Notably, the over-production of filopodia caused by G3BP1 depletion can be alleviated by blocking the formation of the translation initiation complex. We further found that the interaction of G3BP1 with eIF4E is regulated by arginine methylation. Knockdown of the protein arginine methyltransferase PRMT8 leads to elevated protein synthesis and filopodia production, which is reversed by the expression of methylation-mimetic G3BP1. Our study, therefore, reveals arginine methylation as a key regulatory mechanism of G3BP1 during dendritic spine morphogenesis and identifies eIF4E as a novel downstream target of G3BP1 in neuronal development independent of stress response.Communication between neurons relies on neurotransmission that takes place at synapses. Excitatory synapses are located primarily on dendritic spines that possess diverse morphologies, ranging from elongated filopodia to mushroom-shaped spines. Failure in the proper development of dendritic spines has detrimental consequences on neuronal connectivity, but the molecular mechanism that controls the balance of filopodia and mushroom spines is not well understood. G3BP1 is the key RNA-binding protein that assembles the stress granules in non-neuronal cells to adjust protein synthesis upon exogenous stress. Emerging evidence suggests that the biological significance of G3BP1 extends beyond its role in stress response, especially in the nervous system. However, the mechanism underlying the regulation and function of G3BP1 in neurons remains elusive. Here we found that G3BP1 suppresses protein synthesis and binds to the translation initiation factor eIF4E  Excitatory neurotransmission occurs on the postsynaptic neuron at dendritic spines that exist as diverse morphologies. The mushroom-shaped spines with large heads and distinct spine necks are the mature spines for memory storage , but theThe proper development and function of synapses require tight regulation of neuronal protein synthesis. One crucial control point occurs at the level of translation initiation complex formation, which involves the interaction of the translation initiation factors eIF4E and eIF4G with the 5′-capped mRNAs and is regulated by mTOR-mediated phosphorylation of 4EBP-1 . DisruptMany RBPs contain the glycine-arginine-rich (RGG) motif which is a hot spot for arginine methylation , a majorRas-GTPase activating protein SH3 domain binding proteins (G3BPs) are proteins pulled down by PRMT8 from the mouse brain . G3BPs aA). Similar co-immunoprecipitation of the two proteins was observed using another eIF4E antibody that recognized a different epitope . In vitro pull-down experiment was further performed using purified recombinant GST-eIF4E and His-G3BP1, which indicated that the two recombinant proteins could bind to each other directly in cell-free conditions . eIF4E is present near synapses and is implicated in local translation of dendritic mRNAs  . These findings suggest that the translation initiation factor eIF4E could be a downstream effector to mediate G3BP1 function in neuronal dendrites under normal physiological conditions independent of stress granule formation.When the translation is stalled upon the presence of cellular stress, various translation initiation factors that comprise the noncanonical 48S preinitiation complex are present in stress granules . We, theFig. 1ic mRNAs . We, theic mRNAs , 43. We ic mRNAs . Upon imin situ using the puromycin antibody and immunofluorescence microscopy. The high sensitivity, ease of use, and quick labeling make SUnSET a good method to detect the location of proteins undergoing active synthesis. After 10-min incubation, puromycin labeling was detected in both the neuronal cell body and on dendrites of cultured hippocampal neurons. No labeling was observed either in the absence of puromycin or without anti-puromycin antibody. Furthermore, the puromycin signals were largely reduced when neurons were pre-treated with the protein synthesis inhibitor anisomycin, indicating the specificity of puromycin in labeling the newly synthesized proteins (A and B). The decrease in puromycin intensity was not an artifact of over-expressing an exogenous protein because a similar reduction of puromycin staining was not observed in the negative control in which the same amount of GFP plasmid was transfected. G3BP1 undergoes arginine methylation at R433 and R445 located within the C-terminal RGG motif (A and B). Taken together, these findings indicate that G3BP1 suppresses mRNA translation in neurons, and it interacts with the translation initiation factor eIF4E.To ask whether G3BP1 regulates neuronal mRNA translation in dendrites, we performed the surface sensing of translation (SUnSET) assay based on the aminoacylated tRNA analog puromycin . After aproteins . Measuriproteins . Over-exproteins , A and BGG motif , 45. TheGG motif . ConsistGG motif , A and BA). To identify which domain of G3BP1 binds to eIF4E, we generated FLAG-tagged expression constructs with different combinations of G3BP1 domains, followed by an expression in 293T cells and co-immunoprecipitation by FLAG beads to detect eIF4E. The NTF2-like domain but not the other domains was sufficient for the interaction with eIF4E that was comparable to full-length G3BP1 (B). Surprisingly, deletion of the RNA-binding domain renders the remaining G3BP1 (NTF2 + AcidRich + PxxP) unable to interact with eIF4E, although the NTF2-like domain is still present. Therefore, G3BP1 utilizes its NTF2-like domain to bind to eIF4E, but its RNA-binding domain is permissive for this interaction with full-length G3BP1. It is unclear whether the RNA-binding domain by itself can also interact with eIF4E since its expression in multiple experiments was consistently much lower than the other domains of G3BP1 (B).G3BP1 is composed of several distinct domains: the N-terminal nuclear transport factor 2 (NTF2)-like domain, the central acid-rich and proline-rich (PxxP) motifs, and the C-terminal RNA-binding domain consisting of the RNA recognition motif (RRM) and the arginine-glycine rich (RGG) motif A. To ideth G3BP1 B. Surpriof G3BP1 B.Figure C and D).To ask whether the interaction between G3BP1 and eIF4E is functionally important, we transfected hippocampal neurons with the full-length or NTF2-like domain of G3BP1 and examined the consequences on dendritic spine morphogenesis. We reason that the over-expression of the NTF2-like domain will interfere with the interaction between endogenous eIF4E and G3BP1 and hence inhibit their functions in a dominant-negative manner. Indeed, compared to vector control, over-expressing the NTF2-like domain of G3BP1 significantly reduced the density of mushroom spines while the number of filopodia remained unchanged. In contrast, over-expressing the full-length G3BP1 (G3BP1-FL) did not disrupt the mushroom spines. Instead, the filopodia density was significantly reduced , C and DA). In the presence of DMSO (as vehicle control), transfection of neurons with G3BP1-shRNA resulted in the reduction of mushroom spines and the over-production of filopodia, which is consistent with our previous findings (A and B). Taken together, our findings suggest that G3BP1 promotes dendritic spine maturation by inhibiting mRNA translation, which can be achieved, at least in part, by binding to eIF4E via its NTF2-like domain.The NTF2-like domain is essential for G3BP1 dimerization  and bindFig. S4findings . Howeverfindings , A and Bin vitro methylation of purified G3BP1 was induced by incubation with recombinant PRMT1, which has strong arginine methyltransferase activity on G3BP1 in vitro .The interaction between G3BP1 and eIF4E is regulated by the RNA-binding domain , which cin vitro , before thylated A.Figure B). Consistent with our hypothesis, the introduction of the PRMT8-shRNA increased mRNA translation, as indicated by elevated puromycin incorporation on dendrites in the SUnSET assay, suggesting that PRMT8 mimics the role of G3BP1 in suppressing mRNA translation. Notably, co-expression of methylation-mimetic G3BP1 (R433/445F), but not the wild-type nor methylation-deficient G3BP1 (R433/445H), reversed the elevated protein synthesis caused by PRMT8 knockdown (B and C). To ask whether arginine methylation of G3BP1 acts downstream of PRMT8 in regulating dendritic spine maturation, we co-expressed the different constructs of G3BP1 with PRMT8-shRNA and determine the effects on the production of mushroom spines and filopodia. Compared to control shRNA, knockdown of PRMT8 induced filopodia formation and loss of mushroom spines. Notably, co-expression of the methylation-mimetic G3BP1 could reverse the spine phenotypes. On the other hand, co-expressing either the wild-type or methylation-deficient G3BP1 failed to rescue the overproduced filopodia and reduction of mushroom spines (A and B). Collectively, our results indicate that G3BP1 is the major downstream target of PRMT8 in the regulation of protein synthesis, and the methylation of G3BP1 by PRMT8 within the RGG motif is crucial for the proper maturation of dendritic spines in neurons.In non-neuronal cells, G3BP1 function is regulated by arginine methylation during the disassembly of stress granules , 49 and Fig. S4nockdown , B and Cm spines , A and BBesides its established function in stress granule assembly, G3BP1 has been implicated to regulate different aspects of RNA metabolism, including translation, transport, stability, and decay. Additional functions of G3BP1 other than RNA metabolism have also been suggested, such as ribosomal stability and proteasome-mediated protein turnover . Most ofvia metabolic labeling after depleting FMRP , in which G3BP1 is reported to exist as small aggregates in the axons of dorsal root ganglion neurons and negatively regulate mRNA translation. The acid-rich domain of G3BP1 and its phosphorylation on Ser-149 are involved in the regulation of axonal mRNA translation  caused by utations , 61, 62.utations . Argininutations , 65 as wutations . Here weth FXR1P . It is tth FXR1P . It woulhttp://sirna.wi.mit.edu) and were used to create the shRNA after subcloning into the pSUPER vector. The control-shRNA sequence was 5′-GGCTACCTCCATTTAGTGT-3′. For making FLAG-tagged G3BP1, a primer was designed that contained a FLAG tag in-frame with G3BP1 at the N-terminal, and the insert was amplified by PCR from rat hippocampus cDNA and subcloned into the pcDNA3 vector at the BamHI (NEB) and NotI (NEB) restriction enzyme sites. For the construction of individual and combination of G3BP1domains tagged with FLAG, PCR was performed using the full-length rat G3BP1 construct as a template with the primers designed according to the following coding sequences of the rat G3BP1 gene: NTF2 (1–420 bp), NTF2+AcidRich+PxxP (1–1020 bp), RRM + RGG (1021–1395 bp), AcidRich+PxxP (421–1020 bp). To make RNAi-resistant constructs and different variants of G3BP1 [the methylation-deficient constructs (R433/445H) and the methylation-mimetic constructs (R433/445F)], site-directed mutagenesis using PfuUltra II Fusion HS DNA Polymerase  