent-free Tricine buffer . Cellular material was homogenized and centrifuged at low speed (1400 g for 5 min at 4°C) to precipitate nuclear material. Resulting supernatant [homogenate (H)] was collected, mixed with 30% Percoll in Tricine buffer and subjected to ultracentrifugation for 25 min  to collect the plasma membrane fraction (PM). PMs were collected and sonicated (3×3-s bursts). The sonicated material was mixed with 60% sucrose , overlaid with a 5-35% step sucrose gradient and subjected to overnight ultracentrifugation . Fractions were collected every 400 μl from the top sucrose layer and proteins were precipitated using 0.25 volume TCA–deoxycholic-acid in double-distilled water [0.1% (wt.vol) deoxycholic acid] to precipitate proteins. Protein pellets were washed twice with 100% acetone and air dried. Pellets were resuspended in an equal amount of loading buffer; an equal amount of each fraction was run in separate lanes on a western blot.A total of 1×10Brains were collected from P20 wild-type and homozygous twitcher mice, homogenized in Tricine buffer, and processed as above. Male and female twitcher mice were used indistinctly.g for 15 min and the supernatant was transferred to a fresh tube and centrifuged again to clear the lysate at 16,000 g for 30 min. Supernatant was transferred to another tube and protein concentrations were determined using the Pierce MicroBCA Protein Assay kit (cat. no. 23235).Brain tissue was collected, immediately immersed in liquid nitrogen and stored at −80°C until further use. Male and female twitcher mice were used indistinctly. Tissues were thawed on ice, quickly rinsed in ice-cold 1× PBS and processed to obtain whole-cell lysates by homogenization in RIPA buffer  containing protein and phosphatase inhibitors . Lysates were incubated on ice for 30 min and vortexed every 10 min. Lysates were centrifuged at 16,000 Table S2A). Differentiated cells were sent to the Microwestern Array Core Facility (MWAC) at The University of Chicago, USA, where cell extracts were blotted for quantitative analysis of an array of pre-selected antibodies (Table S3). Each antibody was tested in duplicate per sample. For further details on microwestern array methodology, please refer to (http://www.igsb.org/services/mwac-methodology).Neural progenitors were isolated from wild-type and twitcher spinal cords at E12 and grown as neurospheres according to previously published protocols . NeurospPrimary glia were isolated from the cortex of wild-type myelin proteolipid protein (PLP)-GFP pups at postnatal day 3, and grown in glia media according to published protocols . Glial ct-test. *P<0.05; **P<0.01; ***P<0.001.No pre-specified effect size was assumed in experiments. For mouse experiments, 3-5 mice per genotype were used. For cell culture, experiments were repeated at least three times with three technical replicates each. Values are mean±s.d. of at least three independent experiments. Statistical analysis was done using Student's


However, the use of 15O-oxygen has been limited in a clinical environment largely attributed to logistical complexity, in relation to a long study period, and the need to produce and inhale three sets of radiopharmaceuticals. Despite the recent works that enabled shortening of the PET examination period, radiopharmaceutical production has still been a limiting factor. This study was aimed to evaluate a recently developed radiosynthesis/inhalation system that automatically supplies a series of 15O-labeled gaseous radiopharmaceuticals of C15O, 15O2, and C15O2 at short intervals.15O, 15O2, and C15O2; an inhalation controller; and an inhalation/scavenging unit. All three parts are controlled by a common sequencer, enabling automated production and inhalation at intervals less than 4.5 min. The gas inhalation/scavenging unit controls to sequentially supply of qualified radiopharmaceuticals at given radioactivity for given periods at given intervals. The unit also scavenges effectively the non-inhaled radioactive gases. Performance and reproducibility are evaluated.The system consists of a radiosynthesizer which produces C15O-dedicated cyclotron with deuteron of 3.5 MeV at 40 μA, C15O, 15O2, and C15O2 were sequentially produced at a constant rate of 1400, 2400, and 2000 MBq/min, respectively. Each of radiopharmaceuticals were stably inhaled at < 4.5 min intervals with negligible contamination from the previous supply. The two-hole two-layered face mask with scavenging device minimized the gaseous radioactivity surrounding subject’s