E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of  Phosphoribosyl pyrophosphate (PRPP) is an important intermediate in multiple metabolic pathways in cells. It is utilized in the biosynthesis of purine and pyrimidine nucleotides, histidine and tryptophan, and the cofactors NAD and NADP . PRPP isPRPS is essential for maintaining PRPP pool and cellular metabolic homeostasis. In humans, missense mutations of PRPP synthase isozyme 1 (PRPS1) alter enzyme activity or the allosteric regulation and are associated with many severe pathological outcomes . The decE. coli and humans. Phosphate ion (Pi) is necessary to activate class I PRPS, while ADP, an allosteric inhibitor, competes with Pi and ATP at allosteric and active sites, respectively  . The cytal forms , the filE. coli PRPS can form filaments in vitro and in vivo. Using cryo-electron microscopy (cryo-EM), we solve two types of PRPS filaments with near-atomic resolution of 2.3–2.9 Å. Structural and biochemical analyses indicate that the formation of type A filament attenuates the allosteric inhibition. In addition, the conformational changes of the regulatory flexible loop (RF loop) suggest that it plays a role in the regulation of allosteric inhibition and ATP binding. A noncanonical allosteric binding site for AMP and ADP binding is identified in the type A filament, which participates in the regulation of the RF loop. Altogether, our findings reveal a novel mechanism of structural regulation of E. coli PRPS, and provide new insights into PRPS-related human disorders and potential clinical and industrial applications.In the present study, we find that E. coli genome, which has 47.5% sequence identity to human PRPS1 and PRPS2 (There is only one PRPS gene in nd PRPS2 . RecentlE. coli PRPS from E. coli K12 strain in Transetta (DE3) cells, and analyzed its structures under cryo-EM. In order to determine the conditions for inducing PRPS polymerization, we incubated PRPS protein with its known ligands and examine it under negative staining electron microscopy. We provide cryo-EM data and model refinement statistics in We expressed and purified 2+ is known to promote ATP binding at the active site . However, when PRPS was incubated only with ATP or any adenine nucleotide and Mg2+ (10 mM), many PRPS filaments could be observed. On the other hand, we also incubated PRPS at Pi concentrations of 10, 30, and 50 mM, and found that PRPS filamentated at 50 mM Pi  and the second condition is only Pi (50 mM). These two conditions can induce PRPS filamentation without triggering the reaction, and the binding modes of most PRPS ligands are expected to be determined in the models. As a result, two distinct filament structures, type A (ATP and Mg2+) and type B (Pi) filaments were solved  and single-particle analysis. The first condition is the combination of ATP (2 mM) + Mge solved  and 3. Ie solved .Legionella pneumophila PRPS structure (PDB ID: 6NFE), while Pi is located at the binding position of the β-phosphate of ADP at the canonical ADP allosteric site  , which is bound by AMP in the  site 1) .Since we did not add ADP to the mixture for sample preparation, ADP in the model might come from the spontaneous hydrolysis of ATP or be preserved during protein purification. The latter scenario can also explain the unexpected presence of R5P in the model. Meanwhile, in type B filament, R5P binding site and allosteric site 1 are bound by Pi, while ATP binding site and allosteric site 2 are empty .The structural comparison between the two models shows that the PRPS monomers in type A and type B filaments are highly similar except for the regulatory flexible loop region  . The mai2+. One Mg2+ (Mg site 1) coordinates the C1, C2, and C3 hydroxyl groups of R5P with D170 and two water molecules, and the other Mg2+ (Mg site 2) coordinates oxygens of the α- and β-phosphates of ADP (active site) with H131 and three water molecules , which is adjacent to allosteric site 2 bound with SO42+ in multiple human PRPS1 structures (PDB ID: 2H06 and 2HCR) (AMP/ADP), which was formed under the conditions of AMP (2 mM)+ADP (2 mM)  . ParalleP (2 mM) . This stP (2 mM) . In addi related .In the model of type B filament, allosteric site 1 is bound by Pi and allosteric site 2 is incomplete due to the conformational change of hexamer from closed to open . The 5-pBacillus substilis PRPS (PDB ID: 1DKU)  . The B-fxibility . Howeverility  carrying both R302A and Y24A mutations, which may not be able to form filaments. The ability of each PRPS mutant to form type A and type B filaments was evaluated under negative staining electron microscopy. As expected, PRPSR302A/Y24A failed to assemble filaments under all conditions, and PRPSR302A and PRPSY24A could only form type B and type A filaments, respectively  +PRPP → orotidine 5'-monophosphate (OMP) +PPi  in the reaction MP) +PPi . TherefoIn the absence of ADP, we found the substrate concentration used in the assay , PRPS catalyzed the reaction at the maximum rate . Pi is kR302A decreased significantly, while the activity of PRPSR302A/Y24R was higher , filamentous structures of PRPSWT-mCherry could be observed  or inhibit (e.g. E. coli CTPS) activity  cells. After induction with 0.1 mM IPTG at the OD600 range of 0.5~0.8, the cells were cultured at 37 °C for 4 hr and pelleted by centrifugation at 4000 r.p.m. for 20 min. All remaining purification procedures were performed at 4 °C. The harvested cells were resuspended in cold lysis buffer . After ultrasonication, the cell lysate was then centrifuged  at 4 °C for 45 min. The supernatant was collected and incubated with equilibrated Ni-NTA agarose beads (Qiagen) for 1 hr. Subsequently, the column was further washed with lysis buffer supplemented with 50 mM imidazole. Target proteins were eluted with elution buffer containing 50 mM Tris HCl pH8.0, 500 mM NaCl, 250 mM imidazole, and 5 mM β-mercaptoethanol. Further purification was performed in column buffer (25 mM Tris HCl pH 8.0 and 150 mM NaCl) using HiLoad Superdex 200 gel-filtration chromatography . The peak fractions were collected, concentrated, and stored in small aliquots at −80 °C.Full-length of wild-type or mutant 2. ATP was replaced with 2 mM ADP and 2 mM AMP for generating type AAMP/ADP filaments. For type B filaments formation, 6 μM PRPS protein was incubated in a buffer containing 50 mM Na2HPO4 and 100 mM NaCl. All samples were incubated at 37 °C for 30 minutes and then loaded onto the grid. In order to prepare low-temperature cryo-EM grids, protein samples were loaded on a 300-mesh amorphous alloy grids (CryoMatrix M024-Au300-R12/13) with fresh glow discharge. Grids were blotted for 3.5 s with blot force of –1 at 4  °C and 100% humidity before plunge-freezing in liquid ethane with an FEI Vitrobot Mark IV (ThermoFisher Scientific).To generate type A filaments, 6 μM PRPS protein was dissolved in a buffer containing 25 mM Tris HCl pH 7.5, 2 mM ATP, 10 mM MgCle−Å−2, subdivided into 50 frames at 4 s exposure.Micrographs were collected in super-resolution counting mode with K3 Summit direct electron camera (Gatan) on FEI Titan Krios electron microscope at 300 kV. Automated data acquisition was performed with SerialEM  at a nom2.All image processing steps were performed using Relion3.1-beta . Beam-in2, the optimal density map was sharpened to a nominal resolution of 2.9 Å.A similar procedure was performed for the type B filament dataset. A total of 1,186,879 particles were auto-picked from 2776 images. After multiple rounds of 2D classification and 3D classification, 168,218 particles were selected for 3D auto-refinement, and each particle was subjected to CTF refinement and Bayesian polishing. By using a compact mask with a B-factor of 98 Å for the central hexamerAMP/ADP filament dataset, 1066797 particles were auto-picked from 1824 images. After multiple rounds of 2D classification and 3D classification, 53,045 particles were selected for 3D auto-refinement, and each particle is subjected to CTF refinement and Bayesian polishing. By using a compact mask with a B-factor of 51 Å for the central hexamer2, the optimal density map was sharpened to a nominal resolution of 2.6 Å. LocalRes was used to estimate the local resolution for all maps.For the type AE. coli PRPS [Protein Data Bank (PDB) ID: 4S2U] was applied for the initial models of all datasets. The hexamer models were separated and docked into the corresponding electron density map using Chimera v.1.14  is coupled the forward reaction (OA +PRPP → OMP +PPi) of E. coli orotate phosphoribosyltransferase  and the amount of PRPP generated in the reaction was determined by the reduction of in orotate (OA) in the mixture. The concentration of OA was measured by absorbance at 295 nm for 300 s at 25 °C  was dissolved in Tris-HCl buffer  or Pi buffer . After incubation at 37 °C for 1 hr, the protein samples were loaded onto hydrophilic carbon-coated grids and washed twice with uranium formate. Subsequently, the grids were stained with uranium formate. Imaging was acquired with 120 kV electron microscope  with Eagle 4 K × 4 K CCD camera system  at ×57,000 magnification.The purified E. coli cells were fixed with 4% formaldehyde at 37 °C, 220 r.p.m. for 10 min. After fixation, cells were collected by centrifugation at 12,000 r.p.m. for 1 min. Cell pellets were washed twice with PBS and then resuspended in PBS containing Hoechst33342. The cells were then incubated at room temperature for 1 hr. Add 2.5 μL of cell solution and 1 μL of pre-melted 1.2% low melting-point agar was mixed on the glass slide and covered with a coverslip for observation. Images were captured under Plan-Apochromat 63×/1.40 Oil DIC M27 objective on a Carl Zeiss LSM 800 (Axio Observer Z1) inverted fluorescence confocal microscope.E. coli cells were pre-cultured overnight in 2 mL LB medium at 37 °C, 220 r.p.m., and then inoculated in 5 mL LB culture at 37 °C, 220 r.p.m. with OD600=0.05. The cell growth was determined by the OD600 value, which was measured by Eppendorf BioPhotometer D30 every hour after inoculation. This paper provides new insights into how polymerization into two different structures modulates the activity of the enzyme PRPS. The molecular mechanisms proposed are supported by the data, and likely to be of general interest. