Trichostrongylidae, Dictyocaulidae, Chabertiidae, Ancylostomatoidea, and Ascarididae. We characterized these putative PMTs and found that they possess bona fide PMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using an in vitro phosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested against Haemonchus contortus using larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates of H. contortus, with IC50 values  of 4.30 μM (2.15–8.28), 4.46 μM (3.22–6.16), 28.7 μM (17.3–49.5), and 0.65 μM (0.21–1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potent in vitro anthelmintic activity.In humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the families  Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus and Trichuris trichiura , salicylanilides, and cyclooctadepsipeptides –12. Howe–14–S-adenosyl-l-methionine (SAM)-dependent methylation of phosphoethanolamine to phosphocholine for the biosynthesis of phosphatidylcholine has been characterized and found to be essential for the protozoan parasite, Plasmodium falciparum  to phosphocholine  from various families of parasitic nematodes and identified their broad-spectrum inhibitors that we then tested for H. contortus PMT 1 and 2 (HcPMT1 and HcPMT2) in other parasitic nematodes within the families Trichostrongylidae, Dictyocaulidae, Chabertidae, Ancylostomatoidea, and Ascarididae. H. contortus HcPMT1 possesses N-terminal methyltransferase domains that catalyze the first step of methylating phosphoethanolamine (PE) to phosphomonomethylethanolamine (PMME)  with an estimated molecular weight of 37.6 kDa and is here named AcPMT1  with an estimated molecular weight of 47.5 kDa and is here called AcPMT2 (Dictyocaulus viviparus) genome contained coding sequences of both HcPMT1 and HcPMT2 orthologues, namely, DvPMT1  and DvPMT2 (645 bp). DvPMT1 and DvPMT2 code for 483- and 214-amino-acid polypeptides (GenBank accession numbers KJH50371.1 and KJH40940.1), with calculated molecular weights of 53.3 and 23.7 kDa, respectively  . We iden Ascaris . In some Ascaris . HcPMT2  Ascaris , 29. A cd AcPMT1 . In addid AcPMT2 . The lunectively  and 2.Ascaris suum, Oesophagostomum dentatum, and Toxocaracanis, each yielded a single PMT orthologue. The A. suum genome contained a coding sequence (orthologous to HcPMT1) that is 1,383 bp long, encodes a 460-amino-acid polypeptide (WormBase Parasite ID: LK871972.1) with a calculated molecular weight of 47.5 kDa, and is here named AsPMT  with a calculated molecular weight of 41 kDa and is here called OdPMT (T. canis genome contained a HcPMT2 orthologue with a 807-bp coding sequence for a 268-amino-acid polypeptide (GenBank accession number KHN87001.1) with a calculated molecular weight of 29.6 kDa and is here named TcPMT  and cosubstrate (SAM) binding . On the Escherichia coli as N-terminal His6-tagged proteins and purified using nickel affinity column chromatography. Analysis of the purified proteins by SDS-PAGE depicted the molecular weights of AcPMT1, AcPMT2, AsPMT, DvPMT1, DvPMT2, OdPMT, and TcPMT to be approximately 38, 48, 51, 53, 24, 41, and 30 kDa, respectively, consistent with their predicated molecular weights . To analyze the in vitro SAM-dependent methyltransferase activities of the recombinant proteins, we used a fluorescence-based assay with PE as the substrate, SAM as the methyl donor, and individual purified recombinant nematode PMT1 proteins as enzymes. The concentrations of PE and SAM were fixed at 200 and 100 μM, respectively, while the catalytic activity of each protein was measured at various concentrations. All the putative PMT1 proteins that were orthologous to HcPMT1  depicted concentration-dependent increase in catalytic activity , consistent with our previous observations using HcPMT1 as enzyme in the assay (T. canis only yielded a single PMT (TcPMT) that was orthologous to HcPMT2 instead of HcPMT1, we tested this protein for catalytic activity of methylating PE. Interestingly, we found that unlike previous observations with HcPMT2 in which no significant catalytic activity was observed using PE as the substrate  were consistent with Michaelis-Menten kinetics on the substrate, PE , and theN-methyltransferase (PEMT1 and PEMT2) proteins that are required for the three-step methylation of phosphatidylethanolamine (PtdEtn) to phosphatidylcholine (PtdCho). Deletion of both PMETs is lethal for yeast unless exogenous choline is provided in growth medium  in whole-cell lysates of PMT-complemented pem1Δ/pem2Δ S. cerevisiae grown under induction conditions   . In yeasolamine) . No PMT-olamine) .pem1Δ/pem2Δ S. cerevisiae is unable to grow in medium lacking exogenous choline. Therefore, we cultured the nematode PMT-complemented pem1Δ/pem2Δ S. cerevisiae mutant yeast in medium lacking choline but supplemented with 1 mM exogenous ethanolamine in order to determine whether nematode PMT-catalyzed methylation of PE to phosphocholine would lead to de novo biosynthesis of PtdCho and rescue yeast growth. By measuring the growth of the yeast cells over 3 days, we found that the LacZ-complemented pem1Δ/pem2Δ S. cerevisiae strain did not show significant growth over time  analysis of the organic-phase lipid extracts from the yeast showed the presence of both PtdEtn and PtdCho with incorporated radioisotope in the PMT-complemented pem1Δ/pem2Δ S. cerevisiae strains  against the catalytic activity of recombinant HcPMT1 at 40 μM . The rest of the compounds either had <50% inhibitory effect or augmented the catalytic activity of recombinant HcPMT1 and were thus not pursued further. Prior to evaluation of their inhibitory effect on the rest of the nematode PMTs, the identified HcPMT1 inhibitors were tested for mammalian cell cytotoxicity at and above the concentrations that inhibited HcPMT1. Among the 45 HcPMT1 inhibitors, 20 compounds showed no cytotoxicity in HCT-8 cells (American Type Culture Collection CCL244) at the highest concentration (160 μM) tested. Those compounds included NSC12097, NSC87511, NSC133100, NSC56410, NSC177858, NSC333856, NSC620709, NSC35676, NSC24872, NSC99791, NSC156219, NSC186301, NSC145612, NSC177383, NSC335989, NSC113497, NSC227186, NSC328426, and NSC320301. The rest of the compounds displayed significant cytotoxicity against HCT-8 cells at ≤40 μM and were thus not pursued further. The nontoxic 20 HcPMT1 inhibitors were next tested for cross-inhibitory activity against other nematode PMTs from A. duodenale (AcPMT1), A. suum (AsPMT), D. viviparus (DvPMT1), O. dentatum (OdPMT1), and T. canis (TcPMT). For initial screening, all compounds were tested at 40 μM, and seven compounds  showed significant cross-inhibitory activity against all the five PMTs . Subsequently, for each PMT protein, various concentrations of each of the seven compounds were tested, and the enzyme half-maximal inhibitory concentrations (IC50Enzyme) against the various PMTs’ catalytic activities were derived (50Cytotoxicity) derived  from the National Cancer Institute Open Chemical Repository. Because HcPMT1 had earlier been characterized , 27, 29, derived . Alongsi derived . From th derived , and coms tested , indicats tested .de novo efficacy of nematode PMT inhibitors, we performed growth kinetic studies by treating LacZ (control)- and nematode PMT-complemented yeast with various concentrations of the broad-spectrum PMTs inhibitors. We found that compounds NSC56410, NSC62709, NSC177858, NSC133100, NSC35676, NSC87511, and NSC145612  depicted concentration-dependent inhibitory effect on the growth of PMT-complemented mutant yeast  and multiple drug-resistant  isolate of H. contortus. Two assays that measure the effects of increasing concentrations of drug on two distinct phenotypes were employed: a larval development assay (LDA) that assesses the development of nematode eggs from L1 to L3 stages  were screened for 3 stages , 37, and50 values with 96% confidence intervals, goodness of fit (R2) values, and hill slope values for the four compounds are shown in 50 values in the low micromolar range, indicating they possessed potent anthelmintic efficacy. The small differences in IC50 values between compounds NSC133100 and NSC177383 may not be very meaningful with regard to predictions of compound potency given the variability in the Hill slope of the dose response, but these two compounds were clearly more potent than NSC145612 by ~7-fold. On the other hand, compound NSC56410 showed the highest potency among the four compounds, with ~5-fold, 6-fold, and 39-fold higher potency than NSC133100, NSC177383, and NSC145612, respectively. Between the compared compounds, the IC50 values with nonoverlapping 95% CI indicated that they were statistically significantly different and vice versa.Using the LDA, 4 of the 15 compounds tested demonstrated fairly potent anthelmintic activity, with no differences in response between the MDR and drug-susceptible isolates. Dose-response curves for these compounds are shown in H. contortus L3 as a phenotype for detecting anthelmintic activity in candidate compounds. The LMA was performed using the Worminator system  are major constituents , 20. Sig30–Ancylostomatoidea, Ascarididae, Chabertiidae, Dictyocaulidae, and Trichostrongylidae. Four PMT1 proteins containing a putative N-terminal methyltransferase domain were AcPMT1 from A. duodenale, a hookworm that infects humans, AsPMT from A. suum, a common swine parasitic nematode of economic importance, DvPMT1 from D. viviparus, a lungworm of cattle, and OdPMT from O. dentatum, a common swine nematode. Those PMT1 proteins were found to catalyze the methylation of PE to phosphomonomethylethanolamine (PMME), consistent with the enzymatic activity of previously reported orthologous PMT1 proteins in C. elegans and H. contortus , and eventually to PCho  values for SAM that were relatively lower than those reported for C. elegans and H. contortus PMT1 proteins  orthologous to the in vitro . Howeverproteins . At the proteins . In contproteins . It is lon steps , 39, 40.proteins , 25, 28,S. cerevisiae strain lacking the ability to endogenously synthesize PtdCho , we validated that nematode PMTs are critical bona fide enzymes in the biosynthesis of PtdCho. Wild-type S. cerevisiae mainly uses the Bremer-Greenberg pathway in which phosphatidylethanolamine (PtdEA) is converted to PtdCho by two SAM-dependent phosphatidylethanolamine methyltransferases, PEMT1 and PEMT2 (N-monomethylethanolamine (PtdMME), which can then be methylated by PEMT2 to phosphatidyl-N,N-dimethylethanolamine (PtdDME), and eventually to PtdCho , isolated from both human and the yeast, Candida tropicalis , NSC62709 is structurally similar to streptonigrin, which is an antimicrobial and antineoplastic agent isolated from locculus . The diflocculus . NSC3567ak barks , 49. NSC barks 4951. It isopicalis , 53. Theopicalis , 55.H. contortus using two different screening phenotypes. The LDA evaluates the development of nematode eggs to the L3 stage and thus has the ability to detect the anthelmintic activity due to a multitude of effector mechanisms  showed activity in the lower micromolar range when tested against both drug-susceptible and MDR isolates of H. contortus. Since targeting the enzymes in the phosphobase methylation pathway constitutes a novel mechanism of action, activity against the MDR isolate was expected to be similar to the susceptible. As expected, no differences were observed between the two isolates, further confirming this premise. LDA wells with 100% inhibition contained only larvae at the L1 stage when development was stopped at day 7. This suggests that these compounds do not possess ovicidal effect but have a mechanism of action that prevents the development of larvae once they hatch. Nematodes are a phylum of molting animals  and H. contortus phosphoethanolamine methytransferase-2 (HcPMT2) (http://tcoffee.crg.cat/apps/tcoffee/do:regular).Mining of nematode protein and genome databases was performed by a BLAST search using amino acid sequences of the PMTs for the parasitic nematode (HcPMT2) , 29. The(HcPMT2) , 29. TheAncylostoma duodenum PMT1 (AcPMT1), KN730644; Ancylostoma ceylanicum PMT2 (AcPMT2), KE125104.1; Ascaris suum PMT (AsPMT), LK871972.1; Dictyocaulus viviparus PMT1 (DvPMT1), KN716207.1; D. viviparus PMT2 (DvPMT2), KN716960.1; Oesophagostomum dentatum PMT (OdPMT), KN550380.1; and Toxocara canis PMT (TcPMT2), JPKZ01000489.1. The synthesized gene fragments were supplied by IDT cloned in pUCIDT (AMP) vector and lyophilized. Before use, they were reconstituted in sterile molecular-grade nuclease-free water. The primer pairs used for PCR-amplification of the PMT coding gene fragments for directional cloning in the pET15b expression vector (Novagen) in-frame with the N-terminal hexahistidine tag (His tag) are listed in Table S7. The recombinant expression vectors were sequenced to confirm identity of the PMT insert, amplified in the K-12 strain of E. coli cells  and transformed into