was performed, and the PCR products were digested by DpnI (NEB) at 37 °C for 3 h before transformation into E. coli competent cells. Inserts were then subcloned into pcDNA3 vector at BamHI (NEB) and NotI (NEB) restriction enzyme sites. All expression constructs were made by PCR using the high-fidelity PfuUltra II Fusion HS DNA Polymerase. The nucleotide sequence of the insert in each plasmid was verified by Sanger sequencing.To knock down PRMT8, G3BP1 or eIF4E, the 19-nucleotides shRNA derived from rat PRMT8 (5′-GACTACCTCACTGTTCGAA-3′), rat G3BP1 (5′-CCTGTGTCCGACATTCAAG-3′) or rat eIF4E (5′-GAGCGGCTCCACCACTAAA-3′) nucleotide sequences were selected by the online siRNA design program (5 cells/18 mm coverslip) for the analysis of dendritic spines or low density (0.4 × 105 cells/18 mm coverslip) for immunofluorescence staining. The hippocampal neurons were grown at 37 °C, 5% CO2 with Neurobasal medium (Gibco) supplemented with 2% B27 and 0.25% L-glutamine (Invitrogen).All animal experiments were approved and performed in accordance with the Animal Research Ethics Sub-Committee of City University of Hong Kong. Primary hippocampal neurons were dissociated from day 18 embryos of Sprague Dawley rats. Hippocampal neurons were cultured in a 12-well dish on 18-mm coverslips coated with poly-D-lysine  at high density  was in four-fold excess with that of GFP (0.5 μg) in the transfection mix, such that the GFP expression was a reliable indication of neurons taking up the shRNA or cDNA constructs ], eIF4E , FLAG ]. After washing with TBST, the membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibody diluted 1:3000 in 5% non-fat milk in TBST. The following secondary antibodies were used: anti-mouse IgG, HRP-linked antibody , and anti-rabbit IgG, HRP-linked antibody . The HRP signal was detected by the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using the ChemiDoc MP imaging system (BioRAD).Cells or tissues were collected with cold 1X RIPA lysis buffer  plus protease and phosphatase inhibitors . After centrifuging at 4 °C, 13,000 rpm for 10 min, the supernatant was collected. 1X sample buffer  SDS, 25% (v/v) beta-mercaptoethanol, 50% (v/v) glycerol, 0.05% (w/v) bromophenol blue) was added to protein extract which was boiled at 100 °C for 5 min with heat block. Equal amounts of protein samples were subject to SDS-PAGE for separation and transferred onto PVDF membranes . The membranes were blocked with blocking buffer (5% non-fat milk in TBST) for 1 h at room temperature, followed by incubation with primary antibody (diluted with 5% BSA and 0.02% sodium azide in TBST) overnight at 4 °C. The following antibodies were used at the indicated dilutions: G3BP1 , eIF4E , eIF4Eas eIF4E A], FLAG 2, 37 °C incubator. Cells were grown to 70 to 85% confluence and were transfected with Lipofectamine LTX reagent and plus reagent (Invitrogen) in accordance with the manufacturer’s protocols. 24 h post-transfection, cell lysate was collected using either cold 1X RIPA lysis buffer containing various protease and phosphatase inhibitors for immunoprecipitation (IP), or cold 1X NP-40 lysis buffer  containing various protease and phosphatase inhibitors for co-IP.HEK-293T cells were cultured in DMEM medium (Gibco) plus 10% FBS and 1% penicillin-streptomycin in 5% COFor the immunoprecipitation of FLAG-G3BP1, the lysate (1 mg) was incubated with FLAG beads  for 2 h at 4 °C to immunoprecipitate the FLAG-tagged proteins. After washing three times by lysis buffer containing the various protease and phosphatase inhibitors, proteins were eluted with 2.5X sample buffer with 100 μM DTT for Western blot analysis. For the immunoprecipitation of G3BP1, lysate (1 mg) was incubated with G3BP1 antibody  overnight at 4 °C. After incubation with Protein A beads  at 4 °C for 1 h, the beads were washed four times with lysis buffer containing various protease and phosphatase inhibitors, and proteins were eluted with 2.5X sample buffer with 100 μM DTT for Western blot analysis.in vitro binding between G3BP1 and eIF4E, the purified recombinant proteins His-G3BP1  and GST-eIF4E  were mixed in Tris buffer containing various proteinase inhibitors and rotated overnight at 4 °C. GSH beads  were added to the mixture and followed by 2 h rotation at 4 °C. The beads were washed three times with Tris buffer containing proteinase inhibitors and eluted with 2.5X sample buffer with 100 μM DTT. For the in vitro methylation of G3BP1, the purified proteins His-G3BP1 (2 μg), GST-eIF4E (4 μg), PRMT1 , and AdoMet  were mixed in DPBS at 30 °C for 1 h, followed by binding assay by adding the reaction mix and GSH beads (50 ul) into D-PBS with proteinase inhibitors and rotated for 2 h in 4 °C as described above. After elution, samples were boiled at 100 °C for 5 min before SDS-PAGE and Western blot analysis.To detect the 2O, and the coverslips were mounted on slides (Thermo Fisher Scientific) with hydromount medium .Cultured hippocampal neurons were fixed with 4% PFA and 4% sucrose solution for 15 min at room temperature. After washing, the cells were blocked with blocking buffer [0.4% Triton X-100 (v/v) and 1% BSA (v/v)] for 45 min at room temperature, followed by incubation with primary antibodies  and GFPTo examine dendritic spine morphology, the transfected hippocampal neurons were fixed at 16 DIV and incubated overnight at 4 °C with anti-GFP antibody diluted in 1X GDB buffer . On the following day, the cells were incubated with Alexa-488 conjugated secondary antibody for 45 min in dark at room temperature, followed by washing three times with filtered phosphate buffer (20 mM phosphate buffer and 0.5 M NaCl) and mounting.2. In the control experiment with protein synthesis inhibitor, cells were pre-treated with anisomycin (40 μM) for 30 min before addition of puromycin to the medium. Incubation was stopped by two fast washes in PBS , and cells were fixed in PFA-sucrose for 15 min. After fixation and washing three times with D-PBS, cells were permeabilized in buffer (0.5% TX in D-PBS) for 15 min and incubated with blocking buffer (3% BSA in D-PBS) for 30 min. After blocking, the cells were incubated with puromycin antibody  overnight at 4 °C and subjected to immunofluorescence staining on the following day. For the triple staining against G3BP1, puromycin, and GFP, neurons were fixed and blocked as above. They were then incubated overnight at 4 °C with the following primary antibodies: Mouse anti-Puromycin , Rabbit anti-G3BP1 , and Chicken anti-GFP  that were diluted in blocking buffer. On the following day, the cells were washed three times with washing buffer (0.02% Triton X-100 and 0.25% BSA in D-PBS) and incubated with Alexa-488 anti-Chicken, Alexa-647 anti-Mouse, Alexa-546 anti-Rabbit conjugated secondary antibodies diluted at 1:1000 in D-PBS containing 0.02% Triton X-100 and 1% BSA for 45 min at room temperature in dark. After being washed with washing buffer for two times, cells were rinsed with D-PBS and sterilized MilliQ H2O, and the coverslips were mounted on slides with hydromount medium.Cultured hippocampal neurons (16–17 DIV) were incubated with puromycin (1.8 μM) for 10 min in medium at 37 °C with 5% COFluorescence images of dissociated hippocampal neurons were acquired using either the Carl Zeiss LSM 900 with the fast Airyscan mode  or the Carl Zeiss 880 microscopes , with the 63x oil-immersion lens (N.A. 1.4). Images were exported with Zen software and quantified with MetaMorph and Image J software.B) or the control-shRNA group (C).Analysis of dendritic spine morphology was performed as previously reported , 70. SegFig. 2NA group C.To analyse the colocalization of eIF4E and tdTomato-G3BP1, the ImageJ plug-in software “JACoP” was used to calculate both the Manders’ coefficients (M1 and M2) and Pearson’s coefficient. M1 refers to the fraction of pixels in eIF4E which overlap with pixels of tdTomato-G3BP1, whereas M2 refers to the fraction of pixels in tdTomato-G3BP1 which overlap with pixels of eIF4E. Both Manders’ coefficients range from 0 to 1, with 0 indicates no correlation and 1 indicates perfect correlation. Image thresholding using the JACoP plug-in was set manually for each image to calculate the Manders’ coefficients. Pearson’s correlation ranges from −1 to 1, with −1 represents complete exclusion of pixels of eIF4E from tdTomato-G3BP1 and vice-versa; whereas 0 indicates a random correlation and 1 signifies perfect correlation.t test, while data with more than two experimental groups were analysed with One-way ANOVA followed by Tukey post-hoc test. For the groups of data which did not obey the Shapiro–Wilk normality distribution, data with two groups were analyzed by Mann–Whitney test, and results with at least three groups were analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison test. Statistical significance was defined as p <0.05.All values were shown as mean ± SD and analyzed in GraphPad Prism software. Each group of values was analyzed by the Shapiro–Wilk normality test. For the groups of data that passed the Shapiro-Wilk normality test, data with two experimental groups were analyzed by Student’s The authors declare no restrictions on data availability.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Detection analysis of metribuzin in the soil to find time for 50% and 90% metribuzin degradation (DT50 and DT90) was performed using high-performance liquid chromatography (HPLC). Soil microbiota was analyzed by respiration rate (C-CO2), microbial biomass carbon (MBC), and metabolic quotient (qCO2). BC350 °C-amended soil at 10% increased the DT50 of metribuzin from 7.35 days to 17.32 days compared to the unamended soil. Lower application rates (0.1% to 1.5%) of BC550 °C and BC750 °C decreased the DT50 of metribuzin to ~4.05 and ~5.41 days, respectively. BC350 °C-amended soil at high application rates (5% and 10%) provided high C-CO2, low MBC fixation, and high qCO2. The addition of low application rates (0.1% to 1.5%) of sugarcane straw biochar produced at high temperatures (BC550 °C and BC750 °C) resulted in increased metribuzin degradation and may influence the residual effect of the herbicide and weed control efficiency.Biochar-amended soils influence the degradation of herbicides depending on the pyrolysis temperature, application rate, and feedstock used. The objective of this study was to evaluate the influence of sugarcane straw biochar (BC) produced at different pyrolysis temperatures  and application rates in soil (0, 0.1, 0.5, 1, 1.5, 5, and 10%  Herbicides are used for weed control in pre- or post-emergence, and regardless of the mode of application, they can reach the soil and persist with a residual effect for weed control and cause carryover problems in succeeding crops or contaminate non-target organisms and the environment . The negThe soil is the main site where physical, chemical, and biological interactions of herbicides occur . Herbici2 supply [Biochar is a carbonaceous material produced during the thermochemical decomposition (pyrolysis) of biomass under a limited O2 supply . The pyr2 