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Philip Cole as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Allosteric inhibition of PRPS is moderated by filamentous polymerization" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:While all three reviewers found the work significant and of potential broad interest, all felt that the paper needed major rewriting to improve the clarity and language. Numerous examples are given below where improvements are needed.Reviewer #1 (Recommendations for the Authors):The paper needs careful rewriting, and authors should check and review extensively for improvements to the use of English. Here are just three examples out of many that I found:(line 57) "…outcomes in clinical."(line 73) "the ADP" should be "ADP".E. coli individually."(line 352-4) "We fused the sequences of PRPSWT and PRPSR302A, PRPSY24A and PRPSR302A/Y24A mutants were with mCherry and overexpressed them in Reviewer #2 (Recommendations for the Authors):The manuscript reports impressive high-quality cryo-EM reconstructions of two different PRPP synthetase filaments. The detailed structural analysis, combined with structure-based mutagenesis and enzymatic assays reveals a quite complex regulatory mechanism for this important enzyme. I realize the difficulty of the study, given that ADP can bind to three different sites with different effects on the protein activity, and this makes the analysis very difficult. The finding that the assembly of the enzyme in two different filamentous structures and the repositioning of the regulatory loop can affect the binding of ADP to the different regulatory sites, and the binding of the substrates is very interesting.I encourage the authors to review carefully the manuscript to facilitate the comprehension of the main message.Reviewer #3 (Recommendations for the Authors):1. Some language editing would aid in the clarity of the text (throughout the manuscript).2. Species names should be italicized.3. Page 5, line 85: "The cytoophidium is assembled by bunding filamentous polymers of metabolic enzymes" – is this known for all cytoophidia? If so, a reference should be given. If not, the sentence can be rewritten to indicate that this is speculation or based on some evidence in some cases.4. Page 6, line 94: the sentence "The polymerizations of CTPS and IMPDH have been demonstrated to desensitize the proteins to end-product inhibition or allosteric inhibition effects, suggesting its physiological functions in tuning intracellular nucleotide levels". Please provide a reference to support this statement. Also, is this true in all cases of CTPS/IMPDH polymerization?2+ used in the structure with 2 mM ATP (and no π I presume?)5. Page 9, line 150: what was the concentration of Mg6. Page 9, lines 157 and 167, please state the handedness of the twist of the polymers.7. Page 9, line 157, it is stated that R5P is found in the active site, though it was not added to the enzyme before structural studies were conducted. The authors suggest that it may have copurified with the PRPS, but it is not present in the Type B filament structure. The authors also do not show the map around the R5P. The map should be shown to provide confidence in the assignment of R5P to the map in the active site. Could it be formed from the degradation of ATP (just as the ADP and π presumably are?).8. page 9, line 163 and elsewhere: it's awkward to refer to the structure of L. pneumophila as a model. It should be referred to as an x-ray crystal structure (if indeed that is what it is).9. Figure 2 and text regarding panels in figure 2. It is difficult to deconvolute all the structures and alignments without referencing the figure legend constantly. It would be easier if structures were identified in the panels, such as by color coding their name/type in the panel where they are shown in particular colors. When colors are ambiguous . In the text, when a particular structure is being indicated, the color of that structure in addition to the panel id should be given .10. Page 10, line 169: "ATP binding site and the allosteric site 1 are not bound by any ligand". Should this read "allosteric site 2"?11. Page 10, line 183: some object to the use of the word "monomer" in the context of an oligomer. A protein chain or subunit or protomer would be more appropriate.12. It is interesting that the allosteric sites and active sites are found between protein chains in the hexamer, yet this isn't mentioned.13. page 11, line 190. "His131 and the phosphate". please indicate which phosphate (it appears to be the β).14. page 11, 205-206. regarding the second type A filament structure, which was incubated apparently in both AMP and ADP, how is it determined that both AMP and ADP are bound in site 2? Was refinement done with partial occupancy of each? The sentence describing this structure indicates at first that AMP is in site 2, then the next sentence indicates that ADP is bound. Which is it? this should be more clear.15. page 11, line 208. should guanine be adenine?16. page 11, line 211, "and residues responsible for the binding", please indicate which residues. Also, in figure 2, highlight the residues suggested to be conserved. Residues suggested to be involved in binding to ligands include 102, 178, 182, 149, 133, and 147, however, 178, 182, and 149 do not appear to be conserved. Also, are residues of site 1 conserved? if not, what would that mean?17. page 12, lines 229 and 231, why are the B factors negative?18. page 12, line 232, should "figure 2f" be figure 2e?19. Page 12, line 218, it should be indicated that allosteric site 1 is the site being discussed.20. page 12, line 220. Although ATP binds only in the active site, it should be indicated whenever the ATP binding site is mentioned that it is the ATP binding site in the active site (since it is hard to keep track of which site binds which type of nucleotide).21. page 12, line 220, which phosphate binding site of R5P does π bind at?22. page 12, line 233, "In the stable conformation", is this of the type A filament? If so, it should be indicated.23. Use of apostrophes or asterisks on the residue names in the second paragraph (beginning at line 244) of page 13 would help to show that residues are contributed by different subunits.24. page 13, line 250, indicate which atom of Leu23 hydrogen bonds to Arg301 (presumably the carbonyl oxygen of the main chain of Leu23?).25. page 15, line 280: indicate panel c of Sup 5. Also, what are the concentrations of ligands kept at constant concentrations in Sup 5? (e.g. the concentration of ATP when R5P is varied?). What is the concentration of the enzyme?26. page 16, line 315, "the allosteric inhibition", indicate that it is by ADP, i.e. "allosteric inhibition by ADP".27. page 16, line 319, "enhance the inhibitory effect" to "enhance the inhibitory effect of ADP".28. page 17, line 328, "Since AMP is supposedly unable to bind at the allosteric site 1", please provide a reference and/or rationale to support this assertion.29. page 21, line 400, "Although some filamentous polymers of metabolic enzymes were demonstrated to accommodate different states of the proteins, their protomers in various states assemble through the same interfaces and can either boost or suppress the activity". References for CTPS are given here although the interfaces in CTPS filaments that are activated differ from those which are inhibited. This is true when CTPS from different organisms are compared. Please correct or clarify what is meant by this sentence as it appears to indicate that for CTPS, one type of filament forms which can accommodate either active or inactive conformations. Further, there are other enzymes for which different interfaces result in different filamentous forms, with differing activity, for example, ACC enzyme.30. page 22, line 432: "According to our models, both allosteric site 1 and site 2 could be responsible for the binding of the second ADP in these conditions". It seems as though the conclusion of the paper is that ADP binding at allosteric site 1 would block ATP binding to the active site. Is this not true?31. Page 38, line 689, please indicate allosteric site 1 in "while the allosteric site".32. Colors in Figure 2, panels d and f need to be defined in the figure legend.33. page 40, line 708: "negative staining" should read "negative staining electron microscopy" (and elsewhere in the manuscript).34. Please define the meaning of ***, and so on in the figures.35. Please define "S.E.M." in figure 3e legend.36. figure 4b, why is "n.s." shown for the orange line (the double mutant with mCherry)? It is true that the growth of this mutant is similar to wild type without mCherry, but it is much less when compared to the growth of wild type with mCherry.37. π is found in the type A filament, yet it is absent from the type A filament shown in panel 4c. It also competes for binding with ADP in site 1. Should the "+Pi" be located with the other arrow, to type A, and not type B?38. In Figure 4a, the text of the superscripts is very small.39. Please provide some explanation of the model shown in figure 4c in the figure legend.40. Please provide the concentrations of ligands  in each image of Figure S1.AMP/ADP).41. Please indicate somehow that the phosphate ion shown in Figure S2c is derived only from the type A filament (and not the type A Reviewer #1 (Recommendations for the Authors):The paper needs careful rewriting, and authors should check and review extensively for improvements to the use of English. Here are just three examples out of many that I found:(line 57) "…outcomes in clinical."(line 73) "the ADP" should be "ADP".E. coli individually."