protein expression BL21-CodonPlus (DE3)-RIL E. coli (Agilent Technologies). For H. contortus PMTs (HcPMT1 and HcPMT2), the protein expression E. coli cells generated previously were used . For HcPMT1, after induction with IPTG, the culture was incubated at 16°C with shaking for 15 h. For the rest of the PMTs, bacteria were cultured at 37°C for a further 3 h after induction.The coding sequences for each putative PMT were retrieved from GenBank and submitted to Integrated DNA Technologies  for synthesis. The submitted putative PMT coding sequences had the following GenBank accession numbers: ere used . Transfo2PO4, 300 mM NaCl, 10 mM imidazole, 5% (vol/vol) glycerol, 0.5% (vol/vol) Sarkosyl, 1× EDTA-free protease inhibitor cocktail, and 60 kU of rLysozyme (pH 8.0). The resuspended pellets were lysed by sonication and clarified by centrifugation at 20,000 × g for 20 min at 4°C. The supernatant was collected and mixed with 2 mL of prewashed Ni-NTA His Bind Resin suspension (Novagen) and incubated at 4°C for 2 h with agitation. The His-tagged recombinant proteins were purified under native conditions by nickel-affinity chromatography according to the manufacturer’s instructions (Novagen). For HcPMT1, the wash buffer contained 20 mM imidazole, 50 mM Tris base, 500 mM NaCl, and 10% (vol/vol) glycerol (pH 8.0), while the elution buffer was composed of 250 mM imidazole, 50 mM Tris base, 500 mM NaCl, and 10% (vol/vol) glycerol (pH 8.0). For the rest of the PMTs, the wash buffer contained 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8), while the elution buffer was composed of 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 5% (vol/vol) glycerol, and 0.5% (vol/vol) Sarkosyl (pH 8). About 10 mL of the protein eluate was dialyzed in 2 L of a first buffer  at 4°C with stirring overnight, followed by dialysis in 2 L of a second buffer  at 4°C for 3 h. Further buffer exchange and concentration of the eluates was performed using protein concentrators (Fisher Scientific). The sample was first centrifuged for 20 min at 4,000 rpm at 12°C. The exchange buffer  was added to make up the volume to 20 mL, followed by centrifugation for 20 min at 1,500 × g at 12°C. Buffer exchange was repeated seven times, and the final sample volume was reduced to ~1 mL. The purity of the recombinant protein was analyzed by SDS-PAGE, and the concentration was measured using a Qubit 3.0 fluorometer (Life Technologies).Bacterial cells were pelleted by centrifugation. For HcPMT1, 1 g of bacterial pellet was resuspended in 15 mL of lysis buffer  containing a 1× EDTA-free protease inhibitor cocktail (Pierce) and 75 kU of rLysozyme (Sigma). For the rest of the PMTs, 1 g of bacterial pellet was resuspended in 15 mL of lysis buffer composed of 50 mM NaH3 buffer. The washed resin was mixed with 4 mL of solution containing 4 mg of the respective purified recombinant protein, followed by incubation at 4°C for 2 h with shaking. The mixture was centrifuged, the supernatant was removed, and the resin was washed with phosphate-buffered saline (PBS), followed by the addition of 800 μL of glycine  and incubation at 4°C for 1 h. The mixture was washed twice in 50 mL of PBS, 4 mL of crude antiserum was added, and the mixture was incubated at 4°C with shaking overnight. The mixture was packed into a 15-mL column, and after four washes with PBS, the monospecific antibodies were eluted with 15 mL of 0.1 M glycine (pH 12.0). The eluate was collected in 1-mL fractions and neutralized with 150 μL of 1 M Tris (pH 6.8).The experimental animals (rats) procedures in this study were performed according to Tuskegee University Institutional Animal Care and Use Committee approved protocol R1110-18-1. Blood was collected from five rats prior to immunization and used to extract preimmune sera. To raise polyclonal antibodies against PMT proteins, the purified recombinant proteins were emulsified with Freund complete adjuvant (Sigma-Aldrich) and injected into the five rats at 40 μg of protein per rat. Two subsequent booster immunizations were administered at 2-week intervals with 20 μg of the recombinant proteins emulsified in Freund incomplete adjuvant. The rats were sacrificed, blood was collected by cardiac puncture, and sera were isolated from the blood. Pooled preimmune and pooled immune sera were used in Western blotting assays to assess the specificity of the antisera against the recombinant proteins. Purification of the PMT antisera was performed as previously described . BrieflyS-adenosyl-l-methionine (SAM) as the methyl donor, and PE as the substrate, as reported previously  for use of recombinant PMT proteins as enzymes, eviously . To deteTo determine the enzyme kinetic parameters on SAM, fixed concentrations of PMT recombinant proteins  and PE (200 μM) were used in the reactions, with various concentrations of SAM (0 to 160 μM). On the other hand, to determine the enzyme kinetic parameters on PE, fixed concentrations of PMT recombinant proteins  and SAM (100 μM) were used, with various concentrations of PE (0 to 250 μM).S-(5′-adenosyl)-l-homocysteine (SAH) as a positive control, 25 μL of dialysis buffer as a negative control, and 25 μL of 1× transferase assay buffer as a blank control were added to the respective reaction mixtures. The reactions were prepared in triplicate wells using black flat-bottomed 96-well plates. Detection buffer (100 μL) was added into each reaction, yielding a final reaction volume of 150 μL. The plates were covered in aluminum foil and incubated at room temperature with shaking for 30 min, and fluorescence read at 380EX/520EM using a SpectraMax Gemini EM microplate reader (Molecular Devices). Sample readouts were normalized with positive and negative controls.For normalization, instead of purified protein, 25 μL of 100 μM pem1Δpem2ΔS. cerevisiae mutant strain (SKY010) used in this study was generously provided by Ryouichi Fukuda . The strain can only grow in medium supplemented with choline, and as such, was propagated in 1069 YPAD medium (ATCC) supplemented with 1 mM choline chloride (Sigma) at 30°C (pem1Δpem2ΔS. cerevisiae was generated with an S.c. EasyComp transformation kit (Invitrogen) according to the manufacturer’s instructions. The competent pem1Δpem2ΔS. cerevisiae was transformed with recombinant pYES2.1 plasmid harboring respective PMT-coding gene fragments or the negative-control LacZ-coding gene fragment following the instructions in the pYES2.1 TOPO TA yeast expression