However, the in vivo functions of Pik3r1 remain unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone (DEX), a synthetic glucocorticoid. Treating wildtype (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue, and gastrocnemius (GA) muscle. This DEX effect was diminished in GA muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in GA muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced GA myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice, and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle.Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit  Glucocorticoids are steroid hormones that play important roles in regulating whole body metabolism under stress conditions, mainly by mobilizing energy sources to face severe challenges. In skeletal muscle, glucocorticoids inhibit protein synthesis, facilitate protein degradation, and suppress glucose utilization. Amino acids generated from glucocorticoid-induced protein mobilization are the precursors for hepatic gluconeogenesis and inhibiting glucose utilization raises plasma glucose concentrations , 2. WhilPhosphoinositide-3-kinase regulatory subunit 1  is one of these potential GR primary target genes (Glucocorticoids convey their functions through the glucocorticoid receptor (GR), which is a transcription factor that binds to genomic glucocorticoid response elements to modulate the transcriptional rate of its target genes. Thus, GR primary target genes initiate the physiological actions of glucocorticoids. To understand the mechanisms underlying glucocorticoid actions in skeletal muscle, we previously used a combination of global gene expression analysis and chromatin immunoprecipitation sequencing to identify a list of potential GR primary target genes in murine C2C12 myotubes . Phosphoet genes  and encoet genes , 8. Whenet genes , a key set genes , 11. Conet genes . Severalet genes . Alternaet genes . Anotheret genes .Pik3r1 knockout mice (MKO)  with or without a synthetic glucocorticoid, dexamethasone (DEX), and studied the effects on insulin signaling in metabolic tissues, including liver, gastrocnemius (GA) muscle, and epididymal white adipose tissue. We also examined the effect of striated muscle Pik3r1 deletion on systemic glucose and insulin tolerance. In addition, using stable isotope labeling techniques and tandem mass spectrometry, we analyzed the DEX effects on protein synthesis rates in GA muscle of WT and MKO mice. Finally, we investigated the signaling pathways involved in the regulation of protein synthesis and conducted histological analysis of the GA muscle in WT and MKO mice.We previously found that the overexpression of Pik3r1 in C2C12 myotubes reduced cell diameter while reduction in Pik3r1 expression compromised glucocorticoid suppression of insulin signaling . To furtce (MKO) . We treaPik3r1 gene expression was elevated in mouse GA muscle upon DEX treatment (A).We previously showed that reatment . We examreatment A.Figure Pik3r1 gene transcription by GR in vivo, chromatin immunoprecipitation (ChIP) was performed to test the recruitment of GR to the glucocorticoid response element (GRE) of Pik3r1 gene in mouse GA muscle. Eight-week-old male WT mice were injected intraperitoneally with PBS or DEX for 4 days before tissue collection for ChIP given that Pik3r1 had the highest protein expression with 4 days of DEX treatment. The glucocorticoid response element of Pik3r1 has been located between −43,938 and −43,924 upstream of the mouse Pik3r1 gene (Pik3r1 GRE by DEX treatment (approximately 15-fold comparing to IgG ChIP control) (B). Interestingly, GR was also recruited to the Pik3r1 GRE in PBS-treated animals to a lesser degree . This suggests that plasma corticosterone levels are enough to activate Pik3r1 gene transcription through GR.To confirm the activation of 3r1 gene . We founcontrol) B. InterePik3r1 GRE in the GA muscle of PBS and DEX-treated WT mice. The ratios of AcH3/H3 and AcH4/H4 represent the degrees of the histone acetylation in H3 and H4, respectively. As shown in Figure 1C, the level of total histone H3 and H4 was not significantly affected by DEX treatment. However, comparing to IgG control, the ratios of AcH3/H3 and AcH4/H4 were significantly increased by 8 and 10 folds with DEX treatment, respectively (C). In PBS treated animals, the ratios of AcH3/H3 and AcH4/H4 were also significantly higher than the IgG ChIP control . This observation was consistent with the finding that GR was also recruited to the Pik3r1 GRE under PBS treatment. Nonetheless, the ratios of AcH3/H3 and AcH4/H4 were higher in DEX-treated mice than those of PBS-treated mice. This was in agreement with a stronger GR recruitment to the Pik3r1 GRE under DEX treatment . We next examined which histone acetyltransferase is recruited to the Pik3r1 GRE using ChIP. We found that p300 but not Tip60 and GCN5 was significantly recruited to the GRE upon DEX treatment . These results suggested that p300 accounted for the higher histone acetylation status at the GRE upon DEX treatment. Notably, neither p300, Tip60, nor GCN5 were recruited to the GRE upon PBS treatment . These results suggest that histone acetyltransferase(s) other than these three are involved in the acetylation of the Pik3r1 GRE in PBS-treated mice.Transcriptional activation is associated with elevated histone acetylation in the enhancer regions , 19. We ectively C. In PBSPik3r1 gene transcription, C2C12 myoblasts were infected with lentivirus expressing scramble small hairpin RNA  or shRNA against p300 (sh-p300). After puromycin selection, cells were differentiated into myotubes, then treated with DEX or EtOH for 6 h. RNA was isolated from these cells, and qPCR was performed to monitor the expression of Pik3r1. In sh-scrRNA expressing C2C12 myotubes, DEX treatment increased the expression of Pik3r1 approximately 3-fold (E). However, in sh-p300 expressing C2C12 myotubes, such DEX effect was abolished (E). The Western blot showed that p300 was efficiently reduced by RNAi (E).To test whether p300 is involved in GR-activated y 3-fold E. Howevebolished E. The We by RNAi E.Pik3r1 knockout mice (MKO) by crossing Pik3r1flox/flox mice with transgenic mice carrying muscle creatine kinase promoter driving the expression of Cre recombinase (A). Notably, Pik3r1 expression was similar in the liver of WT and MKO mice (A). These results validated the specific deletion of Pik3r1 in the striated muscle. We also tested the ability of DEX to induce the expression of GR primary target gene in GA muscle of WT and MKO mice. WT and MKO mice were treated with PBS or DEX for 7 days. GA muscle RNA was isolated, and qPCR was performed to examine the DEX induction of two previously identified GR primary target genes, Fkbp5 . DEX treatment elevated the expression of Fkbp5 approximately 5-fold in WT mice (B). The basal Fkbp5 expression was lower in MKO mice, though not statistically significant (p = 0.11). DEX treatment also efficiently increased Fkbp5 expression in MKO mice (B). These results indicated that depleting Pik3r1 expression did not affect general GR activity in GA muscle.We generated striated muscle–specific ombinase . In MKO ombinase A. NotablMKO mice A. These s, Fkbp5  and Sesn5 (Sesn1 . DEX treobserved B. DEX tr WT mice B. The baMKO mice B. These B). In MKO mice, insulin treatment significantly increased pAkt/Akt ratio in GA muscle, liver, and epididymal white adipose tissue (A). We found that DEX treatment reduced Akt activity in all three tissues of WT mice (A). In epididymal white adipose tissue and liver of MKO mice, DEX treatment was still able to inhibit Akt activity (A). However, in GA muscle, the ability of DEX to reduce insulin stimulated Akt activity was significantly attenuated (A). These results demonstrate that Pik3r1 deficiency in striated muscle reduced the DEX effect on insulin action in GA muscle but not in other insulin responsive metabolic tissues, such as epididymal white adipose tissue and liver.WT and MKO mice treated with or without DEX for 7 days were injected with insulin for 10 min. GA muscle, liver, and epididymal white adipose tissue were isolated, and the activity of a key molecule in insulin signaling, Akt, was monitored. For Akt activity, we monitored the levels of phosphorylated Akt (pAkt) at serine 473 residue  and totaFig. 2e tissue A. We fou WT mice A. In epiactivity A. Howevetenuated A. These B and C). In contrast, in MKO mice, although DEX treatment still caused hyperinsulinemia (D), DEX-induced glucose tolerance was significantly improved (D). We also performed insulin tolerance test in WT and MKO mice treated with or without DEX for 1 week. We found that DEX treatment resulted in insulin intolerance in WT mice (D). In MKO mice, DEX treatment still caused insulin intolerance, but to with a lesser degree (D). Thus, DEX effect on insulin tolerance was somewhat reduced in MKO mice (D). Without DEX treatment, MKO and WT mice had similar glucose and insulin tolerance and plasma insulin levels were similar (B–D). These results are in agreement with the previous report . The mean GA protein fraction synthesis values were 18.0%, 13.5%, 15.2%, and 12.9% for WT+PBS, WT+DEX, MKO + PBS and MKO + DEX mice, respectively. The overall protein synthesis rate in MKO mouse GA muscle was significantly lower than that of WT mouse GA muscle (15.5% reduction (15.2%/18.0%), p ≤ 0.0001). DEX significantly reduced protein synthesis in both WT and MKO mice. However, in WT mouse GA muscle, the overall protein synthesis rate was decreased by an average of 25.1% (13.5%/18.0%), whereas in MKO mouse GA muscle, the overall protein synthesis rate was reduced by only 15.1% (12.9%/15.2%) with DEX treatment. The relative reduction in the change in GA muscle individual protein fractional synthesis rates were also compared among groups. Experimental fractional synthesis (f) values for the proteins common among each model were plotted in dot plot and sorted from low to high, and then by protein (B). Graphically, in WT+DEX (red line), MKO + PBS (green line), and MKO + DEX mice (purple line), most proteins exhibited a decrease in fractional synthesis as compared with WT (blue line). We then compared the percent change in each protein’s fractional synthesis for WT animals treated with or without DEX (C). Statistical significance was assessed by a binomial distribution of the proportion of proteins showing a negative or positive percent change in fractional synthesis with DEX treatment (C). In WT+DEX, 96.5% or 55/57 proteins showed lower fractional synthesis rates as compared with WT mice (C). Only two proteins, electron transfer flavoprotein subunit beta and peroxiredoxin-1, did not have lower fractional synthesis by DEX treatment (C). In addition, 48/57 or 84.2% of proteins in MKO mice were reduced by DEX treatment. Thus, seven proteins whose protein synthesis rates were reduced in WT mice by DEX were not affected by DEX in MKO mouse GA muscle, whereas two proteins, alpha-enolase and phosphoglycerate kinase-1, had a higher protein synthesis rate  in MKO mice treated with DEX (p = 0.016). Overall, these results indicated that DEX effect on lowering protein synthesis was compromised without Pik3r1 in GA muscle.To determine how DEX treatment in WT and MKO mice leads to muscle atrophy, muscle proteome–wide fractional synthesis rates were measured by LC-MS/MS after t 7 days , 25, 26.t 7 days . Databast 7 days . Global t 7 days A. The me protein B. Graphihout DEX C. Statisreatment C. In WT+ WT mice C. Only treatment . Surprisreatment C. In addwith DEX . FinallyA) to monitor the cross-section area and fiber count of GA muscle of WT + PBS, MKO + PBS, WT + DEX, and MKO + DEX mice, asking whether DEX induced muscle histological changes are compromised in MKO mice. Cross-sectional area of GA muscle fibers significantly decreased from a mean of 2277 μm2 in WT animals to 1382 μm2 in WT + DEX mice (B). In MKO mice, cross-sectional area of GA muscle fibers was 1709 μm2 (B). This is in agreement with the lower protein synthesis rates in MKO GA muscle shown above. Importantly, there was no reduction in cross-sectional area of GA muscle fibers in MKO + DEX mice (1979 μm2) compared with MKO mice. GA muscle fiber count showed similar results. DEX treatment increased fiber count in WT GA muscle, and MKO had higher fiber count than WT mice, but DEX did not alter fiber count in MKO mice (C).We also performed histological analysis A to moniDEX mice B. In MKO1709 μm2 B. This iMKO mice C.Figure D) .Immunohistochemical staining was used to analyze the fiber types of GA muscle of WT and MKO mice. The antibodies against MHC I, MHC IIa, MHC IIb, and MHC IIx were used. MHC I is encoded by Myh7 and represents slow oxidative fibers, whereas MHC IIa is encoded by Myh2 and represents fast oxidative fibers . MHC IIbFig. 