(line 352-4) "We fused the sequences of PRPSWT and PRPSR302A, PRPSY24A and PRPSR302A/Y24A mutants were with mCherry and overexpressed them in We have rewritten the manuscript to improve the clarity and presentation.Reviewer #3 (Recommendations for the Authors):1. Some language editing would aid in the clarity of the text (throughout the manuscript).We have rewritten the manuscript to improve the clarity and presentation.2. Species names should be italicized.Revised.3. Page 5, line 85: "The cytoophidium is assembled by bunding filamentous polymers of metabolic enzymes" – is this known for all cytoophidia? If so, a reference should be given. If not, the sentence can be rewritten to indicate that this is speculation or based on some evidence in some cases.By definition, all cytoophidia are bundled filamentous polymers of metabolic enzymes. Related references have been added.4. Page 6, line 94: the sentence "The polymerizations of CTPS and IMPDH have been demonstrated to desensitize the proteins to end-product inhibition or allosteric inhibition effects, suggesting its physiological functions in tuning intracellular nucleotide levels". Please provide a reference to support this statement. Also, is this true in all cases of CTPS/IMPDH polymerization?Indeed, some differences may present among species or protein isoforms. We specified human CTPS1 and IMPDH2 in this sentence and added references.2+ used in the structure with 2 mM ATP (and no π I presume?)5. Page 9, line 150: what was the concentration of Mg2+ has been added in the text. No π in this condition.The concentration of Mg6. Page 9, lines 157 and 167, please state the handedness of the twist of the polymers.Added (left-handed twist) in the description.7. Page 9, line 157, it is stated that R5P is found in the active site, though it was not added to the enzyme before structural studies were conducted. The authors suggest that it may have copurified with the PRPS, but it is not present in the Type B filament structure. The authors also do not show the map around the R5P. The map should be shown to provide confidence in the assignment of R5P to the map in the active site. Could it be formed from the degradation of ATP (just as the ADP and π presumably are?).We have added a supplementary figure showing the map around R5P.8. page 9, line 163 and elsewhere: it's awkward to refer to the structure of L. pneumophila as a model. It should be referred to as an x-ray crystal structure (if indeed that is what it is).Revised.9. Figure 2 and text regarding panels in figure 2. It is difficult to deconvolute all the structures and alignments without referencing the figure legend constantly. It would be easier if structures were identified in the panels, such as by color coding their name/type in the panel where they are shown in particular colors. When colors are ambiguous . In the text, when a particular structure is being indicated, the color of that structure in addition to the panel id should be given .Revised.10. Page 10, line 169: "ATP binding site and the allosteric site 1 are not bound by any ligand". Should this read "allosteric site 2"?Corrected.11. Page 10, line 183: some object to the use of the word "monomer" in the context of an oligomer. A protein chain or subunit or protomer would be more appropriate.Revised.12. It is interesting that the allosteric sites and active sites are found between protein chains in the hexamer, yet this isn't mentioned.We have now mentioned in the text.13. page 11, line 190. "His131 and the phosphate". please indicate which phosphate (it appears to be the β).It is the α phosphate. Revised accordingly.14. page 11, 205-206. regarding the second type A filament structure, which was incubated apparently in both AMP and ADP, how is it determined that both AMP and ADP are bound in site 2? Was refinement done with partial occupancy of each? The sentence describing this structure indicates at first that AMP is in site 2, then the next sentence indicates that ADP is bound. Which is it? this should be more clear.AMP/ADP filament, only AMP is bound to site 2. We revise the sentence to avoid confusion.In type A15. page 11, line 208. should guanine be adenine?Corrected.16. page 11, line 211, "and residues responsible for the binding", please indicate which residues. Also, in figure 2, highlight the residues suggested to be conserved. Residues suggested to be involved in binding to ligands include 102, 178, 182, 149, 133, and 147, however, 178, 182, and 149 do not appear to be conserved. Also, are residues of site 1 conserved? if not, what would that mean?We revised the figure accordingly .17. page 12, lines 229 and 231, why are the B factors negative?It should be positive. Corrected.18. page 12, line 232, should "figure 2f" be figure 2e?Corrected.19. Page 12, line 218, it should be indicated that allosteric site 1 is the site being discussed.Modified.20. page 12, line 220. Although ATP binds only in the active site, it should be indicated whenever the ATP binding site is mentioned that it is the ATP binding site in the active site (since it is hard to keep track of which site binds which type of nucleotide).Modified.21. page 12, line 220, which phosphate binding site of R5P does π bind at?Pi binds at the 5-phosphate binding site of R5P.22. page 12, line 233, "In the stable conformation", is this of the type A filament? If so, it should be indicated.The sentence is modified.23. Use of apostrophes or asterisks on the residue names in the second paragraph (beginning at line 244) of page 13 would help to show that residues are contributed by different subunits.Modified.24. page 13, line 250, indicate which atom of Leu23 hydrogen bonds to Arg301 (presumably the carbonyl oxygen of the main chain of Leu23?).Added.25. page 15, line 280: indicate panel c of Sup 5. Also, what are the concentrations of ligands kept at constant concentrations in Sup 5? (e.g. the concentration of ATP when R5P is varied?). What is the concentration of the enzyme?We added detail description of the reaction mixtures in the text and legends.26. page 16, line 315, "the allosteric inhibition", indicate that it is by ADP, i.e. "allosteric inhibition by ADP".The paragraph has been modified.27. page 16, line 319, "enhance the inhibitory effect" to "enhance the inhibitory effect of ADP".The paragraph has been modified.28. page 17, line 328, "Since AMP is supposedly unable to bind at the allosteric site 1", please provide a reference and/or rationale to support this assertion.This sentence is removed to avoid confusion.29. page 21, line 400, "Although some filamentous polymers of metabolic enzymes were demonstrated to accommodate different states of the proteins, their protomers in various states assemble through the same interfaces and can either boost or suppress the activity". References for CTPS are given here although the interfaces in CTPS filaments that are activated differ from those which are inhibited. This is true when CTPS from different organisms are compared. Please correct or clarify what is meant by this sentence as it appears to indicate that for CTPS, one type of filament forms which can accommodate either active or inactive conformations. Further, there are other enzymes for which different interfaces result in different filamentous forms, with differing activity, for example, ACC enzyme.We revised the sentences toDrosophila CTPS) or inhibit (e.g. E. coli CTPS) activity . In other cases, enzymes can polymerize into multiple polymer types (e.g. human acetyl-CoA carboxylase) through different interfaces ”.“Filamentous polymers of some metabolic enzymes have been demonstrated to accommodate different states of proteins. In most cases, their protomers in various states are assembled through the same interface, which can enhance .Corrected.34. Please define the meaning of ***, and so on in the figures.p < 0.05, ** p < 0.01 and *** p < 0.001. in figure legends.Added *35. Please define "S.E.M." in figure 3e legend.Edited to Error bars = standard error of the mean (S.E.M.).36. figure 4b, why is "n.s." shown for the orange line (the double mutant with mCherry)? It is true that the growth of this mutant is similar to wild type without mCherry, but it is much less when compared to the growth of wild type with mCherry.Here “wild type with mCherry” means “overexpression of wild-type PRPS fused with mCherry”. We revised figure legends accordingly.37. π is found in the type A filament, yet it is absent from the type A filament shown in panel 4c. It also competes for binding with ADP in site 1. Should the "+Pi" be located with the other arrow, to type A, and not type B?We added “+Pi” to the arrow to type A.38. In Figure 4a, the text of the superscripts is very small.Modified.39. Please provide some explanation of the model shown in figure 4c in the figure legend.Added.40. Please provide the concentrations of ligands  in each image of Figure S1.Added.AMP/ADP).41. Please indicate somehow that the phosphate ion shown in Figure S2c is derived only from the type A filament (and not the type AAMP/ADP model:We added the following sentence to explain why there is no π in the type AAMP/ADP model is empty, supporting that the presence of ADP and π in the former type A filament model were due to spontaneous hydrolysis of ATP”.“In addition, the allosteric site 1 in type A


Atrazine, one of the most commonly used herbicides in the world, is of concern because of its frequent occurrence in various water bodies and the potential threat it constitutes to ecosystems. The transport of contaminants in seasonally ice-covered lakes is an important factor affecting the under-ice water environment, and changes in phase during ice growth and melting cause redistribution of atrazine between ice and water phases. To explore the migration pattern of atrazine during freezing and thawing, laboratory simulation experiments involving freezing and thawing were carried out. The effects of ice thickness, freezing temperature, and initial concentration on the migration ability of atrazine during freezing were investigated. The results showed that the relationship between the concentration of atrazine in ice and water during freezing was ice layer < water before freezing < water layer under the ice. Atrazine tended to migrate to under-ice water during the freezing process, and the intensity of migration was positively correlated with the ice thickness, freezing temperature, and initial concentration. During the thawing phase, atrazine trapped in the ice was released into the water in large quantities in the early stages. The first 20% of meltwater concentration was significantly higher than the average concentration in ice, with the highest case being 2.75 times the average concentration in ice. The results reported in this study are a useful reference for planning possible pollution control measures on such lakes during their freeze-thaw process. Freezing is an important hydrological feature of lakes in high-altitude and high-latitude regions . More thLakes have been extensively studied in the literature, yet there are few studies on ice-covered lakes . HoweverSeveral studies have shown the migration of conventional contaminants (nutrients) during freeze-thaw. For example, Hampton et al.  conducteAtrazine is a well-known chlorotriazine herbicide widely used for weed control in agricultural crops . AtrazinMany organic pollutants in water can be transformed by three primary mechanisms: photodegradation, hydrolysis, and biodegradation ,30. HoweAccordingly, in this study, the migration pattern of atrazine between ice and under-ice water was explored by simulating the top-down ice formation of lakes under natural conditions. The effects of ice thickness, freezing temperature and initial concentration on the migration ability of atrazine during the freezing process were investigated. Moreover, the ice obtained under different freezing conditions was thawed under natural conditions to investigate the migration pattern of atrazine during the thawing process. The results obtained herein can provide the basis for planning possible pollution control measures on ice-covered lakes during their freeze–thaw process.Atrazine was purchased from J&K Scientific , with its purity ≥ 98.8%. Methanol and formic acid were of HPLC grade and were purchased from J&K Scientific  and Kermel Chemical Reagent Co., Ltd. , respectively. A Milli-Q water purification system  was employed. Atrazine was prepared as a stock solution of 20 mg/L. The stock solution was kept in reserve at 4 °C until use.Main testing instruments: Ultra-high-performance liquid-chromatography electrospray triple-quadrupole mass spectrometer ; an ACQUITYTM UPLC BEN C8 chromatographic column . In order to simulate the top-down freezing process of lake water under natural freezing conditions, a self-designed unidirectional freezing simulation device was used in this study a. The waTo study the distribution and migration pattern of atrazine in ice and under-ice water during lake icing, and the effects of different icing conditions on atrazine migration, the design of the experiment was as follows. Before freezing, the initial volume and height of the atrazine solution were 1.8 L and 23 cm, respectively.