kit. For nematode PMTs having both PMT1 and PMT2, the yeast cells were transformed with a mixture of plasmids harboring both , while those with only one PMT were transformed with a single plasmid harboring the respective PMT . The pYES2.1 TOPO vector has a URA3 selection marker which enables the uracil auxotrophic pem1Δpem2ΔS. cerevisiae to grow in uracil-deficient medium. Therefore, uracil-deficient selection plates containing 6.7 g/L yeast nitrogen base without amino acids (Sigma), 1.92 g/L yeast synthetic dropout medium supplement without uracil, 20 g/L agar, 2% glucose, and 1 mM choline chloride were used to grow the transformed yeast colonies. Ensuing colonies were analyzed by PCR to confirm presence of recombinant pYES2.1 plasmid bearing the respective PMT coding sequence. Positive colonies were inoculated and propagated in 15 mL of selection medium  at 30°C with shaking at 275 rpm overnight.The  at 30°C . The PMTpem1Δpem2ΔS. cerevisiae to grow in the absence of choline but in the presence of ethanolamine, 150 μL of the overnight culture was pelleted by centrifugation and resuspended in 10 mL of fresh minimal medium  and cultured at 30°C with shaking for 24 h to starve the cells of choline. The cells were pelleted and resuspended in 1 mL of fresh minimal medium  and 900 μL of the suspension further diluted 10 times with the same medium, with or without 1 mM ethanolamine. The diluted cultures were incubated at 30°C with shaking for 4 days, during which time the optical density at 600 nm (OD600) was measured every 24 h using a SpectraMax Plus 384 microplate reader (Molecular Devices) to monitor growth.To determine the ability of PMT- or LacZ-complemented To determine the expression of PMTs in complemented yeast, 2 mL of the cultured yeast  induced for transgene expression were pelleted and resuspended in 1 mL of PBS. The cells were lysed by repeated freeze-thaw, followed by sonication and the addition of an equal volume of Laemmli sample buffer. The samples were boiled for 5 min and snap-chilled on ice, followed by centrifugation. For each sample, 30 μL of the cleared lysate was fractionated by SDS-PAGE and transferred onto a nitrocellulose membrane. Immunoblotting was done using the respective monospecific-purified rat antisera at a dilution of 1:400 as primary antibodies, and horseradish peroxidase-conjugated chicken anti-rat (Thermo Fisher Scientific) as the secondary antibody at a 1:2,000 dilution. Signal generation was performed using Clarity Western ECL substrate (Bio-Rad), and imaging was performed using a FluoroChem R imager (Protein Simple).pem1Δ/pem2Δ S. cerevisiae cultures were diluted 10× and cultured in minimal medium  containing 1 mM ethanolamine hydrochloride and 0.8 μCi/mL [14C]ethanolamine hydrochloride . Due to the inability of the LacZ-complemented pem1Δ/pem2Δ S. cerevisiae to grow in the absence of choline, 1 mM choline chloride was added to its culture. The cultures were incubated for 72 h at 30°C with shaking, after which the OD600 of cultures was adjusted to the same level by diluting with an appropriate volume of medium. From each culture, 3 mL was harvested by centrifugation, and the pellet was washed in 10 mL of PBS twice. The lipids were extracted as reported previously (After choline starvation (as described above), the complemented eviously , with soEX/520EM. The mean percent inhibition (MPI) of the test compound on enzymatic activity was calculated using the following formula:positive control represents the absolute relative fluorescence units (RFU) for the positive control, which equals RFUpositive control – RFUnegative control; and ΔRFUcompounds represents the absolute RFU value for the sample with the compounds, which equals RFUcompounds – RFUnegative control.The chemical compounds were obtained from the National Cancer Institute/Developmental Therapeutics Program Open Chemical Repository. The library consisted of 419 compounds categorized as Natural Products Set IV (see Table S1). Compounds were individually reconstituted in molecular biology grade dimethyl sulfoxide (DMSO) from Sigma-Aldrich to a stock concentration of 10 mM and stored at −20°C until use. Initial screening was conducted using recombinant HcPMT1 protein as enzyme in the methyltransferase assay, with test compounds at a final concentration of 40 μM. The test reaction mixtures consisted of 40 μM test compound, 2.5 μL of SAM-free methyltransferase reaction buffer, 150 μM PE (pH 7.5), 120 μM SAM (pH 7.8), and 20 ng/μL of HcPMT1 protein, and the final volume made up to 50 μL with 1× transferase assay buffer. The volume of each test compound added did not exceed 1% of the total reaction volume. To the positive reaction mixture, instead of test compound, an equivalent volume of DMSO was added. In the negative-control reaction mixture, instead of HcPMT1 and test compound, equivalent volumes of 1× transferase buffer and DMSO, respectively, were added. Detection buffer (100 μL) containing fluorescence substrate was added into each reaction mixture. The reactions were prepared in triplicate wells in 96-well plates. The plates were covered in aluminum foil and incubated at room temperature with agitation for 30 min, followed by reading the fluorescence at 3804 HCT-8 cells were seeded per well in 96-well plates and grown overnight in 200 μL of RPMI 1640 medium  supplemented with 2.5 g/L of glucose, 1 mM sodium pyruvate, 1.5 g/L of sodium bicarbonate, 10% heat-inactivated FBS (Gibco), and 1× antibiotic-antimycotic (Gibco) at 37°C with 5% CO2 in a humidified incubator. Upon reaching 80 to 90% confluence, cells were treated with various concentrations of the test compounds for 24 h. Control wells received equivalent volumes of DMSO used in the reconstituted compounds. After 24 h of culture, 10 μL of the WST-1 reagent was added to each well, and the plates were incubated in the dark for 1 h at 37°C with 5% CO2. The plates were well agitated, and 150 μL of the medium from each well was transferred to a new clear flat-bottomed black 96-well plate (Corning). The absorbance was read at a test wavelength of 440 nm and a reference wavelength of 690 nm using a multimode microplate reader . All of the reactions were prepared in triplicate. The dose-response curves were generated by nonlinear regression analysis with GraphPad PRISM software using control-normalized data to determine the half-maximal cytotoxicity (IC50) concentrations.Compounds were tested for cytotoxicity in