supply . The app2 supply ,13.50) of herbicide depending on the molecule, pyrolysis temperature, application rate, and feedstock used [50 of 2 days and 23 days) with the addition of rice husk biochar produced at 500 °C, at an application rate of 2%, than in unamended soil [14C-atrazine in Brazilian soil, representing increases of 50% (0.1% w/w), 48% (1.0% w/w), and 46% (5.0% w/w) compared to unamended soil [Biochar-amended soils can directly influence the degradation half-life time (DTock used . For exaock used . In contded soil . Differeded soil . The higded soil .Sw = 10.700 mg L−1 at 20 °C), high mobility in soil , high leaching index (groundwater ubiquity score = 2.96), and low persistence in soil (DT50 = ~20 days) [50 of metribuzin by 4 days when compared to the unamended soil (DT50 = 34 days) [The relationship between the addition of biochar to soil and the degradation of metribuzin is reported in the scientific literature; however, the results are distinct. Metribuzin is a pre- and post-emergent herbicide, a selective residual of the triazinone group, with the ability to effectively control a wide spectrum of eudicot weeds . The her20 days) ,22. This20 days) ,23,24. T34 days) . The deg34 days) . The autTo understand the impact of biochar on the fate of metribuzin in soil, it is necessary to detail the effects of different types of biochar, pyrolysis temperature, and application rate on metribuzin degradation in soil, which is important to assess the risk and fate of this herbicide in the environment. Therefore, the objective of this study was to evaluate the influence of biochar from sugarcane straw produced at different pyrolysis temperatures and application rates in soil on metribuzin degradation and soil microbiota.F = 2.738 and p ≤ 0.002). The regression curves of metribuzin over time in amended and unamended soils with different application rates of biochar  produced at different pyrolysis temperatures  are presented in R2) greater than 0.940 (50) and 90% (DT90) of metribuzin applied in the unamended soil were 7.37 days and 24.49 days, respectively (The sugarcane straw biochar (BC) samples produced at different pyrolysis temperatures  were denominated BC350 °C, BC550 °C and BC750 °C. The interaction between pyrolysis temperature, application rate of biochar in the soil, and degradation time was significant (an 0.940 . The degectively . The temectively . At 150 C0) of metribuzin in amended soil with BC350 °C, BC550 °C, and BC750 °C varied among the application rates . Metribuzin DT50 increased from 7.37 days in unamended soil to 9.62 days and 17.32 days when rates of 5% and 10% of BC350 °C were added, respectively. Regardless of the application rate of BC550 °C and BC750 °C, the DT50 of metribuzin was lower than in unamended soil. However, it was observed that with an increase in the application rate from 0.1% to 10% of BC350 °C and BC550 °C, the DT50 increased on average by 3 days. Lower application rates of biochar produced at different pyrolysis temperatures decreased the DT50 and DT90 of metribuzin, aligned mainly with the higher pyrolysis temperatures (BC550 °C and BC750 °C).The application rates of biochar produced at different pyrolysis temperatures influenced the DT in soil . Applica2 g soil−1 day−1, respectively, at 0 DAA and 150 DAA of metribuzin, respectively. The maximum respiration rate at 0 DAA was at 9 DAI, with respiration ranging from 600 µg CO2 g soil−1 day−1 to 900 µg CO2 g soil−1 day−1, depending on the application rate and pyrolysis temperature. At 150 DAA, the maximum respiration was between 9 and 13 DAI, with respiration rates between 400 µg CO2 g soil−1 day−1 and 900 µg CO2 g soil−1 day−1, depending on the application rate of BC350 °C, BC550 °C, and BC750 °C  and for 150 DAA (F = 3.140 and p ≤ 0.01). The unamended soil presented a total respiration (C-CO2) of 3066 µg CO2 g soil−1 and 2503 µg CO2 g soil−1 at 0 DAA and 150 DAA, respectively (2 was influenced by the pyrolysis temperature only for the highest application rates (5% and 10%) of BC350 °C, BC550 °C, and BC750 °C. In BC350 °C-amended soil, higher C-CO2 was observed by 20.7% (average of rates 5% and 10%) compared to BC550 °C and BC750 °C-amended soil. There was no significant effect between application rates of BC550 °C and BC750 °C in the soil. The application rates of 5% and 10% of BC350 °C provided greater C-CO2 from the soil microbiota, 4424 µg CO2 g soil−1 and 4644 µg CO2 g soil−1, respectively, when compared to unamended soil (3066 µg CO2 g soil−1)  . There w 150 DAA . Rates oand 0.5% .F = 8.565 and p ≤ 0.001) and for 150 DAA (F = 5.549 and p ≤ 0.001). Unamended soil presented an average MBC of 40 µg MBC g soil−1 and 60 µg MBC g soil−1 at 0 DAA and 150 DAA, respectively  when compared to unamended soil and rates of 0.1%, 0.5%, 1.0%, 1.5%, and 10% (averaging 131.87 µg MBC g soil−1). Rates of 5% and 10% of BC550 °C-amended soil increased MBC by 42.2%, on average, when compared to unamended soil, and rates of 0.1%, 0.5%, 1.0%, and 1.5% (The interaction between factors for MBC was significant at 0 DAA (ectively . At 0 DA and 10% . At 150  and 10% . The 5% and 1.5% . qCO2) was significant at 0 DAA (F = 7.727 and p ≤ 0.001) and for 150 DAA (F = 6.914 and p ≤ 0.003). The metabolic quotient (qCO2) of unamended soil at 0 DAA and 150 DAA was 63.7 µg CO2/µg MBC and 39.1 µg CO2/µg MBC, respectively (qCO2 (73.9 µg CO2/µg MBC) when compared to BC550 °C and BC350 °C-amended soil (average of 21.49 µg CO2/µg MBC) at a rate of 1.0%. Rates of up to 5.0% application of BC350 °C showed low qCO2 (average of 23.77 µg CO2/µg MBC) when compared to unamended soil and a rate of 10% (average of 63.7 µg CO2/µg MBC). The highest rates  of BC550° and BC750 °C provided qCO2 ~ 58.4 µg CO2/µg MBC, a value lower than unamended soil (63.7 µg CO2/µg MBC) (qCO2 (11.1 µg CO2/µg MBC) when compared to BC550 °C and BC350 °C-amended soil  . At 150 ded soil . Applicaectively .50 range from 7.03 days to 138 days, depending on climate, soil, and field and laboratory conditions [50 values of metribuzin were dependent on temperature, humidity, and soil physicochemical characteristics, such as OC content [50 from ~385 days to 105 days for soil with a higher sand content (~60%) and lower OC content (0.15%). Metribuzin has high Sw, low sorption, and persistence, suggesting a high potential for movement and ready availability for microbial degradation in the soil. This is particularly true for soils with high OC content [50 values varied significantly with the metribuzin application rate and the physical–chemical characteristics of the soil, with values ranging from 15.17 days to 46.59 days.The values of metribuzin DTnditions ,27,28,29 content ,30,31. M content ,33. The  content . The autC0 may have occurred due to the initial sorption process of metribuzin in soils with different application rates, which directly reflects the amount of bioavailable herbicide in the soil solution for degradation. Mielke et al. [−1) were less sorbed (60%) in soils amended with BC350 °C, especially for lower application rates (0.1% to 1.5%). The authors reported that the amended soil with BC550 °C and BC750 °C showed sorption percentages greater than 80% of metribuzin in the soil. Biochars have different physical and chemical characteristics as the pyrolysis temperature changes during production. Herbicide sorption is directly influenced by characteristics such as porosity, SSA, aromatic structures, carbon contents, surface functional groups, pH, and the elemental composition of the soil [The variation in e et al.  reportedthe soil .The results showed that biochar produced at low temperatures (BC350 °C) negatively influenced the degradation of metribuzin in the soil. The greater degradation of metribuzin in soils amended with BC550 °C and BC750 °C may be related to the physicochemical characteristics of the biochar, as high pyrolysis temperatures produced a material with high pH (9.7), ash content (11%), OC (1.4%), and high nutrient content . These f50 of metribuzin also increased. In most cases, degradation decreases with increasing biochar application rates, associated with greater herbicide sorption [50 of flumioxazin of 11.1, 9.0, 11.1, 13.2, and 15.4 days, respectively.The effect of biochar on the degradation behavior of herbicides in the soil is a complex process that involves the interaction between soil, herbicide, and the physicochemical characteristics of biochar. When an adequate amount of biochar was added to the soil (0.1% to 1.5%), better degradation of metribuzin was observed, which may be related to the inhibition of microbial activity by excess biochar, influencing the richness and abundance of the microbial community and the higher sorption capacity of the herbicide at high rates of application of sugarcane straw biochar produced at high pyrolysis temperatures (>500 °C) ,26,37,38sorption ,40,41. Ssorption . The autKa = 1.3), and under conditions of high pH, it is more in the ionic form, negatively charged, increasing the repulsion of soil colloids, mainly the predominance of negative charges in the organic matter [50 value of metribuzin was lower than in unamended soil, regardless of the application rate of BC550 °C and BC750 °C. Although Mielke et al. [Metribuzin is a strong acid , soil , environmental factors , and physicochemical characteristics of herbicides ,48. In tMicroorganisms can use hydrocarbons on the surface of biochar as a carbon source . OrganicqCO2 values suggest unfavorable conditions for soil microbiota, and low values indicate greater MBC efficiency [50 value of metribuzin was observed in BC350 °C-amended soil when compared to BC550 °C and BC750 °C  [The addition of a large amount of carbon can stimulate enhanced microbial action in the soil and therefore cause greater microbial degradation. In addition, the addition of biochar produced at high pyrolysis temperatures increased soil nutrient levels, especially P, K, and SSA (223 m2 g−1) , which c2 g−1) . The app2 g−1) .qCO2 of soils, regardless of pyrolysis temperature and application rate, were lower than the unamended soil. Possibly, the addition of biochar in the soil boosted the microbiota, reducing the energy expenditure used in maintaining the microbial community and directing resources to cell synthesis, improving microbial growth [At 150 DAA, it was possible to observe that the l growth .50 between 2 days and 23 days compared to unamended soil [Biochar has a high capacity for sorbing and retaining soluble organic matter, gases, nutrients, and water and can therefore provide soil microorganisms with various energy resources, nutrients, moisture, and the formation of macroaggregates ,62,63,64ded soil .In addition, sugarcane straw biochar produced at high pyrolysis temperatures (>550 °C) and at moderate application rates (between 0.5% and 1.5%) boosted the soil microbiota and improved metribuzin degradation. This result reinforces the idea that materials with high sorptive capacity, such as biochar produced at high pyrolysis temperatures, can be used as soil