(1)To explore the effect of ice thickness on the migration of atrazine, four different ice thickness conditions  were set and numbered sequentially as 1–4. Four solutions of atrazine with an initial concentration of 15 μg/L were prepared. The freezing temperature was −5 °C. Freezing ended when the ice thickness reached 3 cm, 6 cm, 9 cm, and 12 cm, respectively.(2)To explore the effect of freezing temperature on the migration of atrazine, three different freezing temperature conditions  were set and numbered sequentially as 5–7. Three solutions of atrazine with an initial concentration of 15 μg/L were prepared. Freezing ended when the ice thickness reached 9 cm.(3)To explore the effect of initial concentration on the migration pattern of atrazine, five different concentrations  of atrazine solutions were prepared and numbered sequentially as 8–12. The freezing temperature was −5 °C. Freezing ended when the ice thickness reached 9 cm.The atrazine solutions were frozen in the freezing device. After freezing, the freezing device was removed from the low-temperature experiment box and the ice and water were separated. The ice melted in the ice melting device and the ambient temperature of the melting was 22 °C. After the ice melted, the concentrations of atrazine in the collected meltwater and under-ice water were measured by UPLC-ESI-MS/MS.To study the migration pattern of atrazine in ice during melting, the freezing experiments numbered 1–12 were repeated and the 12 ice cubes obtained were subjected to the melting experiment. The ambient temperature of the melting was 22 °C. The melt water of each ice block was divided into five batches in turn , and the mass of each batch of melt water was 20% of the total mass of the ice. Melt 1 was the first 20%-fraction of the ice-melt water, Melt 2 was the second 20%-fraction, etc. The freezing and melting experiments were repeated three times.Chromatographic conditions: The mobile phase was methanol (A) and 0.1% formic acid in water (B). The flow rate was 0.2 mL/min. The column temperature was 35 °C and the room temperature for sampling was 15 °C. The injection volume was 5 μL. Mass spectrometry conditions: Electrospray ion source (ESI) as positive ion mode acquisition; multiple reaction monitoring (MRM) mode. The capillary voltage and cone voltages were 3.3 kV and 35 V, respectively. The ion source temperature and desolvation (nitrogen) temperature were 120 °C and 350 °C, respectively. The desolvation flow rate and the cone (nitrogen) flow rate were 500 L/h and 30 L/h, respectively. The MRM parameters are listed in 2 = 0.995). At the low (2.5 μg/L), medium (10 μg/L), and high (25 μg/L) levels of atrazine, the recovery rate of the standard addition was between 96% and 112%, and the relative standard deviation (RSD) of the determination was between 0.39% and 0.92%. Hence, the method was accurate and reliable.In the concentration range of 0~100 μg/L, this method exhibited a good linearity for detecting atrazine  and Cw is the atrazine concentration in the water under ice (μg/L). The value of K can be between 0 and 1. A value of K = 0 indicates that the ice formed is completely pure, and all of the atrazine migrates into the water under the ice. A value of K = 1 indicates that the ice and water have the same concentration of atrazine, and no migration occurs during the process. The lower the K value, the stronger the ability of atrazine to migrate to water under the ice.The distribution coefficient (K) was used to express the migration ability of atrazine during freezing, as defined by Equation (1):m) is used to indicate the effect of atrazine release from ice during melting, as defined by Equation (2):e is the mass of atrazine in each meltwater (μg) and MA is the total mass of atrazine wrapped in ice (μg).The melt-out rate  and atrazine concentrations were measured. The distribution of atrazine in ice and under-ice water at different ice thicknesses is shown in Ice and water samples were collected at different freezing temperatures  and atrazine concentrations were measured. The distribution of atrazine in ice and under-ice water at different freezing temperatures is shown in Ice and water samples were collected at different initial concentrations  and atrazine concentrations were measured. The distribution of atrazine in ice and under-ice water at different initial concentrations is shown in m = 20% means that the mass transfer rate of atrazine is consistent with the melting rate of the ice. It can be seen that the Rm of atrazine in the first 20% fraction of the ice meltwater significantly exceeded 20%, which indicates that atrazine tends to be released from the ice when it first starts to thaw. The amount of atrazine in ice meltwater at the beginning of melting is mainly influenced by the degree of contamination of the ice itself. However, there are differences in the ice obtained under the same conditions. The structure of the ice also affects the release of atrazine in the early stages of melting. This may be responsible for the difference in the amount of atrazine released by ice bodies with similar average concentrations at the beginning of melting. Overall, atrazine remaining in the ice was released from the ice in large amounts during the early stages of melting. As melting progresses, the unmelted ice becomes more ”pure”.The ice bodies obtained under different freezing conditions were melted. The average concentration of meltwater and the change in meltwater concentration during melting are shown in The migration process of atrazine between ice and water could be explained by the formation and growth of ice crystals. The temperature of the upper surface of the solution decreased after the solution was placed at a low temperature. Liquid water becomes metastable when the temperature falls below the equilibrium freezing point. Water crystallization is accompanied by probabilistic nucleation events . When th4) [4 was concentrated in water under the ice, and a small amount of KMnO4 was kept in the ice. 4 in the ice as observed through the microscope. The unescaped KMnO4 was mainly distributed near the bubbles. This may be related to the growth of ice crystals. Atrazine may have a similar distribution and migration pattern to KMnO4.However, there is a limit to the ability of ice crystals to repel impurities during the growth of ice. We observed that a small amount of atrazine is retained within the ice, especially during the early stages of freezing. The reason for this is that when the supercooling of the solution is broken, the system releases heat and randomly generates ice nuclei. Atrazine is trapped in the ice due to the rapid growth of ice nuclei and dendritic ice crystals. This is similar to the process of salt pack trapping during seawater freezing . During 4) . MoreoveThe migration ability of atrazine in ice-water phases is affected by factors including ice thickness, freezing temperature, and atrazine initial concentration . During K value increases with decreasing temperature; hence, the migration ability of atrazine from ice to water under the ice is weakened.The freezing temperature determines the freezing rate, nuclear density, shape, and size of ice crystals . As the For atrazine solutions with different initial concentrations, the freezing point decreases as the initial concentration of the solution increases . The staWhen the ambient temperature is above the freezing point, the surface of the ice begins to melt. The rate of melting is determined by the heat transfer from the environment to the ice surface and corresponds to the droplets’ growth rate at the melting interface . DropletIn this study, the distribution and migration of atrazine in ice and water phases during freezing and thawing under different freezing conditions were investigated. The main conclusions are as follows.(1)Freezing causes redistribution of atrazine in both ice and water phases, and its concentration relationship is expressed as ice < water before freezing < water under the ice. Atrazine continuously migrated from the ice to the water under the ice during the freezing process, which reminded us that we need to pay more attention to the quality of under-ice water in the natural water body during the freezing period.(2)During the initial stages of thawing, atrazine trapped in the ice exhibits a massive release into the water. Moreover, as thawing continued, the concentration of atrazine in the meltwater became lower and lower the unmelted ice becomes purer. In areas where the water environment is prone to freezing in winter, the water environment may deteriorate instantaneously due to the concentrated release of atrazine in the early stages of ice thawing.(3)The K value decreased with increasing ice thickness, freezing temperature, and atrazine initial concentration. The migration ability of atrazine was positively correlated with ice thickness, freezing temperature, and atrazine initial concentration. Atrazine has an extremely low distribution coefficient (K = 0.006) when a solution with an initial concentration of 45 μg/L is frozen at −10 °C to a thickness of 9 cm. The migration ability of atrazine was strongest under this condition.