face, while maintaining the normocapnia during examination periods. Quantitative assessment of net administration doses could be assessed using a pair of radio-detectors at inlet and scavenging tubes, as 541 ± 149, 320 ± 103, 523 ± 137 MBq corresponding to 2-min supply of 2574 ± 255 MBq for C15O, and 1-min supply of 2220 ± 766 and 1763 ± 174 for 15O2 and C15O2, respectively.Using an 15O-labeled radiopharmaceuticals as required in the rapid 15O-Oxygen PET protocol. The production and inhalation were reproducible and improved logistical complexity, and thus the use of 15O-oxygen might have become practically applicable in clinical environments.The present system allowed for automated production and inhalation of series of  Specifically, it has been utilized to quantitatively assess the regional cerebral metabolic rate of oxygen (CMRO2) and the regional oxygen extraction fraction (OEF) [15O-labeled water (H215O) or inhalation of carbon dioxide (C15O2), and cerebral blood volume (CBV) using inhalation of 15O-carbon monoxide (C15O). The technique has contributed to the understanding of pathophysiology and to the development of guidelines for treating patients with stroke and cerebral vascular diseases [18F-fluorodeoxyglucose (FDG) and 15O-oxygen PET imaging are related to malignancy in brain tumor [xygen 15O PET is uxygen 15O PET is uin tumor , and alsin tumor  and morein tumor –6.15O-oxygen inhalation are labor intensive and have not been considered practical if one intends to utilize it in a clinical environment, largely attributed to the need for producing three different radiopharmaceuticals labeled with 15O in parallel to PET imaging [15O2 and H215O in the arterial blood during the PET scan. Naive corrections are needed for radioactivity decay, and for the delay and dispersion of the arterial blood radioactivity curves [Typical procedures in a PET examination with  imaging . Typical imaging  and the  imaging –11 requiy curves , 13. It 15O-gases in 3D PET by means of the improvement of the scatter correction procedures [15O-oxygen has been well validated [15O2 and H215O (or C15O2) are sequentially administered [Recently, significant progress has been reported in the instrumentation and reconstruction software implemented in commercial 3D PET scanners. High-speed electronics offer precise data acquisition with considerably reduced random events. Consequently, the accuracy and signal-to-noise ratio are greatly improved , allowinocedures , suggestocedures . It shoualidated . This alnistered , 26. Suc15O-labeled radiopharmaceuticals. The authors then hypothesized that this may be overcome by using an automated system that enables sequential production and inhalation of the 15O-labeled radiopharmaceuticals with minimal human operation and recording of all signals from the radiopharmaceutical production/inhalation system, and other peripheral equipment utilized in the 15O-Oxygen PET experiment. Recently, a system has been developed to produce and inhale a series of 15O-labeled radiopharmaceuticals of C15O, 15O2, and C15O2 sequentially with minimal human operations, as required in given protocols of the three-step autoradiography [15O-oxygen PET examinations. We then investigated the radioactivity recovery, and chemical and radiochemical purity in the 15O2 and C15O productions for different carrier gas concentrations. We further evaluated impact of reducing the leakage of radioactive gases from the inhalation face mask, and potential ability to estimate the administration dose to individual patients.The logistical complexity remains the major limitation in producing a series of iography  and the iography , 26. The15O-oxygen inhalation following the protocol of Kudomi et al. [15O, 15O2, and C15O2. Data previously conducted as clinical examinations between November 20, 2012 and March 31, 2015 (2.4 ± 1.2 examinations per day) were retrospectively analyzed. The data were from PET examinations on 749 patients, and included (a) the time chart log of the radiosynthesizer, which includes the time-radioactivity curves of the radio-detectors and gas chromatograms; (b) AIF recorded by the functional image calculation software; (c) record of Capnometer; (d) records of the time-radioactivity curves of the area monitor in a PET room, of the air in the exhaust duct from a PET area, and the cyclotron area; and (e) radioactivity curves of a pair of beta-ray detectors placed each of inlet and scavenge tubes of the facemask with the flow values for each tubes. The patients were 433 males and 