amendments to improve soil microbiota as long as the application rate is controlled. However, it is important to note that this result may differ in other soil types, biochar feedstocks, application rates, and pyrolysis temperatures, and specific studies are necessary for each utilization scenario.Latossolo Vermelho-Amarelo).The agricultural soil samples were collected from the top layer (0–10 cm) in Viçosa, MG, Brazil , an area that has not been treated with herbicides for the last three years. The soil samples were air-dried for 10 days, then sieved on 5.0 mm mesh and stored at room temperature. The soil was classified as Oxisol (w/w) representing 0, 1, 5, 10, 15, 50, and 100 Mg ha−1, respectively, assuming a soil density of 1 g cm−3 and incorporation depth of 0.10 m. The physicochemical attributes of unamended and biochar-amended soil produced at different pyrolysis temperatures were reported by Mielke et al. [The sugarcane straw biochar (BC) samples produced at different pyrolysis temperatures  were denominated BC350 °C, BC550 °C and BC750 °C. The soil was amended with sugarcane straw biochar produced at different pyrolysis temperatures  in the application rates of 0, 0.1, 0.5, 1, 1.5, 5, and 10% , and dried in an oven with forced air circulation at 103 ± 2 °C for 72 h. The straw was placed in a sealed reactor to prevent the ingress of Oe et al. , shown iw/w)), and the third factor was the evaluation time . The soil amended with sugarcane straw biochar was added to pots with a capacity of 0.5 kg. The application of metribuzin  was carried out at the maximum recommended dose (1920 g a.i. ha−1) for sugarcane crop, with a control treatment without herbicide application. In this procedure, a CO2-pressurized sprayer equipped with two TT110.02 nozzles spaced 0.5 m apart was used, maintained at a pressure of 1.96 bar and a spray volume of 170 L ha−1. The pots were kept in a greenhouse, and soil samples were collected at 0, 5, 10, 15, 30, 45, 60, 90, 120, and 150 days after herbicide application (DAA). The temperature inside the greenhouse was recorded during the experiment. Soil moisture was adjusted by irrigation of the pots according to the rainfall distribution in 2021 for Viçosa, MG, Brazil [The experimental design was a completely randomized triple factorial scheme 3 × 7 × 10 with 3 replications. The first factor was three pyrolysis temperatures , the second factor was the application rates of biochar in the soil  and the working solution at a concentration of 100 mg L−1, both in acetonitrile (99.9% purity grade). From the working solution, three concentrations of metribuzin  were prepared. The extraction of the herbicide in the soil was performed as described by Mehdizadeh et al. [g for 7 min. The supernatant was collected and filtered through a Millipore filter . An aliquot of 1.50 mL was placed in a vial to be analyzed in high-performance liquid chromatography . The recovery level of metribuzin in fortified soil samples was, on average, 100.2%.The stock solution was prepared at a concentration of 500 mg Lh et al. . The meth et al.  and cent−1). After chromatographic analysis and obtaining the analytical curves, linearity was evaluated by linear regression of the area as a function of metribuzin concentrations and the coefficient of determination (R²). The analytical curve presented an R2 equal to 0.9999 , an injection volume of 30 µL, flow rate of 1.0 mL min−1, wavelength of 254 nm, and column oven temperature of 30 °C. Under these conditions, the retention time was 8.2 min.The quantification of metribuzin was carried out on an HPLC, with a photodiode array detector , stainless steel C18 column . The mobile phase was adapted from López-Piñeiro et al. ,24, comptC is the total concentration (mg kg−1) of herbicide remaining in the soil at time t; C0 is the initial concentration of herbicide at time zero; k is the degradation rate constant (days−1), and t is incubation time in days.The degradation data of metribuzin in the unamended soil and biochar-amended soil were fitted to a first-order kinetics model according to Equation (1).k values, the time required for 50% and 90% of the initial amount of metribuzin to be degraded (DT50 and DT90) was determined (Equations (2) and (3)).From the 2 over a period of time [w/w). After completion of the collections of the unamended and biochar-amended soil for analysis of metribuzin degradation at 150 DAA and 0 DAA (at the same time as herbicide application), the carbon content in soil microbial biomass by induced respiration was based on the measurement of the initial maximum emission of CO of time . In this2 release at 3, 6, 9, 13, 17, 22, 27, and 35 days after the start of incubation (DAI). The C-CO2 released from the soil was transported by a continuous air flow (CO2-free) to a vial containing 10 mL of 0.5 mol L−1 NaOH solution. Precipitation of the carbonate formed was carried out with the addition of 10 mL of BaCl2 0.05 mol L−1 and titrated with 0.25 mol L−1 HCl plus three drops of the 1% phenolphthalein indicator [qCO2) was determined as follows in Equation (4) [The soil collected from the rhizosphere of each experimental unit was homogenized, and 50 g was taken for analysis. Soil samples were sieved (2 mm mesh), moistened (70% field capacity), and incubated in hermetically sealed vials in a Biochemical Oxygen Demand chamber  at 25 °C without light. The respiratory frequency of the soil microbiota was evaluated with the respirometric method of C-COndicator . After 4ndicator . The mettion (4) .(4)qCO2p ≤ 0.05), the degradation curves of metribuzin in soil were plotted in Sigma Plot® , and the parameter data were presented as means and standard deviation (n = 3). For C-CO2, MBC, and qCO2, when significant (p ≤ 0.05) among factors, the means were separated by Tukey’s test and presented as means and standard deviation (n = 4), and the figures were also plotted in Sigma Plot®.The data were subjected to analysis of variance (ANOVA) to evaluate the interaction between factors in each study. The analyses were performed using Sisvar software . When the interaction between factors was significant (50 and DT90) in unamended soil were 7.37 days and 24.94 days, respectively, reflecting the low residual effect capacity of this herbicide in the studied soil.The degradation values of metribuzin (DT50 and DT90 for metribuzin were observed in BC350 °C-amended soil applied at rates of 5% (9.62 days) and 10% (17.32 days).The intensity of metribuzin degradation was dependent on the pyrolysis temperature and biochar application rate in the soil. The highest degradation of metribuzin was observed in soils amended with BC550 °C and BC750 °C when added at lower application rates (0.1% to 1.5%). The highest values of DTThe degradation process of metribuzin in BC350 °C, BC550 °C, and BC750 °C was shown to be related to the negative impact of these carbonaceous materials on the soil microbiota since in BC350 °C-amended soil at high application rates, a higher microbial imbalance was observed, presenting high respiration, low microbial carbon fixation, and high metabolic quotients, unlike BC550 °C and BC750 °C-amended soil. Even though providing greater sorption of metribuzin in the soil, the addition of low application rates of BC550 °C and BC750 °C to the soil may lead to an increase in metribuzin degradation.This material can also become an alternative for environmental remediation and reduce problems related to crop carryover. However, it may negatively influence the residual effect of the herbicide in the soil and consequently reduce the efficacy of the product in controlling weeds in the seed bank and increase the application of post-emergent herbicides.


In addition, a combination of dienophiles and hydroxyl group reagents was examined. For LC separations, reversed-phase C-18 and mixed-mode pentafluorophenyl HPLC columns using different compositions of the mobile phase were compared. With respect to detection sensitivity, the optimum derivatization reagent for the profiling of multiple metabolites was Amplifex. Nevertheless, FMP-TS, INC, PTAD, or PTAD combined with an acetylation reaction showed very good performance for selected metabolites. These reagent combinations provided signal enhancements on the order of 3- to 295-fold depending on the compound. Chromatographic separation of the dihydroxylated vitamin D3 species was readily achieved using any of the derivatization reactions, while for 25(OH)D3 epimers, only PyrNO, FMP, INC, and PTAD combined with acetylation enabled complete separation. In conclusion, we believe this study can serve as a useful reference for vitamin D laboratories, to help analytical and clinical scientists decide which derivatization reagent to choose for their application.The present study systematically compares the sensitivity and selectivity of the analysis of multiple vitamin D metabolites after chemical derivatization using different reagents for liquid chromatography-tandem mass spectrometry (LC–MS/MS). Generally, chemical derivatization is applied to vitamin D metabolites to increase the ionization efficiency, which is particularly important for very low abundant metabolites. Derivatization can also improve the selectivity of the LC separation. A wide variety of derivatization reagents has been reported in recent years, but information on their relative performance and applicability to different vitamin D metabolites is, unfortunately, not available in the literature. To fill this gap, we investigated vitamin DThe online version contains supplementary material available at 10.1007/s00216-023-04753-0. In a3, 3β-25(OH)D3, 3α-25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3. The comparisons were conducted in electrospray ionization (ESI) positive ion mode using multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. The chromatographic separation properties were investigated using four different chromatographic methods.In the present work, we focused on two primary objectives for conducting derivatization reactions for subsequent LC–MS/MS analysis: increased response factors for the analytes of interest and improved separation abilities for isomers and epimers during chromatography. As target metabolites, we chose vitamin D3 and 1,25(OH)2D3 were purchased from Cayman Chemical ; vitamin D3 and (24R)-24,25(OH)2D3 from Toronto Research Chemicals ; 3α-25(OH)D3, 4-phenyl-1,2,4-triazoline-3,5-dione, 2-fluoro-1-methylpyridinium-p-toluenesulfonate, isonicotinoyl chloride hydrochloride (95%), nicotinoyl chloride hydrochloride (97%), acetic anhydride (≥ 99%), pyridine , acetonitrile , and 4-dimethylaminopyridine (99%) from Sigma-Aldrich ; acetic acid and triethylamine (≥ 99.5%) from Carl Roth ; 4-[2-ethyl]-1,2,4-triazoline-3,5-dione from Enzo Life Sciences ; 2-nitrosopyridine from MedChemExpress ; Amplifex Diene Reagent Kit from Sciex ; and 3β-25(ΟΗ)D3- monohydrate from IsoSciences . UHPLC-MS-grade acetonitrile and methanol were obtained from Chemsolute  and formic acid (97%) from Alfa Aesar . Organic-free water was generated