5D) . It appeA). DEX treatment abolished this insulin induction . Interestingly, in MKO mice, neither insulin nor DEX affected S6K Thr389 phosphorylation .We further analyzed the DEX effect on signaling processes that regulate protein synthesis. As described above, WT and MKO mice were treated with or without DEX and were injected with insulin for 10 min before GA muscle was isolated. We first monitored the phosphorylation at threonine 389 of p70S6 kinase (S6K) , which iFig. 6B). Interestingly, the basal levels of 4E-BP1 phosphorylation were lower in MKO mice . Insulin still increased 4E-BP1 phosphorylation in MKO mice . DEX treatment reduced such insulin effect . However, unlike in WT mice, DEX treatment did not abolish insulin effect. Thus, without Pik3r1, some degrees of the insulin effect on 4E-BP1was restored .Next, we monitored the phosphorylation of threonine 37 and 46 of 4E-BP1 . 4E-BP1 Fig. 6C). The basal eIF2α phosphorylation was similar between MKO and WT mice . However, in contrast to the DEX effect in WT mice, DEX treatment did not affect eIF2α phosphorylation in MKO mice .Finally, we monitored the phosphorylation at serine 52 of translational initiator eIF2α. eIF2 consists of three subunits, eIF2α, eIF2β, and eIF2γ, that promotes a new round of translation initiation by exchanging GDP for GTP, which is catalyzed by eIF2B , 34. TheFig. 6In summary, there are three major observations from these results. First, in GA muscle, DEX inhibited insulin induced p70S6K and 4E-BP1 activity. Moreover, DEX treatment increases eIF2α phosphorylation. All these responses could contribute to the suppression of protein synthesis. Second, without Pik3r1, the basal 4E-BP1 phosphorylation was decreased. This phenomenon could contribute to the lower protein synthesis rate in GA muscle of MKO mice. Third, without Pik3r1, the ability of DEX to suppress insulin’s effect on 4E-BP1 was impaired, and DEX was unable to potentiate eIF2α phosphorylation. These results could explain the partial maintenance of protein synthesis rates in GA muscle of MKO mice.Pik3r1 as a GR primary target gene that mediates glucocorticoid responses in murine C2C12 myotubes. In this study, we further demonstrated that the Pik3r1 gene is activated by GR in mouse GA muscle, because GR was recruited to the Pik3r1 GRE, and the levels of histone acetylation were elevated upon DEX treatment. GR was also recruited to the Pik3r1 GRE, and there were significant levels of acetylated H3 and H4 at the Pik3r1 GRE in PBS-treated mice. These results suggest that physiological corticosterone levels at the time we performed ChIP contribute the basal expression of Pik3r1 in mouse GA muscle. As plasma corticosterone levels are modulated by circadian rhythm, we predict that the degree of GR recruitment to the Pik3r1 GRE could vary depending on the time we conduct ChIP experiments. Notably, even during the refeeding stage when plasma corticosterone levels are low, GR is recruited to certain GREs  and Ddit4, are known to play a role in this process  rates at steady-state, where synthesis is balanced by breakdown so high turnover rates and a lower protein pool size can be taken to reflect stimulated breakdown, even in the presence of reduced absolute synthesis rates . Thus, in Pik3r1-depleted GA muscle, the ability of DEX to suppress insulin increased 4E-BP1 phosphorylation was decreased. We also found that DEX treatment increased eIF2α phosphorylation in WT mice GA muscle, which was in agreement with a reduced protein synthesis rate. This DEX effect was not observed in MKO mice. These results could explain a partially restored protein synthesis rate in GA muscle of MKO mice. Interestingly, the induction of eIF2α phosphorylation by DEX was not found in previous studies  were provided by the laboratory of Lewis Cantley  (Pik3r1flox/flox with transgenic mice expressing Cre recombinase under control of muscle creatine kinase (B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J) (Pik3r1flox/flox (WT) mice only ∼310 bps amplified by Pik3r1_loxP_F and Pik3r1_loxP_R primers were observed. Eight-week-old male MKO and WT mice were injected intraperitoneally with 5 or 10 mg/kg body weight of DEX  or PBS (control) for 1, 4, or 7 days. At the end of the treatment period, blood, inguinal, and epididymal adipose tissues, liver, and GA muscle were isolated from mice for protein expression analysis. Mice were housed in ventilated cages with Sanichip bedding along with a cotton Nestlet and a 4gm puck of crinkled paper. They were co-housed and were fed a diet of 18% protein, 6% fat (Envigo 2918) with a 12-h light and dark cycle in a temperature-controlled room of approximately 22 °C. The Office of Laboratory Animal Care at the University of California, Berkeley (AUP-2014-08-6617) approved all animal experiments conducted in this work.Mice with a conditional allele of Pik3r1 gene flanked with LoxP sites at exon7 (College) . Muscle-)5Khn/J) . The folThe protein concentration for samples was measured with Bradford protein dye (BioRad). Proteins (∼30 μg) were mixed with 1× NuPAGE LDS Sample Buffer  and 1× NuPAGE Sample Reducing Agent , boiled for 5 min before being applied to SDS-PAGE. The following are the antibodies we used in this study: anti-Gapdh , anti-Pik3r1 . The intensity of the bands was quantified using Image J software  and normalized to Gapdh.g, 4 °C for 5 min. Pellets were washed with ice-cold PBS, then resuspended in 3 ml buffer S . Samples were incubated on ice for 10 min, then sonicated with the Branson Sonifier 250 sonicator for 50 s . After centrifugation for 10 min at 32,000g, 4 °C, supernatant containing sheared DNA fragments, was collected, and mixed with one sample volume of buffer D . Diluted sample was then incubated with 100 μl of 50% protein A/G agarose beads  for 1 h at 4 °C with gentle shaking to preclean the sample. After centrifugation at 4000g for 3 min at 4 °C to pellet the agarose beads, supernatant was used to set up the IP reactions. The following antibodies were used in this study: anti-IgG , anti-GR , anti-H3 histone , anti-H4 , anti-AcH3 , anti-AcH4 , anti-H3K4me3 , anti-H3K4me1 , and anti-p300 . Samples were allowed to react with antibody for 18 h (overnight incubation) at 4 °C with gentle shaking. Then, 50 μl of 50% protein A/G agarose beads were added into each IP reaction and were rotated for 2 h at 4 °C. Then, agarose beads were washed with the following conditions: 1x low-salt wash buffer , 1x high-salt wash buffer , 1x LiCl wash buffer , and 2x Tris-EDTA buffer. After the last wash, all supernatants were removed, then 400 μl of elution buffer  was added. Samples were rotated at room temperature for 1 h, then centrifuged at 8000g for 1 min. Supernatant was transferred to a new tube and were mixed with 16 μl of 5 M NaCl and were incubated at 65 °C overnight. On the last day, 16 μl of Tris (pH 6.5), 8 μl of 0.5 M EDTA, and 1.5 μl of proteinase K  were added to the sample and were incubated at 55 °C for 3 h. The immune-precipitated DNA fragments were extracted with a PCR clean up kit , then were applied to qPCR to quantify the IP result.WT mice were intraperitoneally injected with 10 mg/kg body weight of DEX  for 4 days. On the last day, GA muscles were harvested and snap frozen with liquid nitrogen. Frozen muscles were ground to fine powder with pestle. Then, tissue powder was cross-linked with 1% formaldehyde in 20 ml PBS at 37 °C for 10 min with gentle shaking. After quenching the cross-linking reaction with 125 mM glycine, samples were centrifuged at 1000Eight-week-old male MKO and WT mice were treated with 10 mg/kg body weight of DEX in drinking water. DEX was dissolved in PBS, which was then added into the drinking water. For control, equal amount of PBS was added into the drinking water. After a 15 h fast, mice were injected with 1 g/kg body weight glucose for an intraperitoneal glucose tolerance test. Tail vein blood was used to monitor blood glucose levels at different time points: 0 (before glucose injection), 15, 30, 60, 90, and 120 min after glucose injection using a blood glucose meter .Fed mice were injected with 1 unit/kg body weight insulin  intraperitoneally. Tail vein blood was used to monitor blood glucose levels at different time points: 0 (before glucose injection), 15, 30, 60, 90, and 120 min after glucose injection using a blood glucose meter .Plasma insulin levels were examined using an ultrasensitive mouse insulin ELISA kit .Mice (as described above) were treated with 10 mg/kg bodyweight of DEX (water-soluble) or PBS (control) for 7 days. After intraperitoneal injection of 1 unit/kg bodyweight insulin for 10 min, mice were euthanized, and GA muscle were collected. The tissue was homogenized in cell lysis buffer  using the BeadBug 6 Homogenizer (Benchmark Scientific.) Homogenized lysate was centrifuged at 13,300 rpm for 15 min at 4 °C. Supernatant was transferred into fresh tubes and used for the following ELISAs.Relative Akt and pAkt levels were measured using the Akt  Multispecies InstantOne ELISA kit  Tissue lysate were loaded with a protein concentration of 0.5 μg/μl. All steps afterward were followed according to manufacturer’s instructions.Relative S6 kinase and p70S6 levels were measured using the p70 S6 Kinase  Multispecies InstantOne ELISA Kit  Tissue lysates were loaded with a protein concentration of 1 μg/μl. All steps afterward were followed according to manufacturer’s instructions.Relative total 4E-BP1 and phosphorylated 4E-BP1 levels were measured using the Phospho-4E-BP1 (Thr36) and Total 4E-BP1 ELISA kit . Tissue lysates were loaded with a protein concentration of 1.25 μg/μl and 1 μg/μl. All steps afterward were followed according to manufacturer’s instructions.Relative total eIF2α and phosphorylated eIF2α levels were measured using the PathScan Phospho-eIF2α (Ser51) and total eIF2α Sandwich ELISA kits  Tissue lysates were loaded with a protein concentration of 0.25 μg/μl.All reagents were purchased or made with the highest quality experimental conditions. 1x PBS (Gibco), hyclone molecular grade water , 0.5 M EDTA (Gibco), 0.1 M PMSF (Sigma), Halt protease inhibitor , 2,2,2-triflouroethanol (Sigma), ammonium bicarbonate (Sigma), DTT (Sigma), IAA (Sigma), trypsin (Sigma), acetonitrile (Fisher), formic acid (Fisher), and deuterium oxide (Cambridge isotope labs).2H2O) (Cambridge isotope laboratories) at the third day of DEX treatment via intraperitoneal injection. The animals remained on DEX and 2H2O for seven more days until time of sacrifice. Animals were maintained on a maintenance dose of 8.0% 2H2O in drinking water to maintain approximately 5.0% excess 2H enrichment in total body water .Whole GA muscle was carefully dissected, placed in a cryomold with an optimal cutting temperature solution and was flash frozen in a liquid nitrogen cooled metal block. Ten micrometer transverse muscle sections were performed using a cryotome at −20 °C, followed by transfer to positively charged microscope slides for hematoxylin and eosin staining. Muscle fiber diameter was recorded using a light microscope, and cross-sectional area was analyzed For immunohistochemistry , 57, 58,2H2O. This maximal enrichment, representing 100% newly synthesized protein molecules, is then compared to the experimentally measured value to determine the fractional synthesis of the protein based on the rise to plateau precursor-product relationship , WT treated with DEX , MKO treated with PBS , and MKO treated with DEX . The rational for biological replicates of n = 5 to 6 were based on prior data for biologic and analytic variability of proteome-wide protein fluxes (p ≤ 0.05) using GraphPad Prism version 8.0 for Mac, GraphPad Software.To determine how DEX treatment in WT and MKO mice leads to muscle changes, we measured GA muscle protein fractional synthesis rates using shot-gun LC-MS/MS analysis after labeling , 24, 26.n fluxes , 24, 26.n fluxes , 24: morMassHunter release, version B.07.00, was the software used for peak list generation. The search engine for proteomic analysis based on MS/MS identifications was Spectrum Mill released version B.04.01. Uniprot (2018) was the sequence database searched for mouse protein identifications . The ove2H from heavy water. MIDA was employed using programs developed by Hellerstein et al. to determine the fractional synthesis of the parent protein (0 isotopomers at time (t)/asymptomatic enrichment of M0 isotopomer as predicted by MIDA) of the parent protein and calculated from the rounded up values of its labeled peptides  for each group and comparisons were analyzed by Student’s The authors declare that they have no conflicts of interest with the contents of this article.