316 females with a body weight of 60.4 ± 11.4 kg and an age of 59.8 ± 26.0 years old. The study was approved by the Ethics Committee at the authors’ institution with the approval number M30-013 for retrospective data analysis on clinical data and M22-089-2 for PET imaging on healthy volunteers.The automated radiosynthesis/inhalation system approved as a medical device in November 2012 in authors’ country has been utilized on more than 2500 clinical scans with i et al. . Basic p15O production from IBA Radiopharma Solutions , is used. The deuteron beam was approximately 3.5 MeV at a beam current of 40 μA on the target. An additional radio water generator (RWG) from HIDEX  was placed to produce 15O-labeled water (H215O) in parallel to the present system.Figure 15O-gaseous radiopharmaceuticals were controlled by a sequencer, which allowed for automatic production. The system was also designed to accommodate changes in the order of sequential radiopharmaceutical productions, the amount of radioactivity, and the period of the inhalation. This study employed a fixed protocol in which where the radiosynthesis and in halation was first C15O followed by a sequential supply of 15O2 and C15O2 at a 4.5-min interval.Each radioactive gas was continuously circulated during the beam bombardment to the target from the cyclotron target to the radiosynthesizer, the inhalation controller, and to a decay tank  using a hybrid flow meter . A calibrated gamma-ray monitor  was placed inside the exhaust duct to monitor the gaseous radioactivity carried from the PET room, and also from the entire facility during the PET examination period.Due to the requirement for radiation safety regulation in the authors’ country, air of the entire PET room was actively exhausted at a flow rate of approximately 2000 m15O-radioactivity and the radiosynthesis of C15O, 15O2, and C15O2 followed the method described by Clark and Buckingham [15O was produced by the 14N15O nuclear reaction in the cyclotron target, in which the target gas to produce 15O2 was nitrogen (N2) containing oxygen (O2) as a carrier gas. The concentration of the carrier gas of O2 was chosen at 0.3% in all clinical studies according to a systematic evaluation in this study (see below). The 15O2 produced in the target was carried to a soda lime column to remove contamination, which was mostly C15O2. C15O2 was produced also in the target filled with N2 containing 1.0% of CO2. The C15O2 produced in the target was funneled to a charcoal column heated to 430 °C to remove contamination of C15O. C15O was synthesized from the 15O2 gas by carrying it away from the target into a charcoal column heated to 1000 °C. A soda lime column was then used to remove contamination, mostly C15O2. Each of the radioactive gases produced were continuously carried to the inhalation controller placed in the PET scanner room during deuteron beam bombardment and then to a compressing tank  was 1000 mL/min by controlling MFC2 and MFC4. The radioactivity supply rate, defined as F2 × R1, was at approximately 1300 MBq/min, 2400 MBq/min, and 2000 MB/min, respectively. The supply period for each radiopharmaceutical was 2 min, 1 min, and 1 min, yielding the total radioactivity supply, defined as (F2 + F4) × R2 × the supply period, was to be 2600 MBq, 2400 MBq, and 2000 MBq corresponding to C15O, 15O2, and C15O2, respectively.While the synthesizer was producing radioactive radiopharmaceuticals, a radioactive gas was carried to the inhalation controller installed in the PET room and circulated to the waste tank (tank 1) in the cyclotron room. This gas was released from tank 1 after sufficient decay. The inhalation controller then supplied radioactive radiopharmaceuticals to a patient’s face mask according to the given protocols. In the clinical protocols at our institution, the supply flow rate defined as , implemented Molecular Sieve 5A (MS) at 120 °C and Pora-Plot Q (PPQ) at 60 °C with capillary columns that are 10 m long, respectively. The gas samples were taken prior to each initiation of the radiopharmaceutical supply.15O inside of the facemask, enabling accurate delineation of the face mask contour [The inhalation/scavenging unit was designed to maintain the normocapnia of a patient during PET examination by introducing fresh air into the face mask while scavenging the gaseous radioactivity inside of the facemask to the decay tank tank 2). The unit consisted of a two-hole two-layered face mask  was used to monitor the end-tidal carbon dioxide partial pressure and the respiration