by a Millipore  Direct-Q8 purification system. Human vitamin D3-free serum (VD-DDC Mass Spect Gold® serum) was purchased from Sigma-Aldrich.Standards of 3β-25(OH)D3 compounds were dissolved in methanol to obtain 1 mg mL−1 stock solutions, which were stored at − 20 °C. Two groups of working solutions for the chromatographic investigations were prepared in methanol/water 90/10 (v/v). Group A included 3β-25(OH)D3 at 20 ng mL−1, vitamin D3 at 30 ng mL−1, and 1,25(OH)2D3 at 10 ng mL−1; group B included 3α-25(OH)D3 at 5 ng mL−1 and (24R)-24,25(OH)2D3 at 10 ng mL−1. Blank working solutions consisted of methanol/water 90/10 (v/v).Solid analytical standards of the investigated vitamin D3-free serum was spiked with vitamin D standard solutions to give final concentrations of 3β-25(OH)D3, vitamin D3, 1,25(OH)2D3, 3α-25(OH)D3, and (24R)-24,25(OH)2D3 of 20 ng mL−1, 30 ng mL−1, 10 ng mL−1, 5 ng mL−1, and 10 ng mL−1, respectively. The spiked vitamin D-free serum samples were divided into two groups. Group A included 3β-25(OH)D3, vitamin D3, and 1,25(OH)2D3, and group B included 3α-25(OH)D3 and (24R)-24,25(OH)2D3. This two-group split ensured that the retention times of the epimers or isobars and the peak areas were readily determined under the applied chromatographic conditions.Human vitamin DThe sample preparation protocol applied was adapted from our previous protocol . Briefly3-d6, which was added at 10 ng mL−1 after every derivatization reaction at the reconstituting stage.For the comparison, it was important that the chosen sample preparation procedure did not influence the results between different derivatization reagents. For this reason, LLE was performed for 50 spiked serum samples and the two organic phases from every LLE experiment were combined to approximately 20 mL total volume. Subsequently, in fresh vials, 380 μL of the combined volume was transferred and evaporated to dryness. The residues were then made to undergo derivatization with the respective reagent. The internal standard was 25(OH)DFor every investigated derivatization reagent, the extraction protocol was applied to 100 μL of the working solution: two samples belonging to group A and two samples belonging to group B. Every working solution sample from each group was measured in triplicate. Three spiked serum samples from group A and three spiked serum samples from group B were prepared for every derivatization reagent. Every sample was measured in duplicate.−1; 50 μL of this solution was added to the dried sample and the mixture was kept at room temperature for 60 min in the dark. After addition of 50 μL of MeOH to decompose any excess reagent, the sample was vortexed and the solvent was evaporated. The sample was reconstituted in 100 μL of a mixture of MeOH:H2O 90/10 (v/v) and transferred to a LC vial.PTAD was accurately weighed and dissolved in dry pure acetonitrile for a final concentration of 0.5 mg mL−1, 33.3 μL) containing 2% of acetic acid was added to the dried sample; the mixture was kept at room temperature for 60 min in the dark. Subsequently, 33.3 μL of a mixture of pyridine:acetic anhydride 67/33 (v/v) including 2 mg mL−1 of DMAP was added and the reaction performed at room temperature for 60 min in the dark. Importantly, the mixture of pyridine:acetic anhydride 67/33 (v/v) should be prepared directly before use. After the addition of 50 μL of methanol, the solvent was evaporated and the sample was reconstituted in 100 μL of a mixture of MeOH:H2O 90/10 (v/v) prior to analysis.For the one-pot reaction, 100 μL of serum was dried and PTAD solution (0.50 mg mL2O 90/10 (v/v) prior to injection.Amplifex diene reagent solution was prepared as described by the manufacturer. To the dried sample, 50 μL of Amplifex diene reagent was added and the mixture was vortexed for 30 s. The reaction was conducted at ambient temperature for 30 min. After adding 50 μL of methanol, the solvent was evaporated and the sample was reconstituted in 100 μL of a mixture of MeOH:H−1) in dry acetonitrile containing 1% triethylamine was added and the mixture was vortexed for 15 s. After incubation for 15 min at 40 °C, the reaction was quenched by adding 50 μL of methanol. The solvent was evaporated, and the sample was reconstituted using 100 μL of a mixture of MeOH:H2O 90/10 (/v/v) before being transferred to an LC vial.The derivatization reaction was adopted from Faqehi et al., who used FMP-TS to derivatize estrogens . To the −1) was prepared in acetonitrile. After ultrasonication (5 min) and centrifugation (5 min), the supernatant was used for derivatization. DMAP solution (10 mg mL−1) was prepared in acetonitrile. The dried sample was dissolved in 100 μL of acetonitrile, and 10 μL of the derivatization reagent was added as well as 10 μL of DMAP solution. The mixture was vortexed for 10 s and subsequently dried. Before analysis, the sample was reconstituted using 100 μL of a mixture of MeOH:H2O 90/10 (v/v).A saturated isonicotinoyl chloride solution (5 mg mL2O 90:10 (v/v) was conducted as the final step prior to analysis.A methanolic solution of PyrNO (2.5 mM) was prepared according to Wan et al.  by weigh−1. To the dried sample, 25 μL of DMEQ-TAD solution was added. The sample was incubated at ambient temperature for 30 min. Subsequently, a second addition of 25 μL of DMEQ-TAD solution was performed, followed by 60 min of incubation. To quench the reaction, 50 μL of methanol was added and the solvent was evaporated to dryness. The sample was reconstituted using 100 μL of a mixture of MeOH:H2O 90/10 (v/v) and transferred to a LC vial.The derivatization reaction was conducted as described by Kaufmann et al. . A solutFor LC–MS/MS experiments, 5 μL of a sample was injected using a 1290 Infinity II LC system . Two different stationary phases and two different mobile phases were investigated for each derivatization reagent. The columns were Phenomenex , Kinetex 2.6 µm C-18 100 Å (100 × 2.1 mm), and Kinetex 2.6 µm F5 100 Å (100 × 2.1 mm). The mobile phases were (A1) water (+ 0.1% formic acid) and (B1) methanol (+ 0.1% formic acid) or (A2) water (+ 0.1% formic acid) and (B2) acetonitrile (+ 0.1% formic acid). The gradient was linearly increased from 50 to 100% B within 15 min and then held for 2 min at 100% B, before returning to the initial conditions for re-equilibration for 3 min. The flow rate was 0.4 mL/min, and the temperature was set to 30 °C for all experiments.+ quadrupole–quadrupole-linear ion trap mass spectrometer equipped with an IonDrive Turbo-V ESI source. ESI was conducted in positive ion mode under MRM conditions. Ion source parameters were as follows: IonSpray voltage: 5500 V; source temperature: 300 °C; curtain gas: 35 psi; ion source gas 1 (nebulizer gas): 30 psi; ion source gas 2 (heating gas): 30 psi; and the collision gas was set to medium. For the MRM experiments, declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized for each vitamin D metabolite derivative separately and the obtained settings are summarized in Table S1 (Supplementary information). The dwell times (ms) were adjusted to obtain a minimum of 12 data points across the chromatographic peaks.The UHPLC system was coupled to a Sciex  QTRAP 6500Data acquisition was performed using Analyst software (Sciex) version 1.7 and MultiQuant (Sciex) version 3.0.3.3 metabolites. In addition, the separation abilities of two common stationary phases under different mobile phase conditions were investigated for the derivatized vitamin D compounds.In this study, we sought to find the optimum derivatization reagent for the determination of multiple low abundant vitamin D metabolites in serum, selected from a wide variety of commercially available reagents. We defined the optimum performance with respect to sensitivity of analysis as well as the chromatographic separation ability for vitamin D isomers and isobars. We purposely chose readily available, commercial derivatization reagents to allow for simple implementation in vitamin D assays from serum/plasma samples. Specifically, the different reagents were compared for LC–MS/MS analysis of five different vitamin D3 metabolite analysis. To our knowledge, FMP-TS has not been used for vitamin D3 metabolite measurements. Finally, the combination of Diels–Alder reaction using PTAD and acetylation of the hydroxyl groups was investigated in comparison to the other reagents.PTAD, Amplifex, DMEQ-TAD, and PyrNO perform a Diels–Alder reaction, where PTAD and Amplifex are firmly established reagents and frequently implemented for metabolite profiling. FMP-TS and INC react with the hydroxyl groups of the analytes and are thus far less specific for vitamin DWe expected Amplifex and FMP-TS to enhance detection sensitivity the most under ESI conditions, based on the permanently charged moiety in the reagent core structures. For the chromatographic separation of the epimers and isomers, we were looking for improved selectivity after derivatization, as some of the underivatized metabolites are sometimes difficult to fully separate under commonly applied chromatographic conditions.In this section, we briefly summarize the observed fragmentation patterns for the products of the investigated derivatization reactions. A more detailed description of the precursor ions and dissociation behavior is summarized in Scheme 1 (Supplementary information).3 metabolites. The positively charged quaternary ammonium group is the common leaving group during fragmentation for all analytes at m/z 59  was Amplifex, which enhanced responses 14–331-fold. This strong enhancement was likely due to the preformed positive charge from the Amplifex reagent 2D3 2D3, depending on the mobile phase. We attribute this difference to different ion suppression effects for the various species. Interestingly, the opposite trend was seen for 25(OH)D3 epimers, where higher detection sensitivity was obtained when H2O-ACN was used as mobile phase 2D3–PTAD products coeluted in contrast to the reaction products of 24,25(OH)2D3. For the PTAD products of 25(OH)D3 epimers, higher sensitivity was shown for the H2O-ACN mobile phase. Higashi et al. observed a 100-fold sensitivity increase after PTAD derivatization of 25(OH)D3 in saliva + or [MPTAD+2×Ac + H-CH3COOH]+. During the reaction of PTAD products with pyridine:acetic anhydride at room temperature, two acetyl groups may be attached to 25(OH)D3, as a result of higher DMAP concentrations or elevated temperatures.For PTAD-Ac products of 1,25(OH)2D3-INC, which can be readily explained by INC addition to the hydroxyl groups at C-1 and C-3, which both give the same precursor ion ([MINC + H]+) at m/z 522.For all other derivatizations, a single product was seen in the chromatograms, except for the two peaks seen for 1,25(OH)Rs) to study the chromatographic effects of derivatization on the two critical pairs of vitamin D metabolites, 3β-25(OH)D3/3α-25(OH)D3 and 1,25(OH)2D3/24,25(OH)2D3. Both peak capacity and resolution were considered to assess the chromatographic quality of the gradient separations [tp1 and tp2 are the retention times of