Exo-β-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-β-mannosidases in biological and biomedical research. via activity-based protein profiling.New cyclophellitol-derived probes covalently label β-mannosidases, enabling the detection of diverse mannosidases  Exo-β-mannosidases, hydrolysing non-reducing terminal β-d-mannopyranosidic linkages, are widely distributed carbohydrate-degrading enzymes.1 In the human gut, exo-β-mannosidases of families GH2, GH5, and GH164 are essential for the fermentation of β-mannans, such as carob or guar endosperm (Ceratonia siliqua) galactomannan, common food additives.2–5 In mammals, the lysosomal glycoside hydrolase family 2 (GH2 6) retaining exo-β-mannosidase, MANBA (EC 3.2.1.25), is responsible for the cleavage of the core Man-β-1,4-GlcNAc linkage during lysosomal turnover of N-glycoproteins.7 Deficiency of this enzyme activity, caused by mutations in MANBA,8,9 results in β-mannosidosis, a rare lysosomal storage disorder that can be diagnosed on the basis of elevated Man-β-1,4-GlcNAc in urine or diminished hydrolysis of 4-methylumbelliferyl (4-MU) β-d-mannopyranoside in plasma.10–12 More recently, MANBA deficiency has been identified as a key risk factor for chronic kidney disease, which affects roughly 1 in 10 adults.13–15exo-β-mannosidases process their substrate using a stereochemically retaining double displacement mechanism that is characterised by the formation of a covalent glycosyl enzyme intermediate . The natural product cyclophellitol, with endocyclic epoxide functionality in place of the acetal group found in a typical glycoside, is a potent irreversible inhibitor of retaining β-glucosidases.17,18 It has been demonstrated that altering the characteristic cyclitol ring to emulate differently configured monosaccharides yields irreversible inhibitors with selectivity towards various retaining exo-glycosidases.19–24 Moreover, it has been shown that substituting the epoxide in cyclophellitol and its configurational isosteres for an N-tagged  aziridine yields effective ABPs for the discovery and visualisation of retaining glycosidases within complex biological samples.19,25–28All of these ate GEI, . This twexo-β-mannosidases using cyclophellitol derivatives presents a unique challenge. GH2 exo-β-mannosidases process their substrate through a 1S5–B2,5‡–OS2 mechanistic conformational itinerary  was epoxidized with mCPBA, furnishing partially protected β-d-manno-cyclophellitol alongside the epimeric epoxide 8. Purification by silica gel chromatography yielded pure 9, which was subsequently deprotected by palladium-catalysed hydrogenolysis in a 9 : 1 mixture of 1,4-dioxane and tBuOH to provide β-d-manno-cyclophellitol 1 in 40% yield after compound crystallisation.To determine whether conformational mismatch precludes d-manno-cyclophellitol aziridine 2 in quantitative yield.To prepare aziridine 2, the intramolecular iodocyclization route was chosen over the epoxide opening (with sodium azide) and closing (under reducing conditions), to take advantage of the stereochemistry of the primary hydroxyl group directing to the beta face. Starting cyclohexene 7 was thus treated with trichloroacetonitrile to furnish the trichloroacetimidate 10 in 73% yield, which was subjected to iodocyclisation to give 11 in 80% yield. Acid-mediated hydrolysis of the resulting iminal and base-induced intramolecular displacement of the iodine with the liberated primary amine gave the partially benzylated aziridine 12. This was reduced under Birch conditions to give β-29 and potassium carbonate in DMF yielded the N-alkyl aziridine 3 in 64% yield. The introduced azide handle was further reacted with Cy5+ alkyne, BODIPY-FL-alkyne30 or biotin–alkyne31 under copper(i)-catalysed azide–alkyne [2 + 3] cycloaddition ‘click’ conditions to afford final ABPs 4, 5, and 6, respectively, in 17 to 36% yield  and aziridine 2 (30% inhibition). CmMan5A showed 20% inhibition with both 1 and 2, and stronger 85% inhibition with 3, indicating that binding is enhanced by the presence of the alkyl group. No significant inhibition of BtMan2A could be detected following incubation with 1 or 2, but the enzyme was 85% inhibited by 3, again indicating performance enhancement attributable to the N-octylazido linker. Assuming a second-order reaction model between the enzyme and inhibitor, these measurements imply extremely low kinact/KI values for the interaction ranging from <0.004–0.034 M−1 s−1.Having candidate inhibitors 1–3 and activity-based probes 4–6 we initially tested 1, 2, and 3 against a diverse collection of well-characterised bacterial CmMan5A bound to compound 3 gave the highest resolution structure at 1.3 Å. Supporting our hypothesized mechanism of reactivity, compound 3 was covalently linked to E330, the known catalytic nucleophile, through its pseudoanomeric carbon  forms a CH/π interaction (3.4 Å) with W285 while the alkyl chain extends out of the catalytic pocket, forming hydrophobic packing interactions with W285, W135, and a hydrophobic groove between N288 and W289. We speculate that these interactions contribute to the observed tighter binding of compound 3 over compound 2. Indeed, the first four carbons of the alkyl linker display well-localised electron density (B factors of 16–25 Å2), indicating rigid binding and suggesting that a shorter linker may hinder binding into the active site pocket.To investigate the structural basis of the observed binding, we prepared covalent crystallographic complexes between each enzyme and its most reactive ligand. c carbon . The rinBtMan2A labelled with 3, solved at 1.85 Å resolution, showed the ligand bound to the catalytic nucleophile in a “relaxed” 4C1-like conformation  and noeuromycin,36 show that the ligand has migrated significantly away from the expected position of the GEI  and part unreacted aziridine (at occupancies 0.4 and 0.6 respectively). We thus conclude that there exists a more fundamental instability in the GEI structure of BtMan2A, possibly explaining earlier failures to trap it.In contrast, the structure of BsGH164 crystals with 1 or 2  resulted in the formation of clear covalent adducts . ABPs 4, 5, and 6 were prepared and applied to the detection of MANBA in mouse kidney tissue, where MANBA is known to be highly expressed,38 or in human plasma, where low levels of detected MANBA activity can be used to rule out a diagnosis of β-mannosidosis. ABPP, via in-gel fluorescence following SDS-PAGE separation, of diluted and pH-adjusted plasma from a healthy donor  showed no evidence for a band at ∼100 kDa, where MANBA is expected to appear . In contrast, mouse kidney extracts treated with Cy5+-labelled ABP 4 yielded one major band at ∼100 kDa  in mouse kidney extracts revealed no apparent inhibition of MANBA by 1 or 2 at 50 μM following two hours of incubation at 37 °C, as did cABPP with cyclophellitol or click-labelled 6-deoxy-6-azido cyclophellitol MDW941.39 However, JJB75, a potent N-alkyl aziridine inhibitor of β-glucocerebrosidase (GBA),26 proved to be a competitor for the active site of MANBA at 3 μM, suggesting that, similar to BtMan2A, MANBA reactivity is significantly driven by affinity for the N-alkyl linker.Consistent with our results with JJB75 26) with both GBA and GBA2, giving detectable labelling with 1 μM compound 4. Competitive ABPP with 1 μM of either cyclophellitol or JJB75 supported the assignment of these bands as GBA and GBA2 since JJB75 and cyclophellitol are known to be efficient inhibitors of GBA and GBA2. Together, these results indicate that the selectivity of an assay for MANBA in mammalian tissue using probe 4 can be enhanced through pre-incubation of the sample with cyclophellitol to eliminate interference from GBA and GBA2 without reducing signal from MANBA.To further test the specificity of probe 4, lysates prepared from HEK293T cells overexpressing non-lysosomal glucocerebrosidase (GBA2) were stained with Cy5-ABP (4) , revealiexo-β-mannosidases can be inhibited covalently and irreversibly with appropriately configured cyclophellitol analogues, particularly those designed with N-alkyl aziridine warheads. Our structural work shows that the epoxide and aziridine inhibitors bind BsGH164 in the same conformation found as 2FMan and that the N-alkyl aziridine binds to CmMan5A in the expected OS2-like conformation of the GH5 exo-β-mannosidase GEI. While exhibiting low potencies, the inhibitors and ABPs presented here react irreversibly with the catalytic nucleophile, displaying sufficient stability and specificity to enable near-quantitative labelling of the catalytic nucleophile. In spite of their limited reactivity, tagged N-alkyl aziridine derivatives were moreover successfully applied to ABPP in both chemical proteomics and in-gel fluorescence formats, as shown by detection, enrichment, and identification of MANBA from mouse kidney extracts.We have demonstrated that, as has been previously shown for other retaining glycosidases, d-Mannopyranose-configured cyclophellitol was synthesized as described in the supplemental synthetic methods and compound characterization section.All chemicals were purchased from Sigma Aldrich unless otherwise specified. β-BsGH164 was expressed and purified as in Armstrong and Davies.5 Briefly, a 1 ml starter culture of pET28-His6-BsGH164 in BL21(DE3) gold cells was used to inoculate NZYTech autoinduction media (NZYTech) containing 50 μg ml−1 of kanamycin. Expression cultures were grown at 37 °C with shaking at 250 rpm for 6 h, the temperature was then decreased to 20 °C and cultures were incubated for an additional 22 h. Expression cultures were harvested by centrifugation  and cell pellets were stored at −70 °C until purification. Cell pellets were resuspended in 120 ml of 50 mM HEPES, 30 mM imidazole, 200 mM NaCl, pH 7.4 with additional protease inhibitor (4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 mM), lysozyme, and DNase. Resuspended cells were then lysed by passage through a cell-disruptor homogenizer at 25 kpsi. Cell lysate was clarified by centrifugation  then loaded directly onto a 5 ml His-tag Excel column . Bound protein was eluted with a linear gradient of 0–100% 50 mM HEPES, pH 7.4, 1 M imidazole, 200 mM NaCl over 20 column volumes, concentrated with a 30 kDa cut-off Amicon centrifugal filter unit, and further purified by gel filtration  in 50 mM HEPES, 200 mM NaCl, pH 7.4. The purity of eluted protein was analyzed by SDS-PAGE and protein-bearing fractions were pooled and concentrated with a 30 kDa cutoff Amicon centrifugal filter unit. Concentrated protein was diluted with 20 mM HEPES, pH 7.4, concentrated again, then diluted to 30 mg ml−1 with the same buffer and flash frozen with liquid nitrogen until use. Protein concentrations were determined spectrophotometrically using a calculated ε280 of 