human ileocecal colorectal adenocarcinoma cells (HCT-8)  by using the cell proliferation reagent WST-1 (Roche) according to the manufacturer’s protocol. The WST-1 assay is a quantitative colorimetric assay for measurement of metabolically active cells. This assay is based on the reduction of the tetrazolium salt (WST-1) by viable cells. About 5 × 1050) using nonlinear regression analysis by GraphPad Prism software.Compounds that had shown more than 50% inhibition on HcPMT1 activity and had no or very low cytotoxicity against HCT-8 cells were further tested for cross-inhibitory effect against the enzymatic activity of other recombinant PMT proteins  at 40 μM. The enzymatic assay was set up as described above, with the respective PMT protein as enzyme. Compounds that depicted cross-inhibitory activity were further tested at various concentrations to derive their half-maximal effective concentrations  supplemented with 1 mM ethanolamine hydrochloride, while the LacZ-complemented yeast cells were cultured in SG medium supplemented with 1 mM choline chloride. Compounds that depicted cross-inhibitory activity against PMT proteins in vitro were added at various concentrations to the cultures. As a negative control, DMSO  was added to yeast cultures designated control cultures. The cultures were incubated at 30°C with shaking at 275 rpm for 4 days. On a daily basis during the course of culture, 200-μL portions of culture aliquots were taken, and their OD600s were measured using a microplate reader. In preliminary work, we had observed that the growth of PMT- and LacZ-complemented pem1Δpem2ΔS. cerevisiae plateaued at 3 to 4 days of culture. Therefore, to determine the inhibitory effect of nematode PMT inhibitors on the growth of PMT- and LacZ-complemented pem1Δpem2ΔS. cerevisiae, measurements taken on day 3 were used. The results were presented as percentage of growth rate using the following formula:Pem1Δpem2ΔS. cerevisiae, considering the growth rate of untreated PMT- or LacZ- complemented Pem1Δpem2ΔS. cerevisiae is 100%; the growth rate = ΔA600Day 3/ΔA600Day 0; ΔA600Day 0 is the absolute value of A600 of PMT- or LacZ- complemented pem1Δ/pem2Δ S. cerevisiae at day 0, which equals the A600 of the culture minus the A600 of the growth medium only; ΔA600Day 3 is the absolute value of the A600 of PMT- or LacZ complemented pem1Δ/pem2Δ S. cerevisiae at day 3, which equals the A600 of the culture at day 3 minus the A600 of growth medium only; the growth rate (plus inhibitor) is the growth rate of PMT- or -LacZ complemented pem1Δpem2ΔS. cerevisiae in the presence of test compound; and the growth rate (minus inhibitor) is the growth rate of the PMT- or LacZ complemented pem1Δ/pem2Δ S. cerevisiae in the absence of test compound, but with a volume of DMSO equal to that of compound added in the treated culture. The dose-response curves were generated with GraphPad Prism software and IC50 concentrations of the compounds in PMT- and LacZ-complemented yeast cells derived.To determine the effect of PMT inhibitors on the growth of PMT- and LacZ-complemented H. contortus isolates used were TxPh-2011-S  and UGA MDR 2020 . TxPh-2011-S  was originally isolated from Pronghorn antelope in West Texas in 2011 and is fully drug susceptible, phenotypically determined by the DrenchRite larval development assay . To establish this as a laboratory isolate, it was initially passaged in goats at Texas A&M University and then at The University of Georgia using both goats and sheep, and it is currently maintained in lambs at Louisiana State University. A nemabiome assay  isolate is resistant to ivermectin, benzimidazoles, levamisole, and moxidectin, as determined by the DrenchRite LDA. To establish this as a laboratory isolate, feces collected from a sheep farm in Georgia with known history of multiple anthelmintic resistance were cultured. Two donor goats were infected with the larvae recovered, which were morphologically  was then performed in the infected goats. First, a combination treatment with albendazole at 15 mg/kg and ivermectin at 0.4 mg/kg was performed, followed by a treatment with levamisole at 12 mg/kg. Later, and to confirm resistance to moxidectin, another treatment was administered using a dose of 0.4 mg/kg of moxidectin. Posttreatment feces were recovered, coprocultures were established, and L3 larvae were identified as 100% H. contortus. Resistance to all drugs was subsequently confirmed using a DrenchRite assay.The me assay  was condogically  identifiNematode eggs were isolated as previously described . After tE. coli OP50 was added to each well. The plates were resealed and incubated for 6 additional days, and the assays were ended with the addition of 20 μL of 50% Lugol iodine to each well. The contents of every well were then transferred to a clean 96-flat well plate, and all eggs and larvae in each well were counted under an inverted microscope as previously described . Plates were prepared in advance and stored at 4°C for periods no longer than 5 days. Prior to use, plates were warmed to room temperature, and 20 μL of water was added to each well to fully hydrate the agar. Between 50 and 100 eggs contained in 20 μL of water were then added, depending on the number of eggs isolated. Plates were sealed with Parafilm to reduce moisture loss and placed in an incubator at 25°C. After 24 h, 20 μL of nutritive media composed of 0.87% Earle’s balanced salts , 1% yeast extract , 0.76% NaCl (Sigma-Aldrich), and 1% escribed . Develop10 transformed, and then IC50 concentrations and dose-response curves were obtained from the log[inhibitor] versus the normalized response variable slope logistic equation for each drug.Data from wells were discarded before Prism analyses if there was evidence of excessive dryness or fungal or bacterial overgrowth. Outliers were identified by using the Z-score outlier detection method. Moreover, Z-score of zero represents a value that equals the mean while a Z-score of 2 indicates that an observation is two standard deviations above or below the mean. Any value outside the Z-score of ±2 for each given concentration was classified as outlier and discarded from further analysis. The statistical analysis of the data was done using GraphPad Prism version 9.4.0. A nonlinear regression model with a variable slope  was the method applied. Between 9 and 12 replicate values were obtained for each drug concentration, previously corrected for control. Drug concentrations