the analytes of the critical pair and wp1 and wp2 are the peak widths at the base of the peaks:In this study, we used peak resolution 2D3 using the different investigated stationary and mobile phase conditions. In general, H2O/MeOH as mobile phase offered better resolving power than H2O/ACN for all the reagents, except for PTAD-Ac products. Equally, the C-18 stationary phase resolved the two derivatized analytes better than the fluorinated column, except for DMEQ-TAD products. In general, the products of most of the derivatization reagents were better resolved than their non-derivatized precursors. Importantly, even in those few cases where the Rs value was lower for the derivatization products than for the non-derivatized metabolites , full baseline separation was achieved.Table Rs ≤ 1.7, separation was readily achieved due to the narrow and symmetric peaks. The one-pot double PTAD-Ac derivatization reaction resulted in lower Rs for this peak pair compared to the single PTAD reaction. Under the investigated chromatographic conditions, PyrNO and DMEQ-TAD derivatization products were the most challenging to separate, because of coelution of several diastereomeric peaks. For PyrNO and DMEQ-TAD, some of the diastereomeric peaks of the analytes were separated, while others coeluted 2D3, the epimeric 3β-25(OH)D3 and 3α-25(OH)D3 species were even more challenging to separate. Table In comparison to 1,25(OH)Rs values were always achieved for the C-3 epimers in comparison to the non-derivatized epimers, on both the C-18 and perfluorinated stationary phases, when H2O/ACN and H2O/MeOH were used as mobile phases. The data demonstrate that the perfluorinated column offered slightly better separation for most of the different derivatization products.A general observation from the current study was that higher Rs = 1.6) of the non-derivatized epimers was also observed on a perfluorinated column in combination with H2O/MeOH (+ 0.1% formic acid) as mobile phase. However, derivatization is crucial for improving the detection sensitivity of 3α-25(OH)D3, as this metabolite is usually present at very low concentration levels. Van den Ouweland et al. successfully measured the underivatized epimers on a pentafluorophenyl column [Baseline separation (l column  using LCl column .Rs ˃ 2.0), as shown in Fig. The one-pot double derivatization reaction using PTAD and acetylation resulted in separation of some of the product peaks D3 species were more than adequately separated using the experimental chromatographic conditions studied here (Rs ˃ 2). This was not always the case for the 25(OH)D3 epimers. Excellent candidates for the epimer separation were PTAD + Ac double derivatization (Rs ≥ 2.9), PyrNO (Rs ≥ 1.9), or INC (Rs ≥ 1.5).In terms of chromatographic selectivity, the most complex spectrum of products was generated by PyrNO, due to the formation of four different products and thus four different chromatographic peaks for every analyte. In general, all the reagents’ derivatization products of the dihydroxylated vitamin DImportantly, the derivatization reactions used here do not require specialized equipment and can be readily performed in routine analytical laboratories. Most of them were conducted at room temperature, except for FMP-TS (40 °C) and PyrNO (70 °C). The fastest reaction (INC) took only 10 s, while the most time-consuming reaction (PTAD-Ac) needed 2 h to complete. While some of the reagents are expensive , most of them are relatively inexpensive in comparison to the cost of the other assay components.In conclusion, unfortunately, there is not a single reagent offering equal enhancement of the detection sensitivity for every vitamin D metabolite, adequate epimer and isomer separation, and low-cost and rapid analysis at the same time. Therefore, we believe that this study can serve as a useful reference for vitamin D laboratories, to help analytical and clinical scientists decide which derivatization reagent to choose for their application. Full validations of the multiple assays compared in this study with respect to linear dynamic range, reproducibility, precision, LOD, and LOQ were beyond the scope of the present investigation, but will be required for the assay using the chosen derivatization reagent.Supplementary file1 (DOCX 40 KB)Below is the link to the electronic supplementary material.


The oxidation of standard oxyhemoglobin forms methemoglobin, causing cyanosis (skin bluish staining). Methemoglobin cannot bind the pulmonary gaseous ligands such as oxygen (O2) and carbon monoxide (CO). As an oxidizing agent, the biochemical approach  is modified in vitro by sodium nitrite (NaNO2). The silver-doped iron zinc oxide (Ag@Fe3O4/ZnO) is hydrothermally synthesized and characterized by analytical and spectroscopic techniques for the electrochemical sensing of methemoglobin via cyclic voltammetry (CV). Detection parameters such as concentration, pH, scan rate, electrochemical active surface area (ECSA), and electrochemical impedance spectroscopy (EIS) are optimized. The linear limit of detection for Ag@Fe3O4/ZnO is 0.17 µM. The stability is determined by 100 cycles of CV and chronoamperometry for 40 h. The serum samples of anemia patients with different hemoglobin levels (Hb) are analyzed using Ag@Fe3O4/ZnO modified biosensor. The sensor's stability, selectivity, and response suggest its use in methemoglobinemia monitoring.Methemoglobinemia (MetHb, Fe Biochemically, oxyhemoglobin (OHb) is a tetrahedral complex having iron [Fe2+] as the central metal atom (CMA) and four pyrrole rings attached to (CMA)3. The transportation of pulmonary gases (O2 and CO) during the metabolic and respiratory cycle is controlled by the iron-containing non-proteinaceous, “heme and prosthetic” group of hemoglobin4. Mutations5 in α and β chains of globular (OHb) cause diseases such as diabetes (HbA1c)6, sickle cell anemia, leukemia, and thalassemia. Congenitally toxic medication, deficiency of cytochrome-b5, and self-disruption of (OHb) oxidize [Fe2+] “prosthetic or heme” to [Fe3+] central metal atom8. The oxidation of ferrous to ferric alters the normal oxyhemoglobin (HbFe2+) as methemoglobin (HbFe3+) or ferrihemoglobin10. This variation leads to chronic methemoglobinemia. Methemoglobin causes the non-binding capability of O2 with [Fe3+], which further leads to the lack of working power of muscles and tissues in vertebrates.Methemoglobinemia is a long-lasting metabolic, idiopathic, genetic, and self-disrupting disorder of oxygen-carrying hemoglobin in the bloodstream of vertebrates11. High and mild (1–5%) levels of (MetHb) lead to the formation of Ar-Amines and Ar-Halides, which cause deficiency of biochemical enzymes (glucose 6-d-phosphate and reductase) and greater concentrations of nitrites and nitrates1. The oxyhemoglobin dissociation curve indicates a higher concentration of allosteric ferric [Fe3+] ions. The induced shift in hemoglobin increases congenital methemoglobinemia through erythrocytes in anemic patients, termed erythrocytosis12. Cyanosis13 (skin bluish staining) establishes rapidly in patients with high levels of MetHb (10–15%) than with normal hemoglobin due to oxygen deficiency2. The clinical trials find that MetHb levels vary from 10 to 70%. An increase in [Fe3+] concentration changes red blood cell (RBCs) color from red to brownish mud, indicating severe cyanosis and hypoxia14. Methylene blue (MB) is the first antidote cofactor for NADH reductase, which lowers chronological methemoglobinemia in respiratory and cardiac patients15.The 8-hydroxy quinone (8-HCQ) in-take causes methemoglobinemia and hemolysis in patients with a deficiency of the (G6DP) enzyme3Fe(CN)6] at specific temperatures and pressure. Redox reactions convert standard hemoglobin to methemoglobin. Hydrogen peroxide (H2O2), hydrogen sulfide16 (H2S), and sodium nitrite (NaNO2) are the strong oxidizing agents catalyzing this reaction17. Several methods have been adopted for detecting hemoglobin and its components. Spectroscopic and separation techniques such as infrared (IR) spectroscopy, mass spectrometry (MS), fluorometry18, fluorescence spectroscopy, gas chromatography19 (GC), and high-performance liquid chromatography (HPLC) are used for the hemoglobin detection. Specific gravity, colorimetry, electrochemical techniques, and Kurt electric resistance20 are also employed for quantitative and qualitative analysis of methemoglobin.Recent studies show that methemoglobin (MetHb) can be synthesized by a controlled reaction of hemoglobin (Hb) with potassium ferrocyanide /KCl solution. The current (I) intensity is higher in Ag@Fe3O4/ZnO, used for the electrochemical detection of methemoglobin (MetHb) in anemic patients. Ag@Fe3O4/ZnO response toward the electrochemical signal depends on the conductivity of this nanomaterial, and the response is studied before and after the activation of the nanomaterial. The strong anodic and cathodic peaks can be seen in the voltammogram after activation, as shown in Fig. 3O4/ZnO modified GCE in 0.1 M K4[(Fe (CN6)]/KCl solution resulted in these prominent anodic and cathodic peaks due to efficient electron transfer. Both current signals, i.e., oxidative and reductive, become stronger after activation. Equations for oxidation (Eq. 5) and reduction (Eq. 6) are given belowThe electrochemical properties of Ag@Fe2+) through (NaNO2) leads to the formation of methemoglobin (HbFe3+) in anemic patients. Electron transfer during this process determines the overall process. Ag@Fe3O4/ZnO are the electrochemical transducers for detecting methemoglobin via CV. Fe3O4/ZnO assesses the electrical conductivity and increment in surface area, and charge transfer is enhanced by Ag NPs43.The chemical oxidation of hemoglobin , T indicates thermodynamic temperature (298.15 K), n represents the number of electrons transferred in rate determining step , and F refers to Faraday constant . Thus, the obtained Tafel slope of the modified electrode is 24 mV/s , determined by the following equation:3O4/ZnO is evaluated by using 0.04 mM [Fe(CN)6]3−/4− and KCl solutions (0.1 M) in a 1:1 M ratio, respectively. To get more precise and comparable results, bare GCE and Ag@Fe3O4/ZnO NPs (SIZO-NPs) modified GCE are analyzed through the CV.ECSA of Ag@Fe3O4/ZnO NPs (SIZO-NPs)/GCE have shown different results. Different scan rates of Ag@Fe3O4/ZnO/GCE are taken in [Fe(CN)6]3−/4− solution to perform the voltammetric analysis and to determine ECSA44. The scan rates vary between 10 to 70 mV/s in redox solution; the outcome is shown in Fig. 3O4/ZnO modified GCE is 0.0791 cm2, and for the unmodified GCE is 0.073 cm2.Both bare GCE and Ag@FeP from the experimentally recorded voltammetry. Thus, the obtained value of the kinetic parameter (ψ) is 0.63.Whereas the kinetic parameter is calculated from the following equation:act, A, and C represents the global gas constant (R = 8.314 JK−1 mol−1), thermodynamic temperature (T = 298.15 K), faraday constant , charge transfer resistance , electrode surface area (A = 0.07), the concentration of the electrolyte