128 480 M−1 cm−1.CmMan5A2 was codon optimized for E. coli, synthesized and cloned into pET24a between the NdeI and XhoI restriction sites by Genscript (Netherlands) to add a C-terminal 6xHis tag, matching the previously crystallized form. The plasmid was transformed into BL21(DE3) gold cells and expressed in ZYM-5052 autoinduction medium40 with 50 μg ml−1 of kanamycin, 2 mM MgSO4 and no added micronutrients, for 20 hours at 30 °C (starting OD600 = 0.03). Harvested cells were resuspended in 0.05 volumes of 50 mM NaPi, 300 mM NaCl, 20 mM imidazole, pH 7.5 supplemented with 0.1 mg mL−1 lysozyme and were lysed by sonication on ice for 5 minutes with 40% amplitude and 30% duty cycle. Lysate was clarified by centrifugation at 18 000g for 10 min at 4 °C, then loaded onto a 5 mL Histrap crude FF column . Protein was washed with 10 CV of binding buffer then eluted with a gradient from 20–500 mM imidazole. Protein-bearing fractions were concentrated using a 10 kDa MWCO Amicon centrifugal concentrator, then purified over Superdex 200 into 20 mM Na-MOPS pH 7.0. The heart-cut of the major elution peak was pooled and concentrated to ∼50 mg mL−1 using a 10 kDa MWCO Amicon centrifugal concentrator, and frozen at −70 °C. Protein was quantified spectrophotometrically using a calculated ε280 of 120 780 M−1 cm−1.The gene for BtMan2A was expressed, and BtMan2A was purified using similar methods to Tailford et al.4 Briefly, an 8 ml starter culture of pET28a-BtMan2A-His6 was used to inoculate 800 mL Luria Broth with 50 μg ml−1 of kanamycin. Expression cultures were grown at 37 °C with shaking at 180 rpm for about 3 hours until an OD 600 of 0.7 was reached. Isopropyl β-d-1-thiogalactopyranoside was added to 1 mM, and the temperature was then decreased to 16 °C and the culture incubated for an additional 21 h. The expression culture was harvested by centrifugation  and the cell pellet was stored at −70 °C until purification. The cell pellet was resuspended in 40 ml of binding buffer, comprised of 50 mM HEPES, 10 mM imidazole, 0.5 M NaCl, 10 mM β-mercaptoethanol pH 8.0, with additional protease inhibitor (4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM) and 1 cOmplete™ Protease Inhibitor Cocktail tablet (Roche). Cells were lysed by sonication, the lysate was clarified by centrifugation at 15 000g for 30 min at 4 °C, and the supernatant was filtered through a 0.22 μM syringe filter and loaded onto a 1 mL HisTrap FF column . The protein was washed with 20 CV of binding buffer, then eluted with a gradient into buffer with 500 mM imidazole over 20 CV. Protein-bearing fractions were concentrated using a 30 kDa MWCO Vivaspin centrifugal concentrator, then purified over Superdex 200 into 50 mM HEPES pH 8.0, 150 mM NaCl, 2 mM DTT. The major elution peak fractions were pooled and buffer-exchanged into 50 mM HEPES pH 8.0, 2 mM DTT, and then loaded onto a 1 ml HiTrap Q column, equilibrated with the same buffer. The protein was eluted using a gradient into loading buffer with 0.5 M NaCl over 20 CV. The main peak fractions were buffer-exchanged into 10 mM HEPES pH 8.1, 5 mM TCEP, concentrated to 15 mg ml−1 and flash frozen with liquid nitrogen until use. Protein concentrations were determined spectrophotometrically using a calculated ε280 of 183 955 M−1 cm−1.The gene encoding BsGH164 and BtMan2A were diluted to 0.2 mg mL−1 in assay buffer 1 . CmMan5A was diluted in assay buffer 2 (20 mM Na-MOPS pH 7.5). Inhibitors 1, 2 or 3 , or 1 μL of water were mixed with 9 μL of each enzyme stock and the reactions were incubated at 30 °C with a heated lid to prevent evaporation. A 2 μL sample taken after 25 hours was diluted with 8 μL of 1% formic acid, 10% acetonitrile and analyzed as described previously.20 A separate 2 μL sample was taken at the same time and diluted with 23 μL of assay buffer 1 (BsGH164), 398 μL of assay buffer 1 (BtMan2A) or 23 μL of assay buffer 2 (CmMan5A). To each of these, in a single well of a black plastic 384-well plate, was added 25 μL of 0.2 mM 4-methylumbelliferyl β-d-mannopyranoside (Sigma) in water. Fluorescence was monitored continuously over 15 minutes via excitation at 360 nm and emission monitoring at 450 nm using a Clariostar (BMG Labtech) plate reader with temperature control set to 25 °C. Fluorescence over time measurements were converted into rates using a 4MU calibration series in each assay buffer and divided by the measured uninhibited control reaction rate to obtain residual activity.BsGH164 were grown in MRC maxi 48-well-plates using the sitting-drop vapor-diffusion method at 20 °C. The protein solution contained His6-BsGH164 at 30 mg ml−1 in 20 mM HEPES pH 7.4 buffer. Well solution contained 100 mM ammonium tartrate dibasic (pH 7.0) and 13% (w/v) PEG 3350 and the protein : well solution ratio was 500 : 500 nl. To obtain an inhibitor-enzyme complex structure the inhibitor  was added to the crystallization drop and incubated for 3 days. The crystals were subsequently cryo-protected in well solution containing 25% ethylene glycol and flash cooled in liquid nitrogen.Crystals of CmMan5A were initially grown from 30 mg mL−1 protein as described previously.2 A seed stock was prepared from needle clusters using the seed bead kit (Hampton research). CmMan5A labelled with compound 3 was prepared by mixing 20 μL of 50 mg mL−1 enzyme with 5 μL of 10 mM compound 3 in 10% DMSO and incubating this for 25 hours at 30 °C. 100 nL of this was sampled and diluted into 10 μL of 1% formic acid, 10% acetonitrile and analyzed as above to assess the degree of labelling. Crystals of CmMan5A labelled with compound 3 were grown in maxi 48-well-plates using the sitting-drop vapor-diffusion method at 20 °C by mixing 100 nL of seed stock into 600 nL of well solution, then adding 900 nL of protein solution. Crystals were apparent after overnight incubation but were allowed to continue growing for 3 days prior to freezing without cryo-protection.Crystals of BtMan2A was crystallized in MRC maxi 48-well-plates using the sitting-drop vapour-diffusion method at 20 °C. The drop comprised 0.65 μl protein  and 0.35 μl mother liquor consisting of 14% (w/v) PEG 3350, 0.2 M sodium bromide, 0.1 M MES (2-(N-morpholino) ethanesulfonic acid) pH 6.7, for the crystal soaked with 2. The drop comprised 1.2 μl protein  and 1.2 μl mother liquor consisting of 12% (w/v) PEG 3350, 0.2 M sodium bromide, 0.1 M MES pH 5.5, for the crystal soaked with 3. The crystal soaks were performed with 10 mM 2 for 4 days, and with 1 mM 3, 1% (v/v) DMSO, for 3 days, and the crystals were fished into liquid nitrogen using a nylon CryoLoop (Hampton Research) via a cryoprotectant consisting of the mother liquor components with 25% ethylene glycol.BtMan2A soaked with compounds 2 and 3 were collected at 100 K out to 2.05 and 1.85 Å, on beamlines I04-1 and I03, respectively. The data were processed using the Xia2 41 pipeline with Dials.42 Diffraction data for CmMan5A in complex with compound 3 were collected out to 1.30 Å at 100 K on beamline I03 at Diamond Light Source (Harwell) and processed using the Xia2 pipeline with XDS.43 Diffraction data for BsGH164 soaked with compounds 1 and 2 were collected out to 1.75 Å and 2.05 Å at 100 K on beamline I03 at Diamond Light Source (Harwell) and processed using the Xia2 pipeline with Dials (see ESI Table 144 or Molrep45 with the known unliganded enzyme structures (PDBID: 2JE8 (BtMan2A), 1UUQ (CmMan5A), 6T5O (BsGH164)) as search models. The resulting solutions showed difference density for the bound ligands within the enzyme active sites. Ligand coordinates and dictionaries were generated using AceDRG46 or jLigand47 and built into the model using Coot,48 followed by alternating rounds of manual model building and density refinement using Coot and REFMAC49 within the CCP4 suite.50 Models were validated using MolProbity51 and the wwPDB OneDep validation server.All diffraction data were collected using a wavelength of 0.9763 Å. Diffraction data for 5 mL of blood was collected from a healthy volunteer into an EDTA-containing collection tube. Cells were removed by centrifugation and plasma was aspirated gently. 450 μL of plasma was pH-adjusted with 50 μL of 500 mM pH 5.5 sodium citrate buffer. 50 μL of the buffered plasma was then diluted with 950 μL of ultrapure water. A negative control sample was prepared by supplementing diluted plasma with 0.05 volumes of 10% SDS and heating to 95 °C for 5 minutes. An additional 0.05 volumes of water was added to the diluted plasma for equivalence.2CO3 was then added and F360/450 was measured using a Clariostar plate reader (BMG Labtech). Free [4MU] was quantified against a calibration series prepared in 200 mM Na2CO3.For activity measurements, 20 μL of each sample was mixed with 20 μL of 200 μM 4MU-β-mannoside (Sigma) in a 384-well plate and incubated at 22 °C for 30 minutes. 10 μL of 1 M NaFor ABPP, buffered and diluted plasma samples were supplemented with probe to a final concentration of 3 μM and incubated at 37 °C for 2 hours. 10 μL  was separated by SDS-PAGE and the resulting gel was imaged for fluorescence using a Typhoon 5 laser scanner .i buffer  Triton X-100, protease inhibitor cocktail ) with 1 mm glass beads and a FastPrep-24 homogenizer . The basic labeling condition consists of equilibrating 25 μg total protein from the homogenate in McIlvaine buffer  in a total volume of 10 μL, and ABP labeling with 5 μL of 3× concentrated ABP prepared in McIlvaine buffer. For concentration dependent labeling, mouse tissue homogenates were equilibrated in McIlvaine buffer pH 5.5 for 15 min at 37 °C, followed by incubation with various probe concentrations up to 10 μM 4  at 37 °C for 1 h. For labeling at varying pH, mouse tissue homogenates were equilibrated in McIlvaine buffer pH 4.0–7.0 for 15 min at 37 °C, followed by incubating with 1 μM 4 (prepared in McIlvaine buffer pH 4.0–7.0) for 1 h at 37 °C. For labeling at varying incubation time, mouse kidney homogenates were prepared as above, and incubated with 1 μM 4  at 37 °C for 2 min to 2 h. For cABPP, mouse tissue homogenates were pre-incubated with either SDS (2% (w/v)), cyclophellitol (3 μM), ABP MDW941 (3 μM), ABP JJB75 (3 μM), 1–3 (50 μM), 5 (3 μM), or 6 (50 μM) at 37 °C for 2 h, followed by incubation with 4 (3 μM) at 37 °C for 2 h. After incubation, samples were denatured with 3.75 μL 5× Laemmli's sample buffer at 98 °C for 5 min, resolved in 7.5% SDS-PAGE gels, and wet slab gels were scanned for Cy5+ fluorescence using a Typhoon FLA 9500 imager . Coomassie staining was carried out to confirm equal loading.Kidneys from wild type mice were homogenized in KP2HPO4). Samples were subsequently denatured with the addition of 125 μL 10% (w/v) SDS and boiling for 5 min at 100 °C. From here on, samples were prepared for pull-down with streptavidin beads as published earlier.52 After