rate during PET scanning. The tube for this Capnometer was also used to supply radioactive gases through a small-diameter tube into a space inside the inner layer of the facemask between the nose and the mouth.15O-oxygen production/inhalation system  via the RWG unit installed in the PET room  was placed in parallel to the All experiments and clinical scans were carried out by a deuteron beam bombardment of 40 μA on a target. The pressure of the target gas was 0.08 MPa. The flow rate of MFC1 in the synthesizer was 500 mL/min, and the supply flow rate to the face mask (MFC2 + MFC4) was 1000 mL/min.2 was evaluated for a production of gaseous 15O2, and consequently synthesized gaseous C15O and H215O in saline. The experiment was carried out for a target gas of N2 containing 0.3%, 0.5%, and 1.0% carrier gas of oxygen (O2). 15O2 and C15O were continuously produced and carried to the inhalation unit. The radioactivity recovery was determined for a 1-min supply for both 15O2 or C15O. Gaseous 15O2 was also carried to RWG to generate H215O saline, and the recovery was determined for 1-min sampling of H215O in the saline. The radioactivity recovery of C15O2 was also assessed similarly using a target gas containing 1.0% carbon-dioxide (CO2) in N2. The radiochemical purity and non-radiochemical purity were assessed for each of C15O, 15O2, and C15O2 productions. These measurements were repeated six times for each radiopharmaceutical.The recovery and the dependency on the carrier gas concentration of O15O2 when the sequential 15O2–C15O2 inhalation protocol at 4.5-min interval was utilized as employed in the clinical examination. The radioactivity was assessed with and without producing the second radiopharmaceutical of C15O2. This experiment was also repeated six times.The radiochemical contamination was determined in the second administration namely C15O, 15O2, and C15O2 inhalation scans according to the fixed protocol, with inhalation periods of 2 min for C15O, and 1 min for 15O2 and C15O2. Radioactivity supply as calculated as an integral of the radioactivity concentration in the supply tube multiplied by its flow rate, and the net administration as calculated as an integral of the radioactivity concentration in the scavenging tube multiplied by its flow rate for each of C15O, 15O2, and C15O2 inhalation scans. The net administration dose was then calculated as a subtraction of those two values for each radiopharmaceutical. Reproducibility of supply and net administration dose among subjects were evaluated. Time-dependent net administration radioactivity was also compared with the arterial input function (AIF) estimated from the continuously monitored arterial radioactivity time-activity curves (TACs).Retrospective analysis was carried out using the data recorded for 749 subjects who underwent all C15O2 inhalation scan and was compared with AUCs of the directly assessed AIF for each patient scan. It should be noted that the delay and dispersion were carefully compensated in AIFs as described in earlier works [15O2 inhalation.The effective administration dose was divided by the body weight of the patient for the Cer works , 13, 30.15O2 and C15O2 inhalation periods. Evaluation was also carried out on functional images of CBF, CMRO2, and OEF calculated according to two mathematical formulations, i.e., the DARG method [215O and 15O2, denoted as V0W and V0O, respectively.Scavenging effectiveness of the two-hole two-layered facemask was evaluated on reconstructed tomographic and maximum intensity projection (MIP) images during G method   and MCF4 (F4)  × R2  because a non-radioactive target gas was carried to the radio-detector RD1, and then gradually increased after 135 s when a new radioactive gas of C15O2 reached to the line. Flow (MFC2) was stopped (arrow b) for approximately 18 s in order to take a sample for the gas chromatography (GC) at 200 s.Figure F4) Fig. c, and th R2 Fig. d among s15O, 15O2, and H215O for three of the target gases with different carrier concentrations of O2  in the N2 gas. Production yields of 15O2 and H215O were not significantly different among the three O2 carrier gas concentrations. However, the C15O production was significantly smaller when a 0.3% concentration of O2 was selected as compared with 0.5% and 1.0% concentrations. The radioactivity yield was 2976 ± 78 MBq for a C15O2 production using a target gas containing 1.0% carbon dioxide (CO2) in the N2 gas with a beam intensity at 40 μA, which was significantly greater than that for an 15O2 