in 0.1 KOH (C), respectively. The catalytic reaction rate constant (kcat) is 1.06 × 10–8.The value of the catalytic reaction rate constant (kcat) is determined by the following equation:−1 mol−1), T denotes thermodynamic temperature (298.15 K), n denotes the number of electrons transferred in the rate-determining step, i.e., 2 and F refer to the faraday constant . The calculated transfer coefficient is found to be 0.71.The transfer coefficient is determined by employing the following equation:Tafel slope (b) can be determined by employing the following equation:The calculated Tafel slope (b) value is 0.24 mV/dec Fig. .Figure 8t, and K are the concentrations of the target analyte, the concentration of interfering species at 35 µM, total current response, and catalytic reaction rate constant, i.e., 1.06 × 10–8, respectively. Σkijamp is the amperometric selectivity coefficient, a measure of the preference of the sensor for the analyte relative to the interferents  to the peak current Ip1 and surface area A1 of blank GCE as shown in Eq. (8). The given formula calculates the roughness factor:The fr is the ratio between peak current Ipa of ferrocyanide [Fe(CN)6]3−/4− through redox couple equaling to blank GCE. The electrode surface area ratio is equivalent to the oxidation ratio between two electrodes, representing the change in actual surface area. The actual dimensions of the electrode followed by an electrochemical pathway and redox cores existing on the surface are responsible for the fluctuation in fr45. The surface areas of Ag@Fe3O4/ZnO (A2) and blank GCE (A1) are 0.0791 cm2 and 0.073 cm2, respectively, and the calculated fr is 1.08.The electrode dimensions and quantity of redox centers on the analyte surface determine the roughness factor's strength (fr). It is intended by I3O4/ZnO/GCE is determined by running 100 cycles with 1 µM of modified hemoglobin, i.e., methemoglobin (MetHb) in 0.1 M PBS of pH 7.4, as shown in Fig. 3O4/ZnO/GCE can reproducibly be used many times. The further steady-state durability of Ag@Fe3O4/ZnO is determined by chronoamperometry, as shown in Fig. The stability of modified electrode Ag@Fe3O4/ZnO using 0.1 M K3[Fe(CN)6] and 0.1 M KCl. The concentration effect of methemoglobin (HbFe+3) on impedance from 10 to 35 µM is given in Fig. EIS assesses the charge transfer mechanism of sensing material and stepwise modification. In Fig. . In the equivalent circuit diagram, Rp is the electron transfer resistance on the surface of an electrochemical sensor, Rs is solution resistance, and Cdl is the component capacitance of the electrochemical sensor.MetHb molecules are bulky, which causes the steric hindrance toward charge transfer. The effect of pH is determined by applying impedance at sequential pH of 6.8. 7.0, 7.2, 7.4, 7.6, and 7.8, as shown in Fig. 3O4/ZnO NPs enhance the electron transfer rate between electrodes due to excess conductivity and reliability. Linearity in signal is due to dispersion at lower frequencies. GCE's electron transfer resistance (Rct) is ~ 27,249 Ω, and Ag@Fe3O4/ZnO is 17,172 Ω, respectively. The relationship is determined between surface resistance and electrical conductivity of Ag@Fe3O4/ZnO.The electrochemical cells work in an electro-catalytic solution under alternating current (AC) potential. Hence, EIS determines the charge distribution on electrodes by applying sinusoidal perturbation in continuous linear and semicircular segment circuits. The interfacial capacitance (CdI), Ohmic resistance (Rs), electron transfer resistance (Rct), and Warburg impedance (Zw) are the major components of a continuous circuit. The electron transfer resistance (Rct) detects electron transference in a redox reaction and is measured by semicircular diameter. Ag@Fe3O4/ZnO NPs is greater compared to bare GCE. The minor difference in conductivity of nanomaterial and GCE is responsible for the change in reduction potential as Ag@Fe3O4/ZnO has a large surface area and higher electrical conductivity. They imply an impact on the selectivity and sensitivity of EIS.−1), Rct is electron transfer resistance , T is the thermodynamic temperature (~ 298.15 K), R is the global gas constant (8.314 J K−1 mol−1), A is electrode surface area (0.073 cm2 for bare GCE and 0.0791 cm2 for Ag@Fe3O4/ZnO/GCE), C is [Fe(CN)6]3−/4− solution concentration (0.1 mM cm−3), and k° is the rate constant of standard heterogeneous electron transfer (cm s−1). k° for Ag@Fe3O4/ZnO/GCE and for unmodified GCE are 1.96 × 10–10 cm s−1 and 1.3 × 10−10 cm s−1, respectively. k° represents the approximate kinetics of redox pairs, and the system having higher k° establishes equilibrium in less time, depicting a faster electron transfer rate.The conductivity of Ag@FeThe alternative, dependent derivative, i.e., the limit of detection (LOD), determines the kinetics and completion of the chemical reaction with actual concentration. LOD refers to the minimum concentration of the analytical sample, which can be distinguished by zero in an analyte. LOD varies with the influence of reaction conditions and the redox pH of analytical components. The following equations can determine LOD:3O4/ZnO NPs show peculiar characteristics as electro-catalyst. The oxidized form of “heme” (Fe3+) in serum samples of methemoglobinemia patients is analyzed by a CV to verify the applicability of the modified Ag@Fe3O4/ZnO sensor. Blood samples  are collected from anemic patients. The samples are diluted in a buffer of pH 7.4, and the recovery spike is denoted over the oxidative and reductive curves of cyclic voltammograms, as shown in Fig. Ag@FeSamples show a maximum oxidation form of standard hemoglobin in anemic patients, which refers to anemia. The recovery percentage ranges from 88.8 to 97.7% of MetHb, as shown in Table 3+) in anemic patients. The redox reaction in cyclic voltammetric (CV) setup verifies the sensing of a biological analyte. All the parameters of CV, i.e., electrical conductivity, stability, selectivity chronoamperometry, pH, concentration, and ECSA, are optimized in phosphate buffer (PBS) and potassium ferrocyanide [Fe(CN)6]3−/4− solutions. The hydrothermal methodology has been adopted for synthesizing Ag@Fe3O4/ZnO electrocatalyst. The redox method is used for preparing methemoglobin from the standard form of oxyhemoglobin. The as-synthesized Ag@Fe3O4/ZnO sensor induces biosensing of methemoglobin oxidation and reduction with high sensitivity of 0.17 µM HbFe3+ with fast response, selectivity, and stability in 0.1 M PBS. Results reveal the potential of Ag@Fe3O4/ZnO NPs as a biosensor for application in clinical trials of detecting methemoglobin.An electrochemical homogeneous deposition route has been adopted for sensing methemoglobin (HbFe


Escherichia coli PRPS (ecPRPS) using cryo-electron microscopy (cryo-EM) 1.PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While human PRPS1 (hPRPS1) and human PRPS2 (hPRPS2) are expressed in most tissues, human PRPS3 (hPRPS3) is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming filaments and cytoophidia is considered as a distinctive mechanism involving the polymerization of the protein. Previously we solved the filament structures of Order to investigate the function and molecular mechanism of hPRPS2 polymerization, here we solve the polymer structure of hPRPS2 at 3.08 Å resolution. hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity.ecPRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.Findings suggest that the regulation of hPRPS2 polymer is distinct from The online version contains supplementary material available at 10.1186/s13578-023-01037-z. Mycobacterium tuberculosis , 3635, 3Bacillus subtilis PRPS (1DKU). Though the percent identity of hPRPS2 and Bacillus subtilis PRPS was 45%, the amino acids interacting with subtrates in active sites and ADP in allosteric sites were very conserved. The ATP active site in 1DKU binds to AMP, and AMP does not contain β phosphate, the residue R104 of Bacillus subtilis PRPS was located in the same position with residue K99 in hPRPS2, they may share same mod for ADP inhibition. Perhaps structures with ADP/GDP in the allosteric site 1 and ATP in the active site solved will help to test our hypothesis in the future.In addition to the structure solved in this study, the reported PRPS structure bound with ADP in the allosteric site in PDB is only E. coli PRPS forms two types of filaments, type A attenuates ADP inhibition, and type B enhances the ADP inhibition. In this study, we found that the active form of hPRPS2 almost lost its activity when disrupting filament formation. In addition, hPRPS2 filament shares the same interface with E. coli PRPS type A filament. These key amino acid residues at the filament interface are conserved in humans, mice, flies, yeast and bacteria, indicating that filament regulation mechanisms may also exist in other species.The structure comparison of hPRPS1 and hPRPS2 shows that they have the same amino acid residues at the filament interface. Previous study found rat PRPS1 and PRPS2, as well as the two so-called PAP-39 and PAP-41 peptides, can form big complex in rat liver . During E.coli CTPS can only form product-bound filament [Filamentation of metabolic enzymes is very ubiquitous and conserved. The assembly of metabolic enzymes filament may affected by ligands binding, molecular crowding and pH , 39.Drosfilament . The polfilament , 45.Drosophila CTPS [E. coli CTPS has an inhibition effect on activity. The filamentation of metabolic enzymes is not only a regulation method of enzyme activity, but has other function. It was reported that forming cytoophidia protects the protein from degradation [hPRPS2 increases in the metastasis and proliferation of many cancers, and decreased in the spermatogenesis deficiency and SCOS [Filamentation of metabolic enzymes can enhance activity, such as human IMPDH, CTPS and ila CTPS , 43, 45.radation , 47. Theand SCOS , 31. AlthPRPS2 driven by Myc interacted with mTOR, leading to tumor growth. Arginylation on N3 (asparagine) of PRPS2 affects its activity and stability [In addition, mTOR pathway and post-translational modification of proteins have been proved to be related to the assembly of cytoophidia –52. The tability . Moreovetability .It is attractive to study whether PRPS2 cytoophidia are related to mTOR pathway or affect post-translational modification. PRPS2 expression is associated with low spermatogenesis and SCOS through p53/Bcl-2/caspases signaling pathway. Another fascinating aspect is whether hPRPS cytoophidia, a large protein machinery formed by or in combination with PRPS2, affects biological function through other signaling pathway. Using our PRPS2 structure information, it will be easier to manipulate the activity and structure of PRPS2 in vivo through CRISPR genome engineering technique. As an important potential diagnostic and therapeutic method for Myc riven Cancers and male infertility, the application of genome engineering therapy in these serious diseases is worth studying.In summary, our study solves the hPRPS2 filament structure with a resolution of 3.08 Å, in which ADP binds to the allosteric site and ATP active site in the hexamer. The key amino acid residues at the filament interface are conserved in humans, mice, flies and bacteraia. Disrupting filament formation of PRPS2 almost loses its activity. K99 in the RF loop may be a key amino acid residue related to the allosteric inhibition of ADP. Therefore, our work provides the basic structural information of PRPS2, and lays a foundation for studying the regulation of PRPS2 in the cell environment and its potential clinical application.E. coli Transetta (DE3) 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 h and pelleted by centrifugation at 4,000 r.p.m. for 10 min. The harvested cells were sonicated under the ice in 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 h. and purified by Ni–NTA agarose beads (Qiagen). Lysis buffer with 50 mM imidazole was used to wash the column. And target proteins were eluted with lysis buffer with 250 mM imidazole. 