were logFor LMAs, the first phase testing using compounds NSC133100 and NSC177383 was done using the Worminator system in parallel with the LDA. Initial stock solutions of 12.5 mM were prepared for each compound using 100% DMSO as a solvent. Initial stock solutions (25 μL) were then diluted in 475 μL of culture media to yield working stock solutions of 625.00, 156.30, 39.10, 9.80, 2.40, 0.60, 0.20, 0.04, and 0.00 μM in 5% DMSO for each drug. In plate, 200 μL of working stock solutions were then diluted in 800 μL of culture media, yielding concentrations of 125.00, 31.25, 7.81, 1.95, 0.49, 0.12, and 0.00 μM in 1% DMSO. For the second phase of testing, compounds showing anthelmintic activity with the LDA were tested in a second round at higher concentrations than in the first phase of testing. Initial stocks of 50 mM were prepared for compounds NSC133100, NSC177383, NSC145612, and NSC56410 using 100% DMSO as a solvent. Initial stock solutions (25-μL portions) were then diluted in 475 μL of culture media consisting of 65% Lauria-Bertani (LB) , 25% NCTC (Sigma-Aldrich), 10% fetal bovine serum , Pen/Strep (Sigma-Aldrich) at a final concentration of 100 mg/mL, and amphotericin B (Sigma-Aldrich) at a final concentration of 2.5 mg/mL. The working stock solutions yielded concentrations of 2,500.00, 625.00, 156.30, 39.10, 9.80, 2.40, and 0.00 μM in 5% DMSO for each compound. In each well in the plate, 200 μL of working stock solutions were then diluted in 800 μL of culture media, yielding final concentrations of 500.00, 125.00, 31.30, 7.81, 1.95, 0.49, and 0.00 μM in 1% DMSO.2 and 37°C. Motility readings were taken at 1, 4, 24, 48, 72, and 96 h. A medium change was conducted at 48 h, with new drug added. Prior to each reading, plates were shaken for 15 min at ambient temperature using a benchtop shaker to stimulate motility of the L3 larvae (previously tested in our lab). The Worminator assay was performed as previously described .For the motility assays, 24-well plates with inserts were used . Portions (640 μL) of culture media were added to each well, and approximately 50 exsheathed larvae contained in 160 μL of culture media in each insert. Once pretreatment readings were taken, 160 μL of the drug solution was added to the well, and another 40 μL was added to the insert, reaching the final concentrations described in the protocol above. All concentrations were tested in triplicate. After addition of the drugs, plates were incubated at 5% COescribed . The perpost hoc test using GraphPad Prism v9.4.0. In addition, Student t tests were performed to evaluate differences between groups. P values of ≤0.05 were considered significant.Data were analyzed using two-way analysis of variance with Tukey’s multiple-comparison


Instrumental variable (IV) estimation is an essential tool to estimate the causal effect of a treatment in randomized experiments when noncompliance exists. In such studies, standard statistical approaches can be biased because compliers and noncompliers can differ in unmeasured ways that affect both the compliance behavior and outcome. Based on a few assumptions such as monotonicity, the IV estimand represents the causal effect of compliers. Profiling compliers and noncompliers has important implications because the IV estimand applies only to compliers. A method for estimating the covariate means for compliers and noncompliers has recently been proposed in political sciences literature. However, this approach requires an assumption that the instrument is randomly assigned, which confines the application of this approach to randomized experiments. In this study, we present two weighting methods for profiling compliers and noncompliers when the instrument and compliance behavior are confounded by several covariates. The proposed approach can be used for both experimental and nonexperimental studies, and hence is more broadly applicable. For the development, an instrumental propensity score is adopted to account for confounded instruments. We demonstrate the utility of the proposed methods based on simulation and real data experiments. Instrumental variable (IV) analysis is used to estimate the causal effect of a treatment in the presence of unmeasured confounders. Angrist et al.  demonstrThe MH approach is useful for studying the profiles of compliers and noncompliers with unconfounded instruments. However, instruments are often confounded, which means that they are random only if several confounders are controlled. For example, the IV analysis of Abadie  studied Before the MH approach, few researchers devised methods for profiling compliers with confounded instruments. For example, Baiocchi et al.  identifiAfter the landmark papers of Imbens and Angrist , and AngThe remainder of this article is organized as follows. We briefly review the MH approach, and then present our weighting approach that accounts for confounded IVs. Based on simulations, we evaluate the proposed IPW and AIPW methods in data with confounded IVs by some covariates. The practical utility of the proposed methods is illustrated by a study of education on future earnings. We end the article with concluding remarks.Z ∈ {0, 1} be the binary instrument, with 1 indicating encouragement toward treatment and 0 indicating encouragement toward control. Let D(z) ∈ {0, 1} be the binary potential treatment value that would be observed if Z = z. The observed treatment is defined as D = ZD(1) + (1 − Z)D(0). We define a column vector of covariates X. To refer to the variables for subject i among N subjects, we add a subscript “i” to these notations. In other words, {Zi, Di(0), Di(1), Xi} denote the instrument, potential treatment values, and vector of covariates for subject i, where Xi = T and Xij is the jth covariate for subject i.We introduce some notations and assumptions to define the profiles of compliers and noncompliers. Let D(1) and D(0): compliers if D(1) > D(0), always-takers if D(1) = D(0) = 1, never-takers if D(1) = D(0) = 0, and defiers if D(1) < D(0). Because defiers are excluded under Assumption 1 below, noncompliers in IV analysis include only the always-takers and never-takers.Angrist et al.  classifiMarbach and Hangartner  presenteAssumption 1(Monotonicity) D(1) > D(0).Assumption 2(Independence of the instrument) {D(0), D(1), X} ⊥ Z.Z is randomly assigned without controlling any covariates, which occurs in randomized experiments. Based on Assumptions 1 and 2, Marbach and