the pull-down procedure, half of the samples were treated by the trypsin digestion buffer  acetonitrile (ACN) and 10 ng μL−1 trypsin) and the bead suspension was incubated in a thermoshaker at 37 °C overnight. The supernatant containing the trypsin-digested peptides was desalted using stage tips, followed by evaporation and redissolving in 75 μL sample solution . The beads containing active-site peptides were eluted with elution buffer (5% (v/v) formic acid, 25% (v/v) ACN, 70% (v/v) H2O, 10 μM biotin) for 30 min at 37 °C, after which the supernatant was desalted using stage tips and prepared for LC/MS analysis using the abovementioned procedures.4.0 mg total protein from mouse kidney homogenates were incubated with either DMSO, 10 μM biotin probe 6, or 5 μM BODIPY-FL probe 5 followed by 10 μM 6, each step being incubated for 1 h at 37 °C in a total volume of 500 μL McIlvaine buffer pH 5.5 (75 mM citric acid/Na2O) on a C18 column , eluted with a 50 min gradient of 10% to 60% phase B (0.1% (v/v) formic acid in ACN), followed by 10 min equilibration to 1% phase B at a flow of 0.4 μL min−1, linked with electro-spray ionization (ESI) via Nano-spray source with ESI emitters (New Objectives) fused silica tubing 360 μm OD × 25 μm ID tapered to 5 ± 0.5 μm (5 nL cm−1 void volume) to a Synapt G2Si mass spectrometer (Waters) operating with Masslynx for acquisition and Ent3 software for polymer envelope signal deconvolution. The following settings in positive resolution mode were used: source temperature of 80 °C, capillary voltage 4.5 kV, nano flow gas of 0.25 Bar, purge gas 250 L h−1, trap gas flow 2.0 mL min−1, cone gas 100 L h−1, sampling cone 25 V, source offset 25, trap CE 32 V, scan time 3.0 s, mass range 400–2400 m/z. Lock mass acquisition was done with a mixture of Leu Enk (556.2771) and Glu Fib (785.84265), lockspray voltage 3.5 kV; Glufib fragmentation was used as calibrant. The PLGS (Waters) program was used for data analysis, protein ID or extraction of mgf files for further Mascot (Matrix Science) search analysis.For the LC/MS analysis, 1 μL of sample was injected with phase A (0.1% (v/v) formic acid in ultrapure H53 18.9 μg of lysates from GBA2-overexpressing HEK293T cells54 were pre-incubated with 1 μM of JJB75 compound 1, 3, 5, 6, or 2% (w/v) SDS (with 5 min boiling at 98 °C when pre-incubation was completed) at pH 5.5 for 1 h at 37 °C, and next incubated with 1 μM ABP 4 at pH 5.5 for 2 h at 37 °C. Samples were denatured and subjected to SDS-PAGE and fluorescence detection. The gel was stained with Coomassie Brilliant Blue G250 to confirm equal protein loading.HEK293T cells were purchased from ATCC and handled according to the published methods.This study was conceived by NGSM, GJD, JMFGA, and HSO. NGSM, GJD, and HSO co-wrote the manuscript with input from all authors. TJMB, and C-SW synthesized all compounds under supervision of JDCC and HSO. NGSM, WAO, and ZA prepared recombinant protein samples, grew protein crystals, and solved protein complex structures. NGSM measured enzyme activity and intact MS. C-LK prepared biological samples and performed ABPP experiments under the supervision of JMFGA, and BIF collected proteomic data.There are no conflicts to declare.OB-020-D1OB02287C-s001


Ovarian cancer is the most lethal gynecological cancer, and it is frequently diagnosed at advanced stages, with recurrences after treatments. Treatment failure and resistance are due to hypoxia-inducible factors (HIFs) activated by cancer cells adapt to hypoxia. IGFBP3, which was previously identified as a growth/invasion/metastasis suppressor of ovarian cancer, plays a key role in inhibiting tumor angiogenesis. Although IGFBP3 can effectively downregulate tumor proliferation and vasculogenesis, its effects are only transient. Tumors enter a hypoxic state when they grow large and without blood vessels; then, the tumor cells activate HIFs to regulate cell metabolism, proliferation, and induce vasculogenesis to adapt to hypoxic stress. After IGFBP3 was transiently expressed in highly invasive ovarian cancer cell line and heterotransplant on mice, the xenograft tumors demonstrated a transient growth arrest with de-vascularization, causing tumor cell hypoxia. Tumor re-proliferation was associated with early HIF-1α and later HIF-2α activations. Both HIF-1α and HIF-2α were related to IGFBP3 expressions. In the down-expression of IGFBP3 in xenograft tumors and transfectants, HIF-2α was the major activated protein. This study suggests that HIF-2α presentation is crucial in the switching of epithelial ovarian cancer from dormancy to proliferation states. In highly invasive cells, the cancer hallmarks associated with aggressiveness could be activated to escape from the growth restriction state. Rising death in EOC is due to the difficulty in early diagnosis and the treatment failure by unable to eradicate the cancer cells. Some cancer cells could survive from treatments due to their strong adaptation to the hypoxic environment developed from aggressive cancer growth or chemotherapy by activating hypoxia-inducible factors (HIFs)4. Hypoxia can modulate the cell response to cancer treatment and result in chemoresistance. Ovarian cancers are highly hypoxia-dependent, which affects the results of chemo- and immunotherapy. Therefore, alleviating hypoxia in ovarian cancer is essential for treatment efficacy. Targeting HIFs can help regulate angiogenesis, change the tumor microenvironment, increase the effect of chemotherapy, and help to overcome chemoresistance5.Ovarian cancer is the most lethal gynecological cancer. The majority of malignant ovarian tumors are epithelial type ovarian cancer (EOC), which is diagnosed at advanced stages. Despite the aggressive surgical and chemotherapeutic treatments, the survival rate of patients with EOC is still low in most countries6, epithelial-mesenchymal transition (EMT)7, invasiveness, metastasis8, and immune surveillance suppression9. It could also adversely affect the effects of chemotherapy with more unsatisfactory survival outcomes11. Many biological events associated with cancer cells occurred under a hypoxic tumor environment, such as the selection of clonal populations resistant to apoptosis12 and anaerobic metabolic switch 13. Intra-tumor hypoxia also contributes to genome instability by suppressing DNA-repair pathways14, DNA methylation15, and generating reactive oxygen species16. Many studies have focused on these fields to identify new hypoxia targets to assist the limitations of cancer treatments17.The biological functions of hypoxia are associated with tumor progressions, such as angiogenesis18. HIF-1α and HIF-2α overexpressions are driven by intra-tumoral hypoxia, growth factor signaling, and genetic mutations in oncogenes and tumor suppressor genes19. These are also associated with increased tumor vascularization and poor prognosis of various cancers such as breast, ovarian, and non-small cell lung20.HIFs play a central role in cellular adaptation to hypoxia. Mammals possess three isoforms of HIFα21. IGFBP3 could also activate Thrombospondin-1 (THBS1), an anti-angiogenic protein, to inhibit tumor angiogenesis, resulting in a temporary tumor growth arrest22.We have previously established an EOC cell line and several sublines with different invasion abilities. Insulin-like growth factor binding protein-3 (IGFBP3) was found as an invasion suppressor that negatively correlated to the invasiveness of EOC cell line. In the animal model, a low IGFBP3 expression was associated with a more significant tumor progression, higher cancer invasion, and distant metastasis, while a high IGFBP3 expression was associated with tumor necrosis and apoptosisThe lack of neovascularization during tumor growth causes tumor hypoxia. In this study, we explored the relationship between invasion and hypoxia in ovarian cancer cells. We also focused on biological events during tumor hypoxia and how cancer cells overcome the hypoxic microenvironment and proceed to tumor progression.21. In addition, P0 inhibits angiogenesis through intracellular regulation of THBS1 expression, an angiogenesis inhibitor22. In our previous study, xenografts with IGFBP3-expressing cell lines, P0, and P4-I  showed growth arrest associated with the inhibition of vasculogenesis and tumor necrosis. This was not observed in xenografts with very low IGFBP3-expressing cell lines, P4, and P4-V 22. A doxycycline-inducible-expressing plasmid, pBIG2i-hIGFBP3, was established to study the chronological reactions after IGFBP3 expression. Breeding P4-pBIG2i-hIGFBP3 or control P4-pBIG2i transfectants on SCID mice, tumor growth was found to be attenuated after IGFBP3 expression. However, the tumors started to grow again after 15 d of tumor cell implantation (IGFBP3 was continuously stimulated for expression for 7 d). Nevertheless, the growth of P4-pBIG2i-hIGFBP3 was retarded compared to that of P4-pBIG2i  staining, these HIF changes became more apparent. The xenograft’s protein expressions at 12, 15, and 28 d after implantation were analyzed  and Von Hippel-Lindau (VHL) were also analyzed. HO-1 is stimulated by hypoxia and is induced by HIFsOverall, the Western blot and IHC results showed that HIFs accumulated when the tumor entered a hypoxic phase. In the transplantation with/without low IGFBP3 expression, a continuous expression of HIF-2α allows the tumor to escape from the growth arrest caused by hypoxia.2 and 5% CO2, 37 °C, 17 h) conditions. Then, the expressions of IGFBP3, HIF-1α, and HIF-2α were analyzed. A549, which is known to express HIF-2α majorly, was cultured as a hypoxic condition and HIF-2α expression control.To confirm the HIF patterns associated with IGFBP3 and related to invasive capabilities, the HIF expressions were studied in these cell lines using a hypoxia chamber. P0 and P4 were cultured on glass slides, and these cells were incubated under normoxia and hypoxia (1.0% O26. Both VHL and HO-1 were expressed under normoxia and increased under hypoxia. These results suggest that the function and the synthesis pathway of HIFs were operating effectively.Immunocytochemistry (ICC) staining showed that A549 highly expressed HIF-2α, P0 had an increase in HIF-1α, while P4 had a markedly increase in HIF-2α under hypoxia Fig. A,B. Base27. As shown in Fig. IGFBP3 expression than P4 in both conditions. IGFBP3 and HIF-1α expressions were increased under hypoxia in both cells. P4 expressed higher HIF-1α and HIF-2α under normoxia, while P0 had a significantly lower HIF-2α expression compare to P4 in both conditions.We analyzed the IGFBP3, HIF-1α, and HIF-2α mRNA expressions in P0 and P4 under normoxia and hypoxia using GAPDH as a controlLuciferase promoter assay was performed to study the effects of IGFBP3 and hypoxia on HIF-1α and HIF-2α expressions in cells with low (P0) or