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. All the experiments were performed at 4 °C.Full-length wild-type or mutant human PRPS2 sequences with a C-terminal 6 × His-tag were cloned into a modified pRSFDuet vector and expressed in 2), and 2 mM ligands  was added to the solution. After incubation at 37 °C for 30 min, the prepared protein samples were applied to glow-discharged carbon-coated EM grids , and stained with 1% uranyl acetate. Negative-stain EM grids were photographed on a Tecnai Spirit G21 microscope (FEI).Wild-type or mutation hPRPS2 proteins were mixed with different substrate conditions. In brief, purified hPRPS2 protein (1 μM) was dissolved in Tris–HCl buffer . Then Grids were immediately blotted for 3.0 s with blot force of -1 and plunge-frozen in liquid ethane cooled by liquid nitrogen using Vitrobot (Thermo Fisher Scientific) at 4 °C and with 100% humidity.6 μM hPRPS2 protein was dissolved in a buffer containing 25 mM Tris HCl pH 7.5, 150 mM NaCl, 2 mM ADP, and 10 mM MgCle−Å−2, subdivided into 50 frames at 4 s exposure. Automated data acquisition was performed with SerialEM [Movies were recorded on Titan Krios G3 (FEI) equipped with a K3 Summit direct electron detector (Gatan), operating in counting super-resolution mode at 300 kV. Each movie stack was acquired in a total dose of 60 SerialEM  at a nom2.All image processing steps were performed using Relion3.1-beta . MotionCThe structure of hPRP2 from AlphaFold was applied for the initial model. The hexamer models were generated and then docked into the corresponding electron density map by Chimera v.1.14. Coot was used E. coli orotate phosphoribosyltransferase . The amount of PRPP generated in the reaction was determined by the reduction of orotate (OA) in the mixture. The concentration of OA was measured by absorbance at 295 nm for 300 s at 37°C [2, 250 mM NaCl, 30 mM Na2HPO4, 1 mM R5P or ATP at concentrations as described in each experiment. ATP or R5P was least added into the mixture to initiate the reaction. All measurements were performed in triplicate.The activity of hPRPS2 was measured by coupled continuous spectrophotometry using SpectraMax i3. The production PRPP of PRPS can be determined by a coupled reaction (OA + PRPP → OMP + PPi) of  at 37°C . ReactioAdditional file 1: Figure S1. Human PRPS2 is assembled into filaments in vitro. Negative staining electron microscopic images of purified human PRPS2 (1 μM) incubated in various conditions. The of nucleotides, phosphate ions (Pi) and Mg2+ are 2 mM, 30 mM and 10 mM, respectively. concentrationsAdditional file 2: Figure S2. Cryo-EM data processing of human PRPS2 filament. A Representative Cryo-EM image of human PRPS2 filament. B Representative 2D averages of human PRPS2 filament in different views. C Local resolution of the type B filament final density map. D FSC curves of central hexamer in human PRPS2 filament density map (dash line shows FSC = 0.143). The final average resolution of hexamer is estimated to be 3.1 Å. E Flow chart of human PRPS2 filament image processingAdditional file 3: Figure S3. The medel of human PRPS2. A Cryo-EM reconstruction of human PRPS2 hexamer. The hexamer is the unit of filament. Each chain is in different color. B The reconstruction structure of human PRPS2 filament. The rise of human PRPS2 filament is 63 Å. When hexamers are aggregated into filament, the adjacent hexamer is twisted by 30°Additional file 4: Figure S4. Catalytic activity of human PRS2 with different concentrations of ligands. Graphs show the catalytic activity of wild-type human PRPS2 in the presence of different amounts of ATP A, R5P B, and phosphate ion C. All tests are repeated three times.Additional file 5: Figure S5. Structure comparison of PRPS. The structure comparison of hPRPS2 (colored in blue), E.coli PRPS type A filament , E.coli PRPS type AAMP+ADP filament , E.coli PRPS type B filament , Bacillus subtilis PRPS . Structure comparison of their hexamers (A), parallel dimmers (B), bent dimmers (C), monomers (D). The RMSD between hPRPS2 monomer and 7XMU, 7XMV, 7Xn3 monomer is 0.712, 0.742, 0.799, respectively


Pseudomonas syringae strains. Our results indicate that the surge of specific free LCBs and ceramides induced by FB1 or an avirulent strain trigger a biphasic ROS production. The first transient phase is partially produced by NADPH oxidase, and the second is sustained and is related to programmed cell death. MPK6 acts downstream of LCB buildup and upstream of late ROS and is required to selectively inhibit the growth of the avirulent but not the virulent strain. Altogether, these results provide evidence that a LCB– MPK6– ROS signaling pathway contributes differentially to the two forms of immunity described in plants, upregulating the defense scheme of a non-compatible interaction.The chemical diversity of sphingolipids in plants allows the assignment of specific roles to special molecular species. These roles include NaCl receptors for glycosylinositolphosphoceramides or second messengers for long-chain bases (LCBs), free or in their acylated forms. Such signaling function has been associated with plant immunity, with an apparent connection to mitogen-activated protein kinase 6 (MPK6) and reactive oxygen species (ROS). This work used in planta assays with mutants and fumonisin B1 (FB1) to generate varying levels of endogenous sphingolipids. This was complemented with in planta pathogenicity tests using virulent and avirulent  Because of their natural mechanisms of resistance to infection, plants are not colonized by the majority of potential microbial pathogens. This capacity is due to different defense mechanisms, including the presence of physical barriers, such as the cuticle and cell wall, and the constitutive production of antimicrobial compounds. In addition, sophisticated schemes of pathogen recognition and defense are displayed . Among t2+ influx, activation of mitogen-activated protein kinases (MAPKs), reactive oxygen species (ROS) production, phytohormone action display and transcriptional reprogramming , pH 5.7 and constantly stirred at 150 rpm to be germinated at 22 °C under a 16 h light/8 h dark photoperiod.Arabidopsis Col-0 wild-type seeds were surface sterilized for 15 min in 10% sodium hipoclorite and 0.05% (w/v) gellan gum, and 5 μg/mL kanamycin). Callus fractions were transferred onto fresh medium every 2 weeks.Cotyledon pieces from Arabidopsis wild-type 2-week-old seedlings grown in sterile conditions were transferred onto callus induction solid medium  cellulose Onozuka RS and 0.2% (w/v) with macerozyme R-10 in 0.5 M mannitol, 5 mM KCl and 2 mM CaCl2. Mixture was gently shaken at 40 rpm for 15 h at 22 °C under dark conditions. After incubation, the suspension was centrifuged at 1000 rpm at 22 °C for 2 min under dark conditions. The fraction over the pellet was recovered and filtered through a nylon mesh of 0.62 µM. The filtrate was collected and centrifuged at 1000 rpm at room temperature. Protoplasts were collected from the surface and counted.Protoplasts were obtained from 7-day-old cell suspension cultures from Arabidopsis wild-type. Cell walls were digested with 1%  per genotype per treatment.A 10 μM FB1 solution was prepared in sterile 10 mM MgClArabidopsis adult plants were exposed to FB1 by infiltration of 20 μL of the FB1 solution on the abaxial right side of the leaves at one point in the middle region using a 1.0 mL needleless syringe. Phenotypical responses were recorded in photographs. Every biological replicate, from a total of 3, consisted of at least of 5–6 treated leaves from independent plants, and 3 independent experiments were performed with a total of 9 plants and 18 leaves per genotype per treatment. v/v) Silwet L-77 was sprayed onto 3-week-old Arabidopsis seedlings grown in Petri dishes (3.5 cm diameter). Control seedlings were sprayed with the Silwet solution. Seedlings were maintained under continuous light at 22 °C during the treatment.A volume of 0.7 mL containing 10 µM SN or PS dissolved in 0.04% (Pseudomonas syringae pv. tomato DC3000 avrRPM1 (Pst DC3000 avrRPM1) and Pseudomonas syringae DC3000 (Pst DC3000) strains stored in 25% glycerol solution at −80 °C were grown in King’s B medium containing 50 µg/mL rifampicin (for Pst DC3000) or 20 µg/mL tetracycline and 50 µg/mL rifampicin (for Pst DC3000 avrRPM1) at 29 °C under dark conditions. Samples were taken from these strains and fresh cultures were obtained to prepare bacterial suspensions in 10 mM MgCl2 with an OD600 nm of 0.1 O.D.U., equivalent to 1 × 108 CFU. A 1 × 107 CFU dilution was prepared and sprayed to the seedlings. They were kept under continuous light at 22 °C and phenotypical responses were recorded in photographs or quantitation of bacterial growth was performed. Every biological replicate, from a total of 3, consisted of at least 2 plates of control and treated seedlings.2 by external pressure with a mortar pestle. Serial dilutions were performed from these homogenates, plated in solid B King medium as described above and incubated at 29 °C for 36 to 48 h, and the bacterial growth was estimated. FB1 had no effect in the growth of Pseudomonas syringae pv. tomato DC3000 avrRPM1 (Pst DC3000 avrRPM1) or Pseudomonas syringae DC3000 (Pst DC3000)  .2O/acetic acid  and photographed with a Motic 100 camera coupled to a Zeigen microscope. The experiments were independently repeated at least 3 times with a total of 6 plants and 12 leaves per genotype per treatment.FB1-infiltrated leaves were stained with 3,3-diaminobenzidine (DAB) solution  under white light for 2 h and then decolored in 96 % ethanol . Then, l2O2) was measured in the medium of the cell suspensions using the procedure described in Sagisaka et al. -2-nitrobenzenesulfonate) at a final concentration of 0.2 mM was added to a preparation of Arabidopsis protoplasts at 1 × 10t-test with an α of 0.05.Data shown are mean values with SE from three independent experiments with three individual replicates (unless indicated otherwise). Significance levels were tested by a two-tailed Student’s