Carcharias taurus and extinct Striatolamia macrota, to reconstruct the size structure for a contemporary locality and four Eocene localities. We compare empirical distributions against expectations from a population simulation to gain insight into potential governing ecological processes. Specifically, we investigate the influence of dispersal flexibility to and from protected nurseries. We show that changing the flexibility of initial dispersal of juveniles from the nursery and annual migration of adults to the nursery explains a large amount of dental distribution variability. Our framework predicts dispersal strategies of an extant sand tiger population, and supports nurseries as important components of sand tiger life history in both extant and Eocene populations. These results suggest nursery protection may be vital for shark conservation with increasing anthropogenic impacts and climate change.Shark teeth are one of the most abundant vertebrate fossils, and because tooth size generally correlates with body size, their accumulations document the size structure of populations. Understanding how ecological and environmental processes influence size structure, and how this extends to influence these dental distributions, may offer a window into the ecological and environmental dynamics of past and present shark populations. Here, we examine the dental distributions of sand tigers, including extant  The enormous spatial and temporal dominance of shark species suggests considerable ecological plasticity, which has likely contributed to their evolutionary success and may be key to understanding the ongoing and future effects of climate change on this diverse group . DocumenBody size has an enormous influence on the structure and functioning of marine communities ,9. FolloCarcharias taurus, Lamniformes: Odontaspididae) grow up to ca 300 cm in length and occupy lower-latitude continental margins around the world, where they primarily hunt for prey along the sea bottom. During the Eocene, sand tigers  is known for its abundant shark fossil record, with locations that ranged from the equator to both poles ,15–17. Tde sites , circle,de sites , diamondde sites , square,To what extent can life-history dynamics drive accumulations of sand tiger teeth, and is it possible to infer such processes from the distributions themselves? Here, we examine sand tiger dental distributions from four Eocene localities that span high- and low-latitudes, as well as a contemporary sand tiger population near Delaware Bay , star. W. 2 (a)Striatolamia macrota teeth are identified by emphasized striations on the lingual side relative to the smooth labial side [S. macrota is the extant sand tiger C. taurus based on the similarities in tooth shape throughout the entire dentition [et al. [S. macrota in Kim et al. [Shark species in the fossil record are largely identified by their tooth morphology  due to tial side . Anterioial side ,23,24. Tentition ,26 to an [et al.  and prevStriatolamia macrota teeth from Banks Island are curated at the Canadian Museum of Nature ; teeth from Seymour Island are curated at the University of California Museum of Paleontology , Paleontological Research Institute , and Swedish Natural History Museum ; teeth from Red Hot Truck Stop locality are curated at the Carnegie Museum of Natural History ; and teeth from Whiskey Bridge locality are curated at the Whiteside Museum of Natural History . Locality descriptions are provided in electronic supplementary material, appendix I. (b)To explore specific ecological mechanisms that may be responsible for the observed dental distributions, we employed a process-based model allowing us to incorporate likely physiological and ecological constraints influencing shark populations. We constructed a two-site size-class model that tracks female shark populations over time, where one of the two sites is designated a juvenile site, or nursery, and the other is designated an adult site . Becauseper capita reproductive rate r was thus set to r = 0 at the adult site, and per capita mortality rate was assumed to be constant across size classes within both juvenile and adult sites at m (g) following the growth trajectory described by West et al. [M at a younger age.We considered four key dynamics influencing changes in population size for both sites: reproduction, somatic growth, mortality and dispersal between sites. In our framework, reproduction takes place only at the juvenile site, whereas mortality occurs at both sites. The e inds/s  at the j9 inds/s . A const9 inds/s  and is mmj, their migration rate to the adult site increases sigmoidally. The juvenile dispersal window ξj describes the flexibility of this mass threshold: a smaller dispersal window (low ξj) means that initial dispersal of juveniles to the adult site operates around a strict mass threshold mj, whereas a large juvenile dispersal window (high ξj) means that initial dispersal of juveniles to the adult site is flexible around mj.In our two-site model, juveniles disperse to the adult site once they have reached a particular mass, and adult females migrate annually from the adult to juvenile site to reproduce. The initial dispersal of juveniles to the adult site and annual dispersal of adults to the juvenile site are considered separately because we assume these events are mass-dependent and time-dependent, respectively. As offspring grow in size to maturation ξa describes the flexibility of this annual dispersal: a smaller dispersal window (low ξa) means that annual adult dispersal to the juvenile site operates around a strict peak day, whereas a large adult dispersal window (high ξa) means that annual adult dispersal to the juvenile site is flexible. We note that the resolution and range of juvenile and adult dispersal windows had to be adjusted from site to site to account for simulation limitations related to population dynamics in different temperature environments. As individuals grow and disperse over time, they drop teeth at a constant rate [We assume that individuals occupying the adult site disperse back to the juvenile site to reproduce annually, such that the adult dispersal rate is a function of time. The adult dispersal window ant rate , such thant rate ,31, the  (c)ξj, ξa). Because simulated distributions were both non-normal and multi-modal, to compare distributions we constructed a single error term taking into account seven shape parameters, including the first two moments, the presence/absence and numerically estimated values of one or multiple modes, and the 25th, 50th (median) and 75th percentiles. Error was then calculated asξj, ξa). Accordingly, the simulated juvenile or adult site dental distribution with a lower ε value will indicate a better match for the observed dental distribution, and the particular combination of  that results in the lower ε value will point to the best-fit dispersal strategy.Our overall goal is to use the known conditions generating simulated dental distributions to gain insight into the unknown conditions generating empirical dental distributions. Specifically, we aim to evaluate (i) whether an observed distribution is better described as a juvenile versus adult site and (ii) which dispersal strategy—strict versus flexible juvenile and adult dispersal windows—may have contributed to the observed distributional geometry. To compare simulated dental distributions to those observed from contemporary and Eocene localities, we first parameterized the model with estimated winter minimum and summer maximum mean ocean temperatures for both juvenile and adult sites. See electronic supplementary material, appendix III for site temperature estimates. We then simulated dental distributions for both juvenile and adult sites across all combinations of (. 3 (a)Carcharias taurus) has been examined extensively in the western Atlantic, in particular for the population near Delaware Bay , with distinct distributional geometries characterizing each locality . The Ban (b)The results of our population simulation reveal that changes in the initial dispersal of younger sharks from the juvenile site to the adult site, and of older sharks from the adult to juvenile site, can drastically change the shape of dental distributions within both sites (C. taurus) are highly migratory along the continental shelf of the western margin of the Atlantic Ocean [ca 150 days, which is roughly a 40-day standard deviation around the peak migration time [Extant sand tiger sharks  by first investigating whether our framework was capable of detecting if the Delaware Bay population more likely represented a juvenile versus adult site. Our assessment reveals that the minimal error for the juvenile site is εj = 1.4, whereas the minimal error for the adult site is εa = 0.74 . We coma = 0.74 . This suξj and ξa) or bimodal (high ξj and low ξa), site identity is relatively straightforward to distinguish based on differences in tooth size means. A more rigorous assessment of our approach is to examine whether the best-fit parameterization of dispersal windows  correlates with our understanding of the Delaware Bay system. We find that, given the adult site identification of the Delaware Bay population, the best-fit dispersal window parameterization indicates a strict mass at which juveniles leave for the adult site. While the lowest absolute error also points to a strict temporal window describing adult dispersal, the lowest 10% error covers a wide range of possible values—from 1 to 40 days (dark red colour in figure 4b). The 40-day standard deviation in the timing of adults to Delaware Bay [ξj) before dispersing to the Delaware Bay site. We therefore suggest that our framework provides support for the role of nurseries with respect to the Delaware Bay population, which we infer based only on the shape of the dental distribution.Except for the scenarios where dental distributions are very similar (high ware Bay  is not i (c)Understanding the nature of shark communities in response to documented changes in Eocene climate may provide insight into the future of shark populations in our changing oceans. Because our primary window into these systems is through the lens of accumulated teeth, interpreting dental distributions from an ecological perspective may permit disentangling aspects of their ecologies, such as life-history mediated dispersal behaviours. We thus use our framework to examine the underlying ecological constraints potentially driving the accumulation of dental distributions.εj = 1.31) relative to adult site (εa = 2.67), and the Seymour Island (Antarctica) site better fits the simulated juvenile (εj = 0.36) relative to adult site (εa = 0.40), though this latter difference is negligible  site better fits the simulated juvenile  compared to adult sites (εa = 3.74), whereas Whiskey Bridge (TX) better fits simulated adult (εa = 1.7) compared to juvenile sites (εj = 2.76)  [For low-latitude localities, we observe that the Red Hot Truck Stop (MS) better fits simulated juvenile (macrota) ,42 found (d)ξj, ξa) values providing similarly good fits (row 2 in The contemporary Delaware Bay sand tiger population as well as those from all Eocene sites except the ambiguous Seymour Island marine locality point to a small juvenile dispersal window, meaning that a strict size threshold initiates first dispersal to the adult site . With rerow 2 in . This erContemporary shark nurseries are thought to enable resource access, promote juvenile growth, and protect vulnerable pups against mortality from potential predators ,14. UponWe find strong support for strict juvenile dispersal windows, and by extension the role of juvenile sites serving as nurseries, for the contemporary sand tiger population as well as three of four Eocene localities. Declines in nursery habitats have been invoked as a potential extinction mechanism for both fossil  and cont. 4We have shown that two important drivers of shark life history—the variability in the size of juveniles first leaving a nursery and the temporal variability marking annual migrations of adults back to a nursery—can result in the diversity of dental distributions characterizing both observed contemporary and palaeontological sand tiger populations. Our perspective is based on the correlation between the known drivers governing a mechanistic model and the unknown drivers governing empirical distributions of shark teeth. Our model demonstrates that differences in juvenile and adult dispersal strategies driven by size and seasonality can account for the observed variation in empirical distributions. Further, the inferences we draw predict expectations from contemporary observations and palaeontological reconstructions. However, our mechanistic model is necessarily limited and so the interpretations that we draw from this comparison must be carefully aligned with the underlying assumptions. While the simplistic model we outline here is meant to be a starting point in modelling ecological processes governing shark dynamics and we admittedly do not test the contribution of alternative mechanisms, future models can specify parameters based on modern shark biology and sedimentological processes.Fossilized dental distributions are likely influenced by many biological, geological and taphonomic processes. The diverse distributions observed across shark species may emerge from differences in survivorship, sexual dimorphism and species’ position within the marine food web. Because fossil teeth are accumulated over long periods of time, differences in some dental distributions may also reflect oceanic conditions and sediment formation, taphonomic dynamics, evolutionary change and/or tectonic processes . Disenta