production.Table 15O, 15O2, and C15O2 productions, in which the target gas was fixed at 0.3% O2 in N2 for C15O and 15O2 productions, and at 1.0% CO2 in N2 for a C15O2 production. Because the target gas was mostly nitrogen gas, the nitrogen concentration is the highest in all three radiopharmaceuticals. It is noteworthy that the CO contamination was the smallest and approximately 0.6% with 0.3% of O2 carrier gas in N2 target gas. The CO contamination increased when higher concentration of CO is used as a carrier gas. The concentration of CO was approximately 1.0% and 2.0% when the target contained 0.5% and 1.0% of O2 as a carrier gas, respectively. No other peaks else than the main peak of the radiopharmaceutical was seen in any of C15O, 15O2, and C15O2 productions, thus the radiochemical purity was effectively 100% for all compounds.Table 15O2–C15O2 supply protocol, which is the same as that shown in Fig. 15O2 production. A rise in the radioactivity concentration after 135 s was attributed to production of the C15O2 after the cyclotron beam bombardment. In contrast, the radioactivity continued to be decreased, when the cyclotron beam was not initiated. The ratio of the two radioactivity curves which decreased monotonically determines the amount of contamination when the rapid protocol was employed, and was shown to be 1% at approximately 280 s, and 0.7 ± 0.1% at 330 s which is the time when the C15O2 inhalation was initiated in the clinical protocol.Figure 15O inhalation (a) and a sequential 15O2-C15O2 inhalation scans (b), respectively. Time-dependent end-tidal carbon dioxide (EtCO2) and respiration rate (RR) curves (c) are also shown for this particular case. The net administration doses, as calculated as a difference of AUC’s of two curves for this particular case, were 451 MBq, 311 MBq, and 430 MBq for the supply of 2747 MBq, 2451 MBq, and 1883 MBq, corresponding to C15O, 15O2, and C15O2 inhalation, respectively. Similarly, supply and net administration doses over all 749 cases were 541 ± 149 MBq, 320 ± 103 MBq, and 523 ± 137 MBq for supply of 2574 ± 255 MBq, 2220 ± 766 MBq, and 1763 ± 174 MBq corresponding to C15O, 15O2, and C15O2 inhalation, respectively  are stable during the PET examination period. The mean EtCO2 was 37.8 ± 5.9 mmHg and 37.9 ± 6.0 mmHg, and RR 14.0 ± 3.6 and 14.0 ± 3.5 min−1, during to the 15O2 and C15O2 inhalation periods, respectively. Difference between the two periods was 0.5 ± 1.0 and − 0.6 ± 2.5 mmHg for EtCO2 and 0.68 ± 0.75 and 0.03 ± 2.38 min−1 for RR, corresponding to healthy volunteers (n = 15) and all subjects including patients, respectively.Figure 15O2 inhalation period in cases 2 and 3 were consistent with the drop in AIF. Irregular shapes of AIF attributed to the pause of respiration were observed in 70 and 85 scans during the 15O2 and C15O2 inhalation scans out of 749 scans, respectively. Figure 15O2 divided by the body weight of each patient as a function of AUC of the invasively assessed AIF. The two values were significantly correlated to each other (p < 0.001).Figure 15O2 and C15O2 inhalation periods. Radioactivity in the facemask relative to the brain was higher during inhalation of 15O2 than that of C15O2. Larger radioactivity was seen in the paranasal sinus area during C15O2 inhalation but not during 15O2 inhalation. The brain uptake relative to the radioactivity in the facemask was higher with C15O2 than that with 15O2 inhalation. Figure 15O, 15O2, and C15O2 inhalation, respectively. Functional images calculated by means of two different mathematical formulations, i.e., DARG [V0W and V0O) and CBV. However, no clear effects of the gaseous radioactivity are visible in both functional images.PET images demonstrated that radioactivity in the facemask was well scavenged and was small compared with that in the brain as typically seen in Fig. e., DARG  and a moe., DARG  methods 15O, 15O2, and C15O2 inhalation on each of two patients. The area radioactivity in the PET room slightly increased during the period when the radioactive gas was circulated to the PET room during the periods indicated by “b” in (a). The radioactivity in the PET room increased immediately after inhalation of each of C15O, 15O2, and C15O2 gases, indicated by “c,” “d,” and “e,” respectively. Sharper peak was observed after 15O2 inhalation as compared with after C15O and C15O2 inhalation. Gaseous radioactivity was observed in the exhaust duct during the second examination, which indicated the leakage from the facemask when gaseous radiopharmaceuticals were