high (P4) invasion-metastasis abilities Fig. B. IGFBP3These suggest that hypoxia itself could increase the synthesis of IGFBP3, which could also regulate HIF synthesis and activate cells with low invasion-metastasis capabilities.22. Abcam’s angiogenesis antibody array was used to identify more targets involved in the production of different HIFs under normoxic and hypoxic conditions. Figure 28. Our experiments show the predominant expression of HIF-1α in P0 and P4-I and that of HIF-2α in P4 and P4-V. Therefore, it is reasonable to infer that P4 induced HIF-2α under hypoxia stress.IGFBP3 could inhibit angiogenesis through THBS1 expressionThis study showed that re-expressing IGFBP3 could transiently suppress tumor growth. The switch from growth inhibition to proliferation occurred by activating HIFs, such as HIF-1α or HIF-2α.IGFBP3 was previously identified as an invasion/metastasis and growth suppressor. IGFBP3 could inhibit angiogenesis by upregulating THBS1, leading to tumor growth arrest. Tumor cells eventually overcame growth arrest status and switched to tumor proliferation after long days. Here, HIF-2α was identified as a critical protein that displays the switch from tumor growth arrest to tumor proliferation.Different isoforms of HIFs were activated in our cell lines with different invasive capabilities. HIF-1α was activated in P0 and IGFBP3-re-expressing P4. HIF-1α and HIF-2α were both activated in P4 and P4 vector-only transfectants. These results imply that the activation of HIF-1α and HIF-2α could be associated with different hallmarks of cancer aggressiveness.29. HIF-1α is expressed in smooth muscle cells surrounding the blood vessels30 and is associated with angiogenesis, EMT, cancer metabolism, invasion, and metastasis33. In contrast, HIF-2α is expressed in endothelial cells of blood vessels30, kidney and lung epithelial cells, bone marrow macrophages, and neural crest derivatives during development34. HIF-2α is also known to induce angiogenesis, vasomotor, and upregulate many transcription factors in cancer35.It was reported that HIF-1α and HIF-2α respond to different hypoxia condition, HIF-1α mediates the acute response (< 24 h), while HIF-2α regulates the cellular response under prolonged hypoxia36. Some studies showed that the overexpression of HIF-1α was associated with poor survival in patients with EOC37. A high expression of HIF-2α was reported to correlate with poor prognosis and high clinical stage38. Both HIF-1α and HIF-2α seemed to be equally crucial in cancer invasion and aggressiveness39.Ovarian tissues commonly express HIF-1α, which could play an important role in the cyclic angiogenesis necessary in developing ovarian follicles and corpus luteum40. The hypoxic environment causes the promoter to be methylated15 and reduces the expression of IGFBP3. The decrease in IGFBP3 and the activation of HIF-2α instead of HIF-1α accelerate the progression in EOC.The unclear clinical significance of HIFs in EOC could be due to the heterogeneity of the composition of cancer tumors and late hypoxic events. This study is unique because it involved cancer cells with a known degree of invasiveness, which is correlated with IGFBP3, and the studies of hypoxic events were in a chronological sequence. Therefore, we could clarify the clinical roles of IGFBP3, HIF-1α and HIF-2α in EOC progression: HIF-1α being important in early hypoxia response and HIF-2α playing in prolonged hypoxia that could be associated with the overcome of tumor growth restriction Fig. . In addi41. IGFBP3 is reported as a direct HIF-1α and HIF-2α target gene and is highly translated under hypoxic conditions43. HIFs transactivates IGFBP3 through the hypoxia response element (HRE), the sequence recognized by HIFs, in the promoter of IGFBP341.The mechanism of how low IGFBP3 expression increase HIF-2α is unclear. It was reported that insulin, the insulin-like growth factor (IGF), IGFPB and HIF could regulate each other in a reciprocal way. Insulin, IGF-1, and IGF-2 induce expression of HIF-1α, and HIF-1α regulated the expression of IGF-2, IGFBP2, and IGFBP3 under hypoxia24, as shown in this study. These clarified that the results showing the shift from HIF-1α to HIF-2α is not through the loss of function in the HIF pathway.This study shows the increase in IGFBP3 promoter activity, mRNA, and protein expression under hypoxia, suggesting that the hypoxia response element (HRE) of IGFBP3 functions normally and that the regulatory function of HIFs is normal. HO-1 and VHL have an increased expression under hypoxiaThe chronological sequence of HIF changes could be more closely observed in IHC and ICC studies in Figs. 45. Many studies have focused on the pathways related to hypoxia. Lacking of blood vessels causing tumors to enter a hypoxic state, and activates HIFs to overcome the hypoxic stress, resulting in treatment resistance. Therefore, drugs targeting on HIF-2α, such as PT235846 and Belzutifan47, to be potential new drugs for ovarian cancer treatment.Targeted therapy, in additive to current chemotherapeutic regimens, much improved survival outcome in advanced stage ovarian cancer. One of the target drugs currently used is bevacizumab, which inhibits angiogenesisOur research shows that the activation of HIF-1α or HIF-2α by cancer cells is related to the invasion ability of ovarian cancer cells. Highly aggressive ovarian cancer cells show lower IGFBP3 and tend to express HIF-2α under hypoxic conditions. The expression patterns of IGFBP3 and HIFs can be used in the future as diagnostic markers on the invasiveness of ovarian cancer, and on the design for the strategy of target therapy.This study provides a cell model as a basis for future studies on the chronological sequence of HIF changes and effects on tumor cell functions, and in clinically, provides new candidate for target therapy. More studies are needed to determine the mechanisms of the shift between HIF-1α and HIF-2α in tumorigenesis.In conclusion, this study showed the importance of HIF-1α and HIF-2α in tumor growth arrest and proliferation. HIF-1α could react with IGFBP3 associated with tumor arrest signals, while HIF-2α assists tumor growth and aggressiveness. Highly aggressive cancer cells have more cancer hallmarks to escape from the growth restriction states. This study also provides more detailed events during tumor hypoxia and supports the development of a better diagnostic and treatment for EOC.21 were maintained in a DMEM solution with 5% FBS. P0 was the original OVTW59 cell line, while P4 was a subline selected sequentially from P0, was more invasive than P0. The A549 cell line , which expresses HIF-2α under hypoxic conditions, was used as the study control.The EOC cell line OVTW59 P0 and P4 sublines 48 . Transfectants with plasmid without the IGFBP3 cDNA was labeled as -pKG3226 (-V). The transfection of the plasmids followed the protocol of Arrest-In Transfection Reagent .In the inducible IGFBP3-expressing cell line, which was used only in the in vivo study, the full-length human IGFBP3 cDNA was constructed in a doxycycline-induced expression plasmid pBIG2i (provided by Dr. Lin from NTUH) and was labeled as -pBIG2i-hIGFBP3. Transfectants with plasmids without the IGFBP3 cDNA were labeled as -pBIG2i. IGFBP3 expression was induced by 2 mg/mL of doxycycline in drinking water during heterotransplantation. In the constant IGFBP3-expressing cell line, the full-length human IGFBP3 cDNA was constructed in the expression vector pKG3226 that contains the human β-actin promoter and was labeled as -pKG3226-hIGFBP3 (-I)7 of P4-pBIG2i-hIGFBP3 or P4-pBIG2i-transfected cells. The sizes of tumor growth were measured using vernier calipers. The formula to calculate the tumor volume: length × width × width × 0.52, approximates a solid elliptical mass volume.Severe combined immunodeficiency (SCID) mice, aged 6–8 weeks, were transplanted subcutaneously with 2 × 10In addition, 2 mg/mL of doxycycline and 2% sucrose were added in the drinking water of animals, starting on d 8 when the diameter of the tumor exceeded 0.5 cm. The mice were autopsied on 12, 15, 28, and 36 d after the tumor implantation, and the xenograft tumors were examined.Female SCID mice were weighed and randomly divided into two groups, transplanted subcutaneously either with P4-pBIG2i-hIGFBP3 or with P4-pBIG2i-transfected cells. Five mice in each group were sacrificed on 12, 15, 28, and 36 days after tumor implantation (or at humane endpoints) by cervical dislocation after anesthesia by Isoflurane inhalation. The primary humane endpoints selected in the study were weight loss, reduction of activities, the presence of open wounds at heterotransplantation site, infection, or behavior change. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at National Taiwan University College of Medicine and College of Public Health . All methods were carried out in accordance with IACUC guidelines and regulations. This study was carried out in compliance with the ARRIVE guidelines.2 and 5% CO2 for 17 h.The tumor cells were cultured in dishes, well plates, and glass slides under normoxic conditions until 70% confidence; then, these were moved to a hypoxia chamber containing 1.0% of OIn IHC, paraffin blocks of the xenograft tumors were cut into 4-μm sections. The slides were incubated with primary antibodies: IGFBP3 , HIF-1α , HIF-2α , HO-1 , or Von Hippel-Lindau (VHL)  at 4 °C overnight (about 17 h).5 cells were cultured on 25 mm × 25 mm glass slides and incubated under hypoxic or normoxic conditions for 17 h. Then, the slides were incubated with primary antibodies: IGFBP3 , HIF-1α , or HIF-2α  at 4 °C overnight (about 17 h).In ICC, 0.2 × 10A chromogen in IHC and ICC was developed, following the protocol of UltraVision Quanto Detection System HRP DAB . The sections or slides were stained with hematoxylin for examination, and photos were taken using Moticam X3 Plus  microscope camera by Motic Images Plus 3.0 software. The signals of IHC and ICC were quantified and analyzed by ImageJ 1.53 k software .The total protein from the cells was purified using a Triton X-100 Lysis Buffer , and the total protein from the xenograft tumors was purified using a Tissue-PE LB Buffer . These lysis buffers contained Halt Protease and Phosphatase Inhibitor Cocktail . The final protein concentrations were determined using a Bio-Rad Protein Assay Dye Reagent . Then, the proteins were mixed with a sample buffer T-Pro Laemmli (SDS sample) Reagent (reducing 4 ×)  in final concentrations of 40 μg per 20 μL and incubated at 100 °C for 10 min. The samples were separated by electrophoresis in a gradient (4 to 15%) SDS-PAGE gel  in a ProSieve EX Running Buffer  with 60 mA for 45 min. Then, they were transferred from the gel to PVDF membranes  by Semi-Dry EBU-4000 Blotting System  in a ProSieve