IgE+ plasmablasts develop following allergen exposure and B cell activation. They secrete IgE and therefore are directly linked to maintain the mechanisms of IgE-mediated allergies. Here, we show that the presence of IgE+ plasmablasts in peripheral blood not only coincides with clinical allergy, but also predicts the upcoming development of clinical disease.Using an equine model of naturally occurring allergy, we compared the timing of allergen exposure, arrival of IgE+ plasmablasts in peripheral blood, and onset of clinical disease.in vitro culture. We also compared the IgE secretion by IgE+ plasmablasts with total plasma IgE concentrations and found that while IgE secretion consistently correlates with clinical allergy, total plasma IgE does not.We found that IgE+ plasmablasts predict the development of clinical allergy by at least 3 weeks and can be measured directly by flow cytometry or by IgE secretion following Together, we describe IgE+ plasmablasts as a reliable and sensitive predictive biomarker of allergic disease development. AllergeIgE is produced when allergen-specific B cells are activated by allergen in the presence of the cytokines IL-4 and IL-13 –10, leadCulicoides (Cul) hypersensitivity (Culicoides (Cul) hypersensitivity, which is an IgE-mediated allergic response  once a year in December. All horses were on the same diet. They were kept full time on large pastures with run-in-sheds, free access to water, mineral salt blocks, were grazing in the summer and fed grass hay in the winter.All experiments were conducted on samples from Icelandic horses with and without clinical allergy Table 1.Cul hypersensitivity. Cul hypersensitivity was confirmed, as previously described  in comparison to injections with saline and histamine as negative and positive controls, respectively. Allergic horses developed immediate reactions to Cul WBE, while healthy horses did not. Allergic horses (n=7) included 5 mares , 1 gelding (age 10) and 1 stallion (age 9). All 7 allergic horses and 6 of the healthy horses were also used in our previous study which characterized IgE+ plasmablasts in horses  before this study took place. Healthy horses (n=10) included 5 mares  and 5 geldings .Icelandic horses in this study were either clinically healthy or had seasonal, recurrent escribed , by intrBlood samples were obtained from the V. jugularis using the BD Vacutainer system . Samples were collected from all horses weekly from March 22-April 26, twice weekly from May 3-June 10, twice monthly from June 17- November 15, and once in February and December. Midges were first observed in the environment for a few days during the weeks of April 5 and April 19. April 26 was the last day in the spring when the temperature dropped to freezing temperature. Daily minimum and maximum temperatures were recorded from wunderground.com at a weather station <1 mile from the horse pasture.At every blood collection day, clinical allergy scores were assigned to each horse as previously described . Scores All animal procedures were carried out in accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee at Cornell University (protocol #2011–0011). The study also followed the Guide for Care and Use of Animals in Agricultural Research and Teaching.2.2Heparinized blood samples settled at room temperature for at least 30 minutes to allow erythrocytes to separate from the cell-rich plasma. Cell rich plasma (1 ml), containing peripheral blood leukocytes (PBL), was collected and centrifuged at 500 xg for 10 minutes at 4°C. PBL were then treated with an acid wash as previously described . All othIn addition, peripheral blood mononuclear cells (PBMC) were isolated by layering cell-rich plasma over Ficoll  at a 2:1 (v:v) ratio. Density gradient centrifugation and PBMC isolation was performed as previously described . PBMC we2.37 PBL or 6x105 PBMC were incubated for 15 minutes with an antibody master mix in PBS-BSA (0.5% (w/v) BSA, 0.02% (w/v) NaN3, all from Sigma-Aldrich, St. Louis, MO, USA). Three horse-specific monoclonal antibodies (mAbs) were used in the master mix: IgE mAb 176  non-essential amino acids, 2mM L-glutamine, 50µM 2-mercaptoethanol, 50µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin  and 10% FCS ), and then centrifuged at 100 xg for 5 minutes at room temperature. PBL were resuspended to 1 ml in cell culture medium, and 100 µl of PBL from each individual was mixed with cell culture medium to a final volume of 200 µl and incubated in 96-well flat-bottomed plates  at 37°C, 5% COSecreted equine IgE was measured in undiluted supernatants using a fluorescent bead-based Luminex assay. The assay was set up as previously described , 34 with2.5The data were not normally distributed as confirmed by D’Agostino and Pearson tests. Therefore, non-parametric tests were used for data analysis. To compare clinical scores, IgE+ plasmablast frequencies, CD23+ frequencies, and IgE concentrations between allergic and healthy horses at different timepoints, a Holm-Sidak multiple comparisons test was used. A nonparametric Spearman rank correlation was calculated for all horses to compare the frequency of IgE+ plasmablasts, total IgE+ cells after acid wash, secreted IgE concentrations, plasma IgE concentrations and clinical scores. All graphs plot means and standard deviations unless specified otherwise and p values <0.05 were considered significant. Analysis was performed with GraphPad Prism software version 8 .33.1sp= 0.9583, p<0.0001). Spontaneous secretion of IgE after in vitro culture of PBMC or PBL for 72 hours also resulted in similar concentrations of IgE in the cell culture supernatants .IgE+ plasmablasts were quantified in peripheral blood by a short lactic acid wash (pH 3), followed by incubation with fluorescent antibodies against IgE and CD23, and quantification by flow cytometry . To quan3.2To determine when IgE+ plasmablasts enter peripheral blood in comparison to the onset of clinical allergy, a group of allergic (n=7) and healthy horses (n=10) living under identical environmental conditions were monitored for one year. Heparinized blood was collected frequently from each horse . The frequency of IgE+ cells out of total PBL after acid treatment also described the increase in IgE+ plasmablasts that occurred during the “Preclinical Phase” on April 26  after allergen exposure. In our study, allergen exposure was dependent on the outdoor temperature, where warmer humid days have higher midge burden, and therefore higher allergen exposure through midge bites. We found that the frequency of IgE+ plasmablasts increased after each peak in warm weather, showing that IgE+ plasmablasts rapidly develop and enter circulation following allergen exposure and thereby continue to promote clinical signs of allergy.via ELISA, or testing sensitization of mast cells with allergen-specific IgE, via skin prick tests, are the primary means to diagnose and monitor allergies (Currently, detection of allergen-specific IgE in serum, llergies –4. Both In addition, IgE+ plasmablasts can also be measured by their IgE secretion, and we showed that this IgE secretion also distinguishes allergic from healthy individuals. This contrasted with measurement of total plasma IgE concentrations. We showed that, in horses, plasma IgE levels do not distinguish allergic horses until later in the summer during chronic allergy, and even then, not all allergic individuals exhibit elevated plasma IgE. There are multiple explanations why the influx of IgE+ plasmablasts, which secrete IgE, do not always result in an increase in plasma IgE. First, secreted IgE by plasmablasts may rapidly bind to IgE receptors and therefore not contribute to the plasma IgE concentration. Second, IgE+ plasmablasts may be short lived  or only While there are candidate biomarkers to diagnose pre-existing allergies , predictCul hypersensitivity (This study was performed in horses already allergic for 3-8 years with a naturally occurring, seasonal IgE-mediated allergy called sitivity , 27. Oursitivity , 39. Howsitivity . It is tWhile IgE+ plasmablasts have been identified in humans using complex staining and identification procedures –44, theyIgE-mediated hypersensitivities begin in a silent sensitization phase, when allergen-specific B cells are activated but there are no clinical signs. Following subsequent allergen exposure, IgE-switched memory cells, and IgE+ plasmablasts, are then activated. While this study shows the reactivation of B cells in individuals with pre-existing allergy, we have shown here for the first time that the appearance of new IgE+ plasmablasts in peripheral blood happens in the critical window between a new allergen exposure and before the onset of clinical signs of allergy.In addition to serving as a predictive biomarker of allergy, IgE+ plasmablasts also provide insight into the mechanism of allergen-specific IgE production and maintenance in an individual. There are two mechanisms of IgE class switching in mice, directly from IgM to IgE or sequentially through an intermediate class switching to IgG1 , 48. SeqSequential class switching to IgE can occur both in germinal centers and also in local tissue-associated lymphatic tissues , 56. It What happens to IgE+ plasmablasts following allergen exposure also remains to be explored. We have shown that IgE+ plasmablasts exist transiently in peripheral blood and are maintained by recurrent allergen exposure. Plasmablasts have also been studied in other diseases, including viral infection  and LupuIn conclusion, we have identified IgE+ plasmablasts as a novel peripheral blood biomarker of allergy that precedes recurrent clinical allergy, is rapidly responsive to changes in allergen exposure, and is easily identifiable after an acid wash approach. We propose IgE+ plasmablasts as a predictive biomarker during allergen sensitization before first onset of disease, and as a treatment success marker during allergen immunotherapy treatment.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Cornell University.Conceptualization and methodology were designed by ES and SB. ES, SB, and JT performed experiments. Formal analysis, writing, and editing were all done by ES, SB, and BW. Funds were acquired by BW. All authors contributed to the article and approved the submitted version.