Hangartner . However, this extension has very limited applicability, which provides motivation for our weighting approach.The approach of Marbach and Hangartner  can be dIn this section, we propose two weighting methods to identify the profiles of compliers and noncompliers when the IV and potential treatment values are confounded by several observed covariates.To develop an IPW method, we first replace Assumption 2 with the following, which is adopted from Abadie , FrolichAssumption 3 {D(0), D(1)} ⊥ Z|X.X. Therefore, Z and {D(0), D(1)} can be confounded by X. We address this confounding by adopting an IPS, e(X) = P[Z = 1|X]. The IPSs are unknown unless the study is a randomized experiment. In addition to Assumption 3, we impose an assumption that the IPS for every subject is strictly between 0 and 1.Assumption 3 means that the instrument is independent of the potential treatment variables conditional on Assumption 4(Positivity) 0 < e(X) < 1 for every subject.fj(x) = P[Xij = x] represent the probability mass function or probability density function of Xij. To refer to the densities of Xj for compliers, always-takers, and never-takers, we add superscripts “c”, “a”, and “n” to fj(x), respectively. In other words, these densities are denoted byXij can be expressed asWe introduce additional notations as follows. Let P[Di(1) > Di(0)] = E[Di(1) − Di(0)]. This proportion of compliers can be identified by E[Di(1) − Di(0)] = E[Di|Zi = 1] − E[Di|Zi = 0] if Assumption 2 holds. However, when the instrument is confounded by X, this identification result does not hold because E[Di(z)] ≠ E[Di|Zi = z] for z = 0, 1. To identify E[Di(1) − Di(0)], we express the expectation of each potential treatment as the IPW mean based on the result of Lunceford and Davidian , it is worthwhile to note that ei = E because Zi and Di(1) are independent conditional on Xi under Assumption 3. Then, it follows thatTo identify Di(1)Zi = DiZi and the law of total expectation, Based on that E[XijDi(0)] can be identified byXj for always-takers and never-takers are defined asIn a similar way, Theorem 1(IPW representations) Under Assumptions 1, 3 and 4, the means of Xijfor compliers, always-takers, and never-takers are identified by the following IPW representations, respectively:β: ei = e. In practice, we use a logistic regression model to estimate e:β is estimated by maximum likelihood (ML). Let For estimation, we assume that the IPS is parameterized with a regression parameter vector In Theorem 2, we present the asymptotic properties of the proposed IPW estimators. Their asymptotic variances are presented in Appendix B.Theorem 2(Asymptotic distributions of the IPW estimators) Under Assumptions 1, 3 and 4, the random quantitiesandconverge in distribution to mean-zero normal random variables.Robins et al.  proposedmi1 and mi0 denote the mean models of D for subject i if that subject had been assigned to Z = 1 and Z = 0 groups:mi1 = E[Di(1)|Xi] and mi0 = E[Di(0)|Xi]. We augment these treatment models into the IPW representations in Theorem 1, which gives the following theorem for the doubly robustness of the AIPW representations.To develop the AIPW estimators, we augment treatment models into the IPW estimators. Let Theorem 3(Doubly robustness of the AIPW representations) Under Assumptions 1, 3 and 4, the following AIPW representations correctly identify the means of Xijfor compliers, always-takers, and never-takers if either the IPS or treatment model is correctly specified:mi1 and mi0 are parameterized with regression parameter vectors, α1 and α0, as follows:z = 0, 1, α1 and α0, respectively. These ML estimators are obtained by fitting the logistic regression models of D on X to the groups of Z = 1 and Z = 0, respectively. We denote by For estimation, we assume that In Theorem 4 below, we present the asymptotic properties of the AIPW estimators. Their asymptotic variances are presented in Appendix D.Theorem 4(Asymptotic distributions of the AIPW estimators) Under Assumptions 1, 3 and 4, the random quantitiesandconverge in distribution to mean-zero normal random variables.X1 and X2, were generated from a multivariate normal distribution with a zero mean vector and a compound symmetric covariance matrix with unit variance and a correlation coefficient of 0.5. Once these normal covariates were generated, X2 was dichotomized to be a binary variable: I(X2 > 0).The proposed IPW and AIPW estimators were evaluated based on Monte Caro simulations with unconfounded and confounded IVs. Because the MH approach is valid for unconfounded IVs, we also included the MH estimators in the simulations with an unconfounded instrument. Two covariates, β1, β2):  =  for no confounding,  =  for mild confounding, and  =  for moderate confounding. The intercept term βo was selected to have the marginal mean of Z be approximately equal to 0.30 for all three confounding scenarios for the instrument.The instrument Z was generated from the following logistic regression function:U was defined such that U = 0, 1 and 2 indicated a never-taker, an always-taker, and a complier, respectively. The probability of being a complier was a logistic regression function:P[U = 2|X])/2. Then, D was a deterministic function of Z and U: D = ZI(U = 2) + UI(U ≠ 2). These probability models for compliers and noncompliers were used across all confounding scenarios for the instrument. Our simulation models yielded approximately P[U = 2] = 0.32 and P[U = 0] = P[U = 1] = 0.34.A compliance class mi1 and mi0. However, our simulation results demonstrated that the AIPW estimators were robust to this mild model misspecification. The reason the true treatment model was not a logistic model is as follows. The stratum  contains compliers and always takers, and therefore mi1 = P[Ui = 2|Xi] + P[Ui = 1|Xi]. The stratum  contains only always takers, and hence mi0 = P[Ui = 1|Xi]. Because P[U = 0|X] = P[U = 1|X] = (1 − P[U = 2|X])/2 under our simulation models, we have mi1 = (1 + P[Ui = 2|Xi])/2 and mi0 = (1 − P[Ui = 2|Xi])/2, which are not logistic functions.By construction of our simulations, the true treatment model was not exactly a logistic function; hence there was a model misspecification for the treatment model because logistic models were used to estimate X1 and dichotomous X2. The true means of X1 and X2 for compliers and noncompliers were calculated based on a very large sample size with several iterations. These true means are determined by the probability