EX Transfer Buffer  with 375 mA for 25 min. The membranes were blocked with Trident Universal Protein Blocking Reagent  and were incubated with antibodies: IGFBP3 , HIF-1α , HIF-2α , VHL , HO-1 , GAPDH , cyclophilin A , or HSP90 . HSP90 was used as the control. Signals were developed, following the Trident Femto Western HRP Substrate  manufacturer’s recommendations, and were photographed using UVP BioSpectrum 600 . The signals of Western blots were quantified and analyzed by Image Studio Lite version 5.2 .27. The total RNA from P0 and P4 was purified using a Novel Total RNA mini kit . The reverse transcript to cDNA followed the protocol of SuperScript III Reverse Transcriptase . Gene expressions were analyzed using ABI7900  with SYBR Green Real-time PCR Master Mix . The specific PCR primer sequences of these genes were as follows: IGFBP3 forward, 5′- TGGGCCATGACTGAGGAAA -3′ and reverse, 5′- TGCCGACCTTCTTGGGTTT -3′; HIF-1α forward, 5′-AGTGCCACATCATCACCATATAGAGAT-3′ and reverse, 5′- CTGTTCTATGACTCCTTTTCCTGCT-3′; HIF-2α forward, 5′- AGCCTCCATCTGCCATCAGTC-3′ and reverse, 5′- CTTGCCATGCCTGACACCTTG-3′; GAPDH forward, 5′- TGGTATCGTGGAAGGACTCA-3′ and reverse, 5′- AGTGGGTGTCGCTGTTGAAG-3′. The quantitative real-time PCR data were analyzed using the 2−ΔΔCt method.The expressions of IGFBP3, HIF-1α, and HIF-2α mRNA were detected, and GAPDH was used as the normalizing controlIGFBP3 , HIF-1a , and HIF-2a  were generated by chromosomal DNA extraction from P0, amplified by Q5 High-Fidelity DNA Polymerase , and constructed into pGL3 . The pGL3-IGFBP3, pGL3-HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins  or IGFBP3 neutralizing antibodies  were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega  measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit . Luciferase activities were calculated relative to the empty pGL3 transfectants.The promoter fragments of The expression of angiogenesis-related proteins among P0, P4, P4-I, and P4-V under normoxic and hypoxic conditions (17 h) were identified using Human Angiogenesis Antibody Array-Membrane (43 targets) . The membrane blots were photographed using iBright CL1000 Western Blot Imaging Systems  and analyzed by Image Studio Lite version 5.2 .The animal study protocol had been reviewed and approved by the Institutional Animal Care and Use Committee(IACUC) , National Taiwan University College of Medicine and College of Public Health .The animal study was carried out in accordance with IACUC guidelines and regulations, and compliance with the ARRIVE guidelines.The research plan containing the gene recombination and transfection of the plasmids was approved by National Taiwan University Hospital and was carried out according to National Taiwan University Hospital's gene recombination and biological materials experimental regulations.IACUC Approval No: 20130524. The animal use protocols listed below had been reviewed and approved by the Institutional Animal Care and Use Committee(IACUC), National Taiwan University College of Medicine and College of Public Health.Supplementary Information.


Therefore, the number density, Nanoparticle agglomeration in the transition regime (e.g. at high pressures or low temperatures) is commonly simulated by population balance models for volume-equivalent spheres or agglomerates with a constant fractal-like structure. However, neglecting the fractal-like morphology of agglomerates or their evolving structure during coagulation results in an underestimation or overestimation of the mean mobility diameter,  Incipiec plumes . At highc plumes  or durinc plumes  and atmoc plumes , these avd, with nanoparticle agglomerates [Agglomeration dynamics could be simulated by population balance models if an accurate collision frequency, employed . For exa regimes  based ong and dm , changesg and dm , diameteg and dm . Accountg and dm ,14. Ther, Df=1.8 ,16 often=1.8 [dp ,18. Simiomerates .g,mσ = 1.48 ± 0.03 [2[2 nanoparticle agglomerates in the transition regime [With mesoscale discrete element modeling (DEM), the evolution of particle size distribution , morphol8 ± 0.03 , that is8 ± 0.03  and in e± 0.03 [2 and soot± 0.03 [2,24 agglo± 0.03 [2. This DEn regime . FurtherSectional models have been interfaced with computational fluid dynamics (CFD) in order to explain soot formation in diffusion flames ,14 or pri et al.  interfacparable) . In thisparable)  was coupThe objective of this work is to develop an accurate but computationally affordable MPBM for nanoparticle agglomeration in the transition regime accounting for the evolving agglomerate structure and size distribution. Thus, the DEM-derived quasi-self-preserving size distributions of agglomerates  and data2 [P, is increased from 1 to 5 bar to simulate conditions close to those in combustion engines [Ballistic and Brownian coagulation dynamics of nanoparticles in the absence of rotation, convection, deposition, van der Waals, electric or hydrodynamic forces are simulated by DEM of agglomeration in the free molecular and transition regimes . This is soot [2 . Particl soot [2 . The tim soot [2 . Particl engines . The geored soot  and metared soot  primary The mobility diameter, nd dp by :(1)dmdp=, dg, by :(2)dmdg=2, ZrO2, Au and Ag aerosols [These power laws are obtained for soot particles formed in flames with maximum soot volume fractions spanning two orders of magnitude . Furtheraerosols .md distribution from the free molecular to the transition regime has been validated with data of organic and inorganic nanoparticles from premixed [md distribution as a function of the normalized mean, md/p,d of agglomerates, with primary particles having P = 1 , 3 (b: dotted line) and 5 bar (b: solid line). Small 2 [2 [P from 1 to 5 bar accelerates the attainment of the The evolution of the detailed DEM-derived premixed , diffusipremixed  and sprapremixed . Figure g., soot ), while g., SiO2  and ZrO2 SiO2 [2 ) that ate growth . At dm/d8 ± 0.03  dNAgdnic mean :(4)βm=βfr regime :(5)βfm=4m regime :(6)βco=8ure on β , mAg is to βm by :(9)βp= residence times spanning 6 and 8 orders of magnitude, respectively (m regime . Increasressures . This reat 1 bar a that tatructure . The MPBectively . and (2) , solid len line) . Ramifiee) [dp=9  and 19.6e) [dp=9 , respectT = 295 K (symbols). Soot nanoparticles are sampled from the premixed flames with EQR = 2  or 2.4  shown in The impact of the agglomerate morphology on the coagulation dynamics of nanoparticles in the transition regime is investigated next. In particular, (symbols ) are comApproximating agglomerates with volume-equivalent spheres underpredicts their A simple monodisperse population balance model (MPBM) is derived here for the agglomeration of nanoparticles in the transition regime. The MPBM uses relations derived from detailed discrete element modeling (DEM) simulations in order to obtain the evolving structure of agglomerates quantified by their mobility and gyration diameters. As a result, the DEM-derived collision frequency,


Amitriptyline and nortriptyline were found to inhibit OCT1 in recombinant cells with IC50 values of 28.6 and 40.4 µM. Thirty other antidepressant and neuroleptic drugs were also found to be moderate to strong OCT1 inhibitors with IC50 values in the micromolar range. However, in 35 healthy volunteers, preselected for their OCT1 genotypes, who received a single dose of 25 mg amitriptyline, no significant effects on amitriptyline and nortriptyline pharmacokinetics could be attributed to OCT1 genetic polymorphism. In contrast, the strong impact of the CYP2D6 genotype on amitriptyline and nortriptyline pharmacokinetics and of the CYP2C19 genotype on nortriptyline was confirmed. In addition, acylcarnitine derivatives were measured as endogenous biomarkers for OCT1 activity. The mean plasma concentrations of isobutyrylcarnitine and 2-methylbutyrylcarnitine were higher in participants with two active OCT1 alleles compared to those with zero OCT1 activity, further supporting their role as endogenous in vivo biomarkers for OCT1 activity. A moderate reduction in plasma isobutyrylcarnitine concentrations occurred at the time points at which amitriptyline plasma concentrations were the highest. In a second, independent study sample of 50 patients who underwent amitriptyline therapy of 75 mg twice daily, a significant trend of increasing amitriptyline plasma concentrations with decreasing OCT1 activity was observed (p = 0.018), while nortriptyline plasma concentrations were unaffected by the OCT1 genotype. Altogether, this comprehensive study showed that OCT1 activity does not appear to be a major factor determining amitriptyline and nortriptyline pharmacokinetics and that hepatic uptake occurs mainly through other mechanisms.The tricyclic antidepressant amitriptyline is frequently prescribed but its use is limited by its narrow therapeutic range and large variation in pharmacokinetics. Apart from interindividual differences in the activity of the metabolising enzymes cytochrome P450 (CYP) 2D6 and 2C19, genetic polymorphism of the hepatic influx transporter organic cation transporter 1 (OCT1) could be contributing to interindividual variation in pharmacokinetics. Here, the impact of OCT1 genetic variation on the pharmacokinetics of amitriptyline and its active metabolite nortriptyline was studied  Amitriptyline (AT) is a tricyclic antidepressant that has been in use for the therapy of major depression and other psychiatric disorders since the 1960ies. It is still frequently used today but usually as second-line therapy, due to its risk for severe adverse reactions. In addition, AT and its active metabolite nortriptyline (NT) show large interindividual variation in pharmacokinetics and, accordingly, there is substantial interest in therapy individualisation by drug monitoring and using molecular genetic biomarkers for polymorphic drug membrane transport and biotransformation . The mecUpon systemic absorption, AT is subject to extensive hepatic metabolism, with less than 5% excreted unchanged in urine . AT is ma of 9.4, approximately 99% of AT is positively charged at physiological pH and, despite its lipophilicity (logD7.4 of 3), it may traverse cell membranes more efficiently by carrier-mediated transport than by non-ionic diffusion. One possible influx transporter with particular relevance for hepatic uptake could be the organic cation transporter 1 , a member of the Solute Carrier (SLC) family with a very broad substrate profile .The inhibition of OCT1 by different psychotropic drugs was studied in transport experiments using HEK293 cells stably transfected with wild-type OCT1. The cells were generated by targeted chromosomal integration using the Flp-In system , as has been described in detail before . The cel5 cells/well in 12-well plates coated with poly-D-lysine and