Recently, several theories have been developed to understand the reaction mechanism for OER, though there is an  uncertainty in most of the cases, due to the complex surface structures. Several key factors such as lattice oxygen, defect, electronic structure, oxidation state, hydroxyl group and conductivity were identified and shown to be important to the OER activity. The contribution of each factor to the performance however is often not well understood, limiting their impact in guiding the design of OER electrocatalysts. In this work, we showed mixed B-site pyrochlore Y2MnRuO7 catalyst exhibits 14 times higher turnover frequency (TOF) than RuO2 while maintaining a low overpotential of ~ 300 mV for the entire testing period of 24 h in acidic electrolyte. X-ray photoelectron spectroscopy (XPS) analysis reveals that this B-site mixed pyrochlore Y2MnRuO7 has a higher oxidation state of Ru than those of Y2Ru2O7, which could be crucial for improving OER performance as the broadened and lowered Ru 4d band resulted from the B-site substitution by Mn is beneficial to the OER kinetics.This paper describes the development of mixed B-site pyrochlore YThe online version contains supplementary material available at 10.1186/s40580-022-00311-z. This catalyst was designed based on the analyses above for the optimal TOF on a precious metal base, that is, (1) controlling the size in the nm-scaled regime, (2) using Mn to create nonstoichiometric compound and reducing the Ru usage per unit formula, (3) changing the oxidation state of Ru, and (4) regulating the surface density of hydroxyl groups through defect engineering (on B-site). Our results indicate that the YMRO exhibited much higher TOF value than Y2Ru2O7 (YRO) and the reference RuO2 despite YRO had a relatively higher surface density of hydroxyl groups. The YMRO electrocatalysts also maintained a stable low overpotential at a current density of 10 mA/cm2 for over 24 h in a chronopotentiometry study. X-ray photoelectron spectroscopy (XPS) analysis indicates that oxidation state of Ru increases after the B-site metal substitution, which could be a key contributing factor for the observed enhanced OER performance, suggesting the electronic configuration plays an important role in this mixed B-site pyrochlore OER catalysts.In this paper, we describe the preparation of mixed B-site Y3)3·6H2O, 99.9%), ruthenium (III) nitrosyl nitrate solution (Ru(NO)(NO3)x(OH)y, x + y = 3), Nafion® 117 solution (~ 5%), ruthenium (IV) oxide , and sodium carbonate monohydrate  were purchased from Sigma-Aldrich. Iridium (IV) oxide , manganese (II) nitrate tetrahydrate (Mn(NO3)2·4H2O, 99.9%), iridium(III) chloride hydrate , and manganese(IV) oxide  were purchased from Alfa Aesar. Citric acid monohydrate  and sodium hydroxide  were from Fisher Chemical. Veritas® double distilled perchloric acid  was obtained from GFS Chemicals. Yttrium(III) chloride hexahydrate  was purchased from Stream Chemicals. Tetrahydrofuran (THF) was obtained from Macron Fine Chemicals. Vulcan carbon XC-72 was purchased from Cabot Corporation. Hydrogen  and oxygen  were supplied by Airgas, Inc. All these chemicals and gases were used without further purification.Yttrium (III) nitrate hexahydrate (Y(NO2[MnxRu1−x]2O7 was prepared using a sol-gel method 2O7, were prepared by following the protocols described elsewhere 2O7 (YMRO) was prepared using a modified method reported previously 2O7 electrocatalysts. Among these samples, Y2[Mn0.5Ru0.5]2O7 exhibits the highest TOF, which is around 1.7 times higher than Y2Ru2O7 and 14 times higher than the RuO2 reference catalyst. Oxidation state of Ru appears to play an essential role (vs. surface density of hydroxyl group) in regulating OER performance in the mixed B-site pyrochlore OER catalysts. Change of electronic structure due to B-site substitution by a 3d transition metal (Mn) cation favors the formation of broadened Ru 4d band width and lowered band center, resulting in the enhanced TOF of OER electrocatalysts. This B-site substitution strategy works for both Ru- and Ir-based pyrochlore (Y2MnIrO7 and Y2MnRuO7) OER catalysts. Thus, the approach presents a general strategy for enhancing the OER performance of electrocatalysts of complex oxides.In summary, we prepared a series of nm-scaled, mixed B-site pyrochlore YAdditional file 1: Figure S1. SAED pattern of Y2MnRuO7 (YMRO). Figure S2. Geometric activity of Y2MnRuO7 (YMRO) and RuO2 electrocatalysts. Figure S3. Representative SEM image of the reference RuO2 electrocatalyst. Figure S4. Electrochemical properties of Y2MnRuO7 (YMRO) and RuO2 electrocatalysts:  CVs in a non-faradic current region (1.1-1.2 V vs. RHE) at scan rates of 10, 20, 30, 40 and 50 mV/s, respectively; (c) linear fitting curves of the capacitive current versus CV scan rate; and (d) intrinsic activity normalized by Cdl. Figure S5. Representative SEM images of (a) Y2Ru2O7 (YRO), (b) Y2Mn0.2Ru1.8O7 (YMRO-0.1), (c) Y2Mn0.4Ru1.6O7 (YMRO-0.2), (d) Y2Mn0.6Ru1.4O7 (YMRO-0.3), (e) Y2Mn0.8Ru1.2O7 (YMRO-0.4) and (f) Y2Mn1.2Ru0.8O7 (YMRO-0.6) powders. Figure S6. Representative XRD patterns of Y2Mn1.4Ru0.6O7 (YMRO-0.7) and the references. Figure S7. Representative XRD patterns of Y2Mn2O7. Figure S8. Geometric activity of Y2[MnxRu1-x]2O7 (YMRO-x) and reference RuO2 electrocatalysts. Figure S9. Polarization curve of the Y2Mn2O7 electrocatalyst. Figure S10. XPS survey scans of (a) YMRO and (b) YRO electrocatalysts. Table S1. The amount of precursors used in the synthesis of Y2[MnxRu1-x]2O7. Table S2. XRF analysis of as-made YMRO-x. Table S3. XPS analysis of Ru 3d region of YMRO and YRO. Table S4. XPS analysis of Y 3d region of YMRO and YRO. Table S5. XPS analysis of O 1s region of YMRO and YRO.


Introduction:Selaginella doederleinii Hieron is a traditional Chinese herbal medicine, the ethyl acetate extract from Selaginella doederleinii (SDEA) showed favorable anticancer potentials. However, the effect of SDEA on human cytochrome P450 enzymes (CYP450) remains unclear. To predict the herb-drug interaction (HDI) and lay the groundwork for further clinical trials, the inhibitory effect of SDEA and its four constituents  on seven CYP450 isoforms were investigated by using the established CYP450 cocktail assay based on LC-MS/MS.Methods: Appropriate substrates for seven tested CYP450 isoforms were selected to establish a reliable cocktail CYP450 assay based on LC-MS/MS. The contents of four constituents  in SDEA were determined as well. Then, the validated CYP450 cocktail assay was applied to test the inhibitory potential of SDEA and four constituents on CYP450 isoforms.Results: SDEA showed strong inhibitory effect on CYP2C9 and CYP2C8 (IC50 ≈ 1 μg/ml), moderate inhibitory effect against CYP2C19, CYP2E1 and CYP3A (IC50 < 10 μg/ml). Among the four constituents, Amentoflavone had the highest content in the extract (13.65%) and strongest inhibitory effect (IC50 < 5 μM), especially for CYP2C9, CYP2C8 and CYP3A. Amentoflavone also showed time-dependent inhibition on CYP2C19 and CYP2D6. Apigenin and Palmatine both showed concentration-dependent inhibition. Apigenin inhibited CYP1A2, CYP2C8, CYP2C9, CYP2E1 and CYP3A. Palmatine inhibited CYP3A and had a weak inhibitory effect on CYP2E1. As for Delicaflavone, which has the potential to develop as an anti-cancer agent, showed no obvious inhibitory effect on CYP450 enzymes.Conclusion: Amentoflavone may be one of the main reasons for the inhibition of SDEA on CYP450 enzymes, the potential HDI should be considered when SDEA or Amentoflavone were used with other clinical drugs. On the contrast, Delicaflavone is more suitable to develop as a drug for clinical use, considering the low level of CYP450 metabolic inhibition. Numerous studies have shown that drug-drug interaction (DDI) can cause serious adverse reactions, drug ineffectiveness and even death . InductiAngelica dahurica root extract inhibited CYP450 enzymes such as CYP2C, CYP3A and CYP2D1, and caused pharmacokinetic and pharmacodynamic interactions with Tolbutamide and Diazepam (Rhizoma coptidis (L.)] has obvious inhibition on CYP2D6  showed unique pattern pf enzyme inhibition  should be considered as well . In the Diazepam . ShenmaiDiazepam . Researcn CYP2D6 . Newbouln CYP1A2 . Licorichibition . The moshibition .Selaginella doederleinii Hieron belongs to Selaginella genus of the Selaginellaceae family and is commonly used as a traditional Chinese herbal medicine, which was reported to have anti-hyperglycemia, anti-virus, anti-cancer and other pharmacological activities, and used in the treatment of nasopharyngeal carcinoma, lung cancer and trophoblastic tumor  had anti-cancer effect  or human recombinant CYP450 enzymes (etc.)  . Based os (etc.) . The cocon study . Therefoin vitro cocktail CYP450 assay by LC-MS/MS to detect the inhibitory effects of SDEA and its four constituents  on seven human CYP450 isoforms: CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A. To determine whether SDEA and its components are the potential inhibitors of any of those important human CYP450 isoforms. Our research could reveal the metabolic interactions between CYP450 enzymes and SDEA constituents, which could clarify the clinical safety issues and promote the drug development of SDEA and its constituents.In summary, we established an Pooled mixed gender human liver microsomes from 50 donors were purchased from XenoTech , β-nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from solarbiobiotech , Omeprazole (HPLC purity> 99%), Taxol (HPLC purity> 99%), Tolbutamide (HPLC purity> 99%), Chlorzoxazone (HPLC purity> 99%), Dextromethorphan Hydrobromide (HPLC purity> 98%), Alpha-Naphthoflavone (HPLC purity> 98%), Fluconazole (HPLC purity> 98%), Quercetin (HPLC purity> 98%) and Ketoconazole (HPLC purity> 99%) were purchased from Dalian Meilunbio. Co. Ltd , Phenaceti (HPLC purity> 99%), Sulfaphenazolum (HPLC purity> 99%), Quinidine (HPLC purity> 99%) were purchased from Shyuanye Biotechnology Co. Ltd , Testosterone (HPLC purity> 98%) were purchased from Derick Biotechnology Co. Ltd , 4-Methylpyrazole (HPLC purity> 97%) were purchased from J&K Scientific .S. doederleinii and the structure was fully elucidated by MS, UV, 1H-NMR and 13C-NMR, which was confirmed by comparison with the literatures (The SDEA extract was prepared following our previously described procedure . Delicaferatures . AmentofMethanol and acetonitrile (HPLC grade) were purchased from Merck KGaA , formic acid (HPLC grade) was purchased from Aladdin , ethanol  was obtained from Sinopharm Chemical Reagents , and ultrapure water was prepared by a Millpore Milli-Q system .Amentoflavone, Delicaflavone, Apigenin, Palmatine