supplied (b). The radioactivity curves in the exhaust duct corrected for background (b), and decay of 15O were plotted in (c). The %leakages were summarized in Table 15O, 15O2, and C15O2 inhalation scans. Leakage was not detected in more than a half of scans, namely 417, 395, and 529 scans with C15O, 15O2, and C15O2 inhalations, respectively. Three examinations demonstrated that the leakage reached 1%, but the average leakage was 0.17 ± 0.65% when corrected for the radioactivity decay.Figure 15O-labeled radiopharmaceuticals of C15O, 15O2, and C15O2 by combining it with the 15O-dedicated small cyclotron of C3D when a deuteron beam was accelerated at a beam current of 40 μA , and the standardization of PET examination.The present study demonstrated that the novel radiosynthesis system was able to produce and supply a sufficient amount of a series of 15O-labeled radiopharmaceuticals may enable the determination of AIF from radioactivity concentration curves in the carotid arterial region. This may further provide opportunity of a non-invasive quantitation of functional parametric images without the arterial blood sampling [15O-oxygen PET scans.An important outcome of the inhalation/scavenging unit implemented in this system was the ability to remove radioactivity from the area inside the FOV of PET imaging by effectively scavenging the non-inhaled radioactive gases from the outer layer of the two-hole two-layered face mask Fig. , while asampling . Systema2 causes a change by 6% in CBF [2, which is an indirect marker of PaCO2, between 15O2 and C15O2 inhalation periods, e.g., 0.5 ± 1.0 and − 0.6 ± 2.5 mmHg, corresponding to healthy and patient populations, respectively. The variations in EtCO2 of ± 1.0 mmHg between the 15O2 and C15O2 inhalation periods in healthy controls were practically the same. In the entire subjects including the patient population, the variation was larger and reached to ± 2.5 mmHg, which was attributed to the unstable breathing. As typically shown in Fig. 2 curves which were unstable in patients. The transient change in PaCO2 during the PET examination would still be in an acceptable range in order to reliably quantify CBF and CMRO2 in clinical settings. Continuous supply of a fresh air to the two-hole two-layered facemask assisted easy breathing despite of prevention from the leakage of radiopharmaceuticals, as seen in Fig. The scavenged gaseous radioactivity was compressed and stored in the decay tank  does not define the upper limit of radioactivity nor a radioactivity concentration at the exhaust duct for short half-life radio-nuclei in gaseous form. This is because the public safety may be guaranteed by stopping the room exhaust if significant leakage occurs. In such a situation, the workers in the facility would have to leave from the area to prevent from a high risk of radiation. However, this is not practically adequate, because clinical examinations are carried out on patients inside the facility. To reduce the radioactivity released from the exhaust duct to the public, the use of decay tank was shown to be effective, in which use of the facemask that prevents from the leakage to the room was essential. The present inhalation/scavenging unit which reduced the leakage to the order of 0.1% was essential when utilizing the gaseous 15O-oxygen radiopharmaceuticals in clinical PET examinations.The present study demonstrated that the average leakage was 0.17 ± 0.65% from the facemask as estimated in this study including corrections for backgrounds and the decay Fig. . Due to 15O-labeled gaseous radiopharmaceuticals produced enough amount of C15O, 15O2, and C15O2 at a reasonably short interval with sufficiently high radiochemical purity as required for clinical use. It is also important to note that logistical complexity in terms of producing and inhaling three sets of 15O-Oxygen-labeled radiopharmaceuticals has been considerably improved. This is particularly essential when a recently proposed rapid protocol of Kudomi et al. [2, and OEF can therefore be obtained from a short scan period of 8 min, with considerably reduced work load of clinical staff as compared with previous PET examination systems. Effective scavenge allow for accurate quantitation using a commercial PET scanner in 3D mode. Minimal leakage from the facemask should contribute to reduction of unnecessary radiation for workers and for public.The present study demonstrated that the recently developed system for automated production and inhalation of a series of i et al.  is utili