incubated as described above. On the day of the experiment, the cells were washed twice with prewarmed (37°C) Hank’s Balanced Salt Solution (HBSS). They were subsequently incubated for 3 min at 37°C with 1 µM of the fluorescent OCT1 substrate ASP+ (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide) and increasing concentrations of the antidepressants in 500 µl HBSS. The reaction was stopped by adding 2 ml ice-cold HBSS. This was subsequently removed and the cells were lyzed in 500 µl radioimmunoprecipitation assay (RIPA) buffer for 10 min under shaking. The cell uptake of ASP+ was quantified using a Tecan Ultra microplate reader  at excitation wavelength 485 nm and emission wavelength 612 nm. The intracellular ASP+ concentrations were normalised to the total amount of protein in the sample that was determined using the bicinchoninic acid assay  and Prism 5 .Approximately 48 h before the transport experiments, recombinant OCT1-expressing cells and empty vector-transfected control cells were seeded at a density of 4 × 10id assay . IC50 vaClinicaltrials.gov (NCT02054299) and EudraCT (number 2012-003546-33).In this open-label study, the pharmacokinetics of 25 mg AT were analysed in relation to OCT1, CYP2D6, and CYP2C19 genotypes. In total, 35 unrelated healthy volunteers participated in this study. When considering the frequent OCT1 alleles *2, *3, *4, *5, and *6 to be functionally deficient, approximately 25% of Europeans are carriers of at least one deficient OCT1 variant and about 7% are homozygously deficient with respect to OCT1 . In ordeHealthy male and female volunteers aged between 18 and 50 y with a body mass of at least 48 kg and a body mass index of 17–32 were eligible for inclusion. Volunteers who underwent regular drug treatments other than oral contraceptives or who suffered from any relevant chronic illness, as well as pregnant or lactating women, were not included. All subjects were healthy according to a detailed medical history, medical examination, electrocardiogram, urine status and clinical chemistry, and haematology parameters .®, Mylan dura GmbH, Darmstadt, Germany) was orally administered to each subject. Blood samples were taken before AT administration and at 1, 2, 4, 6, 8, 12, 24, and 48 h after administration. The blood samples were centrifuged at room temperature for 10 min and the plasma was stored at −20°C before the concentration analyses. Blood pressure and heart rate were measured, a resting electrocardiogram was taken, pupillometry measurement was taken, and the participants were asked to report on any adverse events and possible symptoms  using visual analogue scales 1 h before AT administration and at the following time points after administration: 65 min, 3, 5, 7, 11.5, 23.5, and 47.5 h.After overnight fasting, a single dose of 25 mg AT , and the plasma was stored at −20°C. For determining the plasma concentrations of AT, NT, IBC, 2-methylbutyrylcarnitine, and propionylcarnitine, plasma samples were mixed with twice the volume precipitation reagent of 10% (v/v) methanol and 90% (v/v) acetonitrile that included the corresponding deuterated internal standards AT-d6 , NT-d3, IBC-d6, 2-methylbutyrylcarnitine-d9, and propionylcarnitine-d3 and d9  and shaken for 15 min. After centrifugation , two-thirds of the supernatant were transferred to a new reaction tube and evaporated at 40°C under nitrogen flow. The residue was reconstituted under shaking in 0.1% methanoic acid and briefly centrifuged before quantification using a Nexera UHPLC system  coupled to an API 4000 tandem mass spectrometer . Separation was done using a Brownlee SPP RP-Amide column  with a Phenomenex C18 pre-column . For AT and NT, the mobile phase consisted of 0.1% (v/v) methanoic acid, 5.3% (v/v) methanol, and 31.7% (v/v) acetonitrile in water. For the carnitine derivatives, it consisted of 0.1% (v/v) methanoic acid, 0.43% (v/v) methanol, and 2.57% (v/v) acetonitrile in water. The lower limit of quantification was 0.5 ng/ml for AT and 0.1 ng/ml for NT. Precision and accuracy were controlled by additional control samples spiked with 2 and 20 ng/ml of AT and NT, resulting in coefficients of variation of 6.0 and 3.8% (means of 2.02 and 19.5 ng/ml) for AT and of 3.8 and 2.7% (means of 1.91 and 18.8 ng/ml) for NT. The mass spectrometry detection parameters are listed in The peripheral venous blood samples of the healthy volunteers were treated with ethylenediaminetetraacetic acid (EDTA) for anticoagulation, centrifuged within 30 min after withdrawal , as described before . The following genetic variants were analysed using single-base primer extension using fluorescence-labelled dideoxynucleotides . AUC of NT was calculated from the time of dosing until the last measurement at 48 h using the linear/log trapezoidal rule, as a decline in NT concentrations was not observed in some subjects and extrapolation to infinity thus not possible. Further parameters that were studied included the total plasma clearance after oral administration (CL/F) and the terminal half-life (t1/2), which were calculated as CL/F = dose/AUCinf and t1/2 = ln (2)/lambda z.For the study in healthy volunteers, the primary endpoints were the AUCs of plasma AT and NT concentrations. Secondary endpoints were the other pharmacokinetic parameters of AT and NT, as well as heart rate, blood pressure, pupil size effects, and possible adverse events . Pharmacokinetic parameters were calculated by non-compartmental analysis using Phoenix 64 WinNonlin version 6.3 . AUCa priori ordering (or trend) in gene activities from zero to normal to ultra-rapid (for CYP2C19 and CYP2D6). To do so, the genotypes were categorized into 0, 1, or 2 active alleles for OCT1, into 0, 0.5, 1, 1.5, 2, 2.5, or 3 active alleles for CYP2D6, and into 0, 1, 1.5, 2, 2.5, or 3 active alleles for CYP2C19, depending on their known effects on transporter/enzyme activity. OCT1 alleles *2, *3, *4, *5 were classified as being zero active. However, given the substrate-dependent effects of OCT1*2, calculations were repeated with OCT1*2 classified as being fully active. A semi-quantitative gene dosage was calculated for CYP2D6, as has been described earlier  or mean plasma concentration per dose unit (study in depressive disorder patients) and OCT1, CYP2D6, and CYP2C19 genotypes were calculated using the Jonckheere-Terpstra non-parametric analysis, which takes into consideration the  earlier . For calEM = 100/(0.1 × AUCEM/AUCPM + 0.4 × AUCEM/AUCIM + 0.5) and for CYP2C19 EM as DEM = 100/(0.03 × AUCEM/AUCPM + 0.27 x AUCEM/AUCIM + 0.7). The dose adjustments for the poor (PM), intermediate (IM), and ultra-rapid (UM) metaboliser phenotypes were calculated as follows: DPM or IM or UM = DEM x AUCEM/AUCPM or IM or UM. The multipliers in the EM calculations account for the typical population frequencies of the respective genotypes in European populations (e.g. 0.1 for 10% of CYP2D6 PM). The rationale behind these calculations is that the average recommended drug dose usually determined without considering the genotypes was chosen as the average optimum for populations with the given genotype frequencies styryl)-N-methylpyridinium iodide) in OCT1-overexpressing cells. As shown in 50 value of 28.6 ± 18.9 µM and NT of 40.4 ± 16.2 µM, which is in agreement with previously reported data  allele, nine subjects carried one active allele (OCT1*1) and one allele with no or reduced activity , and 12 subjects were carriers of two OCT1 alleles with no or reduced activity . There were no significant differences in demographic data between the OCT1 genotypes  received 25 mg of AT as a single dose. The study participants were between 18 and 48 y of age, with a mean age of 27 y. The mean body mass index was 23.0 kg/menotypes .inf of AT varied about fourfold (range: 109.9–429.9 h*µg/L) and the AUC48h of NT approximately sevenfold (range: 39.3–283.7 h*µg/L). However, these variations were apparently not a result of OCT1 polymorphism, as differences in AUC between carriers of two, one, or zero active OCT1 alleles were not statistically significant , which was almost twofold higher in the group comprised of the carriers of two active OCT1 alleles as compared to the other two groups. However, this difference is likely explained by one subject with particularly high plasma NT concentrations, who had low CYP2D6 activity and very high CYP2C19 activity (48h of the ‘active moiety’ (sum of the AUC48h of AT and NT) between the OCT1 genotypes were not significant .Large variation was seen in the pharmacokinetics of AT and, even more so, for its therapeutically active metabolite NT. The AUCnificant . The onlactivity . Any difmax, Cmax, and AUC48h for NT differed significantly based on OCT1 genotype , whereas any differences in AT pharmacokinetic parameters were still statistically not significant.Interestingly, if OCT1*2 would be considered as being fully active, Tinf and AUC48h as well as a longer plasma half-life and a lower AT clearance . Statistically significant time- and concentration-related adverse effects like dry mouth, visual or hearing impairment, restlessness, headache, nausea, dizziness, or a sensation of cold reported using visual analogue scales as well as potential anticholinergic effects studied by pupillometry were not observed after the 25 mg AT dose.AT was generally well-tolerated and no serious adverse events occurred during the entire study. Using visual analogue scales, the participants reported symptoms of fatigue, which peaked at 3 h after AT administration at which plasma AT concentrations were generally the highest . Howeverp = 0.018, Jonckheere-Terpstra test). In contrast, mean plasma NT concentrations per dose unit were relatively similar for all OCT1 genotypes. Differences in the ‘active moiety’ (sum of AT and NT plasma concentrations) between OCT1 genotypes were significant . Multiple linear regression analysis showed significant effects for OCT1 and CYP2C19 on AT and highly significant effects for CYP2D6 on NT mean plasma concentrations per dose unit , and NT , but a trend of increasing plasma concentrations with decreasing OCT1 activity was still seen for AT.If OCT1*2 would be considered as being fully active, 44 patients would be carriers of two active OCT1 alleles, five patients would be carriers of one active OCT1 allele, and one patient would be a carrier of zero active OCT1 alleles. With this classification, the mean plasma concentrations per dose unit were not significantly different between OCT1 genotypes for both AT . At time points 2 and 4 h after AT administration, at which plasma AT concentrations were generally the highest, plasma IBC concentrations were reduced to 72 and 67% of the baseline IBC concentrations  are relevant for intestinal absorption, hepatic uptake, and passage across the blood-brain barrier.