and Rutin  were precisely weighed and dissolved in methanol to obtain a stock solution with a concentration of 1 mg/ml, which was stored in refrigerator at 4°C. Before use, the stock solution was diluted with methanol into standard working solution and quality control working solution.The concentrations of Amentoflavone and Apigenin standard curve working solution were: 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ng/ml; the concentrations of Amentoflavone and Apigenin quality control (QC) working solution were as 30, 150 and 650 ng/ml. The concentrations of Palmatine standard curve working solution were: 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 and 200 ng/ml; three level QCs were as 3, 30, and 150 ng/ml. The concentrations of Delicaflavone standard curve working solution were: 3.125, 6.25, 12.5, 25, 50, 100 and 200 ng/ml; and three QCs were as 8, 30 and 150 ng/ml. SDEA solid was accurately weighed and dissolved with methanol, then sonicated until complete dissolution. Diluted it with methanol to 1, 5, 25, 40 μg/ml and mix with the 40 ng/ml internal standard (Rutin) respectively. The mixture was vortexed for 2 min, then centrifuged at 13000 rpm for 10 min at room temperature. The supernatant was acquired for LC-MS/MS quantitative analysis.Potassium phosphate buffer  containing 1 mM NADPH, 0.5 mg/mL human liver microsomes, and a cocktail assay of seven probe substrates  or a single substrate (≤Km) were incubated at 37°C for 15 min . The conTo determine the optimal incubation time for each CYP substrate, human liver microsomes (0.5 mg/mL) were incubated at 37°C for 0, 5, 10, 20, 30 and 60 min with each CYP substrates. All substrates concentration were 1 µM. After quantitative analysis using LC-MS/MS, the linearity of metabolite formation was evaluated.For the determination of inhibition curves using single substrate and the cocktail, seven selective CYP inhibitors were used at different concentrations as follows: 0.05–1 µM α-Naphthoflavone for CYP1A2, 1–50 μM Fluconazole for CYP2C19, 0.1–15 µM Quercetin for CYP2C8, 0.01–5 µM Sulfaphenazole for CYP2C9, 0.005–2 µM Quinidine for CYP2D6, 0.1–5 µM 4-Methylpyrazole for CYP2E1, and 0.005–1 µM Ketoconazole for CYP3A .50 value. The amount of metabolites produced by incubation without SDEA (control group) was taken as 100%, then draw inhibition efficiency curve.Inhibitory effect of SDEA on CYP450 isoforms was determined by cocktail assay. SDEA at 10 concentrations from 0 to 200 µg/ml were used to measure ICSingle-point inhibition was assessed by Delicaflavone, Amentoflavone, Apigenin and Palmatine at 10 µM, using the same protocol as described in In order to explore whether SDEA, Delicaflavone and Amentoflavone have time-dependent inhibition on CYP450 isoforms, the cocktail inhibition assay . was useAll metabolites and surrogate standards were analyzed by using LC-MS/MS on a Shimadzu  LCMS-8040 triple quadrupole mass spectrometer. Chromatographic separation was performed using an Ultimate® XB-C18  with gradient elution of water (0.2% formic acid) and methanol at 0.3 mL/min (gradient B). Column maintained at 40°C. 5 μl of supernatant was injected for LC-MS/MS analysis.The following optimized MS parameters were used: ion spray voltage: 6.0 kV; Nebulizer gas flow: 3 L/min; Drying gas flow: 12 L/min; DL temperature: 250°C; Heatblock temperature: 400°C.The gradient elution procedure is: 0–1 min, 30%–45% B; 1–3 min, 40%–75% B; 3–5 min, 75%–85% B; 5–8 min, 85%–95% B; 8–12 min, 95% B; 12–13 min, 95%–30% B; 30% B for equilibration.m/z 536.90 → 375.00 (−) for Amentoflavone; m/z 538.9 → 256.95 (+) for Delicaflavone; m/z 352.00 → 336.10 (+) for Palmatine; m/z 271.15 → 153.05 (+) for Apigenin; m/z 611.00 → 302.85 (+) for Rutin (IS). The collision energies (CE) of the four components were 33 V, −45 V, −35 V, −35 V and −35 V respectively.Detection was carried out using electrospray with polarity switching, collision-induced dissociation, and multiple reaction monitoring (MRM). The MRM mode transition: A gradient condition was applied with the following program: 0–1 min, 10%–20% B; 1–1.01 min, 20%–75% B; 1.01–3 min, 75%–77% B; 3–5 min, 77%–80% B; 5–8 min, 80%–85% B; 8–9 min, 85%–95% B; 9–12 min, 95% B; and then 10% B for equilibration. For specific MRM parameters, please refer to 50 values were determined by using the GraphPad Prism 5.0 software .Quantitative LC-MS/MS data were analyzed using Shimadzu LabSolutions software . The Km and IC2 weighting. Palmatine showed a linear relationship in the range of 0.391–200 ng/ml, and Delicaflavone showed a linear relationship in the range of 3.125–200 ng/ml. The four SDEA constituents showed a good linear relationship over their concentration range with coefficient of determination r2 ≥ 0.99  were prepared separately and used to evaluate those quantitative methods, all samples were analyzed by three replicates. The related standard deviation (RSD) of the tested compounds ranged from −8.88% to 12.60% . MRM mas1 μg/ml, 5 μg/ml, 25 μg/ml and 40 μg/ml SDEA samples were prepared and injected into LC-MS/MS respectively. Using the established quantitative method, the content of four constituents in SDEA was determined. The average w/w % (Weight compound/weight extract) showed that Delicaflavone, Palmatine, Apigenin were 2.21%, 0.019% and 0.024% respectively, and Amentoflavone reached 13.65%, which was the most abundant one in those four constituents .in vitro marker reactions to evaluate the activity of CYP3A , Chlorzoxazone (CYP2E1), Paclitaxel (CYP2C8) remained linear for first 20 min and metabolic rates of Tolbutamide (CYP2C9), Omeprazole (CYP2C19), Phenacetine (CYP1A2) were linear for up 15 min, Testosterone (CYP3A4) remained linear only for first 10 min and then flattened out. Trend diagram of the generation of metabolites for seven enzyme substrates with time was included in Considering the linear curve of metabolic reaction, the final concentration of substrate is usually lower than its Km value in order to ensure linear relationship of metabolic rate range and high affinity. What’s moreover, enzyme-substrate affinity data illustrated that the Km value should be similar for a specific enzyme and substrate, regardless of enzyme source . Since t50 values obtained by cocktail assay was compared with a single substrate using known inhibitors of each enzyme reaction alone, so as to verify the feasibility of this assay. a-Naphthoflavone was inhibitor of CYP1A2, our cocktail assay showed that IC50 value of a-Naphthoflavone on CYP1A2 was 0.18 µM, and the value was 0.31 µM from single substrate assay, which indicated similar results. The IC50 value of Quercetin on CYP2C8 was 5.43 µM in cocktail assay, while the number was 6.07 µM in single substrate experiment, all of them were located in desirable range by literature  on cocktail assay and single substrate method were also performed and compared. Inhibition curves were shown in 50 values were summarized in The ICterature . IC50 va50 values were 1.04 μg/ml and 1.06 μg/ml. SDEA also showed moderate inhibitory ability on CYP2C19 (IC50 was 2.22 μg/ml), CYP2E1 (IC50 was 8.90 μg/ml) and CYP3A (IC50 was 5.18 μg/ml for Testosterone and 7.97 μg/ml for Dextromethorphan). The inhibition activities for CYP1A2, CYP2D6 were mildly, both IC50 values were above 10 μg/ml  was more than 50%. Palmatine inhibits CYP3A (Testosterone) by more than 50% and has a weak inhibitory effect on CYP2E1. Delicaflavone has a weak inhibitory effect on CYP2E1 and CYP3A, but it had no obvious inhibitory effect on the other CYP450 isomers at this concentration .50 values were less than 5 μM for the seven cytochrome P450 isoforms, especially for CYP2C9, CYP2C8 and CYP3A, the IC50 values were even less than 0.5 μM  demonstrated a time-dependent inhibition on CYP2C19, CYP2D6, CYP3A (Testosterone) , and AmeBased on these results, we have determined the inhibition effect of incubation time with SDEA or Amentoflavone on CYP2C19, CYP2D6, CYP3A (Testosterone) activity. When HLM was co-incubated with SDEA (0.5 μg/ml) in presence of NADPH for 0–35 min, the metabolic activity of CYP2C19 decreased from 95.5% to 69.5%; the activity of CYP2D6 decreased from 84.5% to 63.5% and CYP3A (Testosterone) decreased from 98.5% to 63% . As for In vitro model of human CYP450 enzymes inhibition cocktail assay used in this study can discover the potential of interaction between herb and drugs, reduce the risk of using clinical drug, lay a foundation for clinical medication. Incubation time (15 min) and substrate concentration were determined by combining literature data with experimental conditions , more importantly, after 35 min co-incubation of SDEA with NADPH, CYP3A activity was reduced from 98.5% to 63%, which significantly increases the risk of herb-drug interaction with serious consequences. Some oral hypoglycemic agents, such as Glyburide, Rosiglitazone, and Repaglinide, are mainly metabolized by CYP2C8 or CYP2C9, it may increase hypoglycemia risk when those medicines are used together with SDEA. For epilepsy patients, SDEA should be used with caution when taking Phenytoin and Valproic acid (metabolized by CYP2C19) at the same time, in order to avoid serious adverse reactions.The experiment results showed that SDEA had a strong concentration-dependent inhibition effect on CYP2C9 and CYP2C8 , time-deal drugs . In addial drugs , and it The contents of Amentoflavone and Delicaflavone in SDEA were 13.65% w/w and 2.21% w/w respectively, which were similar to results of previous studies  Table 1Table 1. Similar to SDEA, Amentoflavone also has time-dependent inhibitory effects on CYP2D6 and CYP2C19 . AlthougBecause the content determination and single concentration inhibition showed that Palmatine and Apigenin contents were low , and theThrough the established reliable cocktail assay, SDEA was found to have concentration-dependent inhibition on several CYP450 enzymes, and inhibited CYP2D6, CYP2C19, CYP3A in a time-dependent manner. The inhibition activity may be mainly due to the higher content of component: Amentoflavone (13.65%). Amentoflavone has inhibitory effects on all tested CYP450 enzymes, the inhibitory effects on CYP2C9, CYP2C8 and CYP3A were even greater than the corresponding specific inhibitors. Amentoflavone also has time-dependent inhibition on CYP2D6 and CYP2C19. The other two constituents from SDEA, Apigenin and Palmatine, both showed concentration-dependent inhibition. For Delicaflavone, no significant inhibitory effect on CYP450 enzymes was observed. Since Delicaflavone owns excellent anti-cancer ability and low HDI potential, it was more suitable to develop as a new anti-cancer drug.