models for the compliance classes; hence they were the same for all confounding scenarios for the instrument. The true means of X1 for compliers, always-takers and never-takers were 0.133, -0.064 and -0.064, respectively. The true means of X2 for those compliance classes were 0.457, 0.520 and 0.520, respectively. The sample sizes were from 500 to 6000. The number of simulations was 500 for a given sample size.We presented the performance of the profiling estimators for continuous To compare different methods, we calculated the bias, empirical standard error (ESE), and 95% confidence interval coverage rate (CR). These performance measures were calculated based on the 500 simulated data sets under each simulation scenario at a given sample size. Bias was obtained by taking the absolute value of the average of the 500 values of . ESE was the empirical Monte Carlo standard error of the 500 estimates. The standard error estimates of the MH estimators were obtained by using the R package ivdesc . Those oX1 and X2 for never-takers were slightly less variable than the corresponding MH estimators , originally analyzed by Card , 14. In X using the approach of Abadie  = E[Di(0)]. As a result, the denominator of E[(1 − Zi)Di/(1 − ei)]. Based on The always-taker mean of Xij is Di(1) = 0 implies Di(0) = 0. This leads toP[Di(1) = Di(0) = 0] = E[1 − Di(1)]. Because E[Zi/ei] = 1, we can identify the denominator of (The never-taker mean of i(0)=0].Under moBased on Sβ is the likelihood score equation for the IPS model. The last element of N−1A−1BAT−, with N−1A−1BAT−. The asymptotic normal distributions of We can express the IPW estimator for ribution . The varE[Di(1) − Di(0)] [E[Di(1) − Di(0)] if either the IPS or treatment model is correctly specified. Therefore, it is sufficient to show that the AIPW representation for the numerator of E[XijD(1)] − E[XijD(0)].We demonstrate that the AIPW representation of − Di(0)] , 26, 27,Zi − ei)Xijmi1/ei] = 0, and hence the above equation becomes E[XijD(1)]. In addition, we can express mi1 is correctly specified, then E[Zi(Di − mi1)Xij/ei] = 0 and E[Xijmi1] = E[E[XijDi(1)|Xi]] = E[XijDi(1)]; hence E[XijDi(1)].We can express the first term in the numerator of E[XijDi(0)]. E[(Zi − ei)Xijmi0/(1 − ei)] = 0, and hence E[XijDi(0)]. In addition, we can express mi0 is correctly specified, then E[(1 − Zi)Xij(Di − mi0)/{1 − ei}] = 0 and E[Xijmi0] = E[E[XijDi(0)|Xi]] = E[XijDi(0)]; hence, E[XijDi(0)]. This completes the proof.In a similar way, we can demonstrate that the second term in the numerator of S1 and S0 are the likelihood score equations for mi1(α1) and mi0(α0), respectively. The last element of N−1C−1DCT−, with N−1C−1DCT−. The asymptotic normal distributions of We can express the AIPW estimator for ribution . The varS1 Data(CSV)Click here for additional data file.


Methylorubrum extorquens NBC_00036 and Methylorubrum extorquens NBC_00404. The genomes were sequenced using the Oxford Nanopore Technologies MinION and Illumina NovaSeq systems. Both genomes are circular, with sizes of 5,661,342 bp and 5,869,086 bp, respectively.Here, we report the complete genome sequences of  Methylorubrum extorquens, NBC_00036 and NBC_00404.Soil-derived bacteria have served as a source of many important secondary metabolites. Yet, methylotrophs, common in soil, have not been widely studied for their biosynthetic gene clusters (BGCs) . Here, wMethylorubrum extorquens NBC_00036 and NBC_00404 were isolated from Danish and Spanish soil on ISP-2 medium (Streptomyces Project 2 (ISP-2) medium. Single colonies were then picked and used to make glycerol stocks. The strains were inoculated from the glycerol stock directly into liquid ISP-2 and grown at 30°C and 140 rpm for genomic DNA purification using a modified Qiagen Genomic-tip 20/G kit protocol, as described in  and then sequenced on an R9.4.1 flow cell using a MinION device. Barcode 23 was used for NBC_00036, and barcodes 46 and 87 were used for NBC_00404.N50 value of 26,081 bp and a coverage of 34×, and 118,436 raw reads for NBC_00404, with an N50 value of 11,876 bp and a coverage of 163×.The data were basecalled and demultiplexed using Guppy  applying the high-accuracy model, excluding reads smaller than 1 kb. This yielded 15,270 raw reads for NBC_00036, with an Next-generation sequencing (NGS) DNA prep set (Novogene) libraries with size selection from 250 to 400 bp were used to generate Illumina NovaSeq data. This yielded 6,310,138 and 7,033,369 2 × 150-nucleotide (nt) read pairs for NBC_00036 and NBC_00404, providing coverages of 324× and 374×, respectively (as reported by Polypolish). The reads were trimmed using Trim Galore with Cutadapt V2.10 to a minimum length of 100 bp with a quality of at least 20  using GTDB-tk V1.7.0 . The genomes were annotated using PGAP V6.3  and SAMN31331782 (NBC_00404). The genome sequences can be found under GenBank accession numbers CP110131 and CP110130.The data are available under NCBI BioProject accession number


Oryza sativa L.) and cucumber (Cucumis sativus L.) were grown in hydroponic culture to explore their physiological responses to Mg deficiency stress. Both Mg-deficient rice and cucumber plants exhibited lower biomass, leaf area, Mg concentration, and chlorophyll content (Chl) compared with Mg-sufficient plants. However, a more marked decline in Chl and carotenoid content (Car) occurred in cucumber. A lower CO2 concentration in chloroplasts (Cc) was accompanied by a decrease in the maximum rate of electron transport (Jmax) and the maximum rate of ribulose 1,5-bisphosphate carboxylation (Vcmax), restricting CO2 utilization in Mg-deficient plants. Rice and cucumber photorespiration rate (Pr) increased under Mg deficiency. Additionally, for cucumber, Car and non-photochemical quenching (NPQ) were reduced under lower Mg supply. Meanwhile, cucumber Mg deficiency significantly increased the fraction of absorbed light energy dissipated by an additional quenching mechanism . Under Mg deficiency, suppressed photosynthesis was attributed to comprehensive restrictions of mesophyll conductance (gm), Jmax, and Vcmax. Cucumber was more sensitive to Mg deficiency than rice due to lower NPQ, higher rates of electron transport to alternative pathways, and subsequently, photooxidation damage.Magnesium is an essential macronutrient for plant photosynthesis, and in response to Mg deficiency, dicots appear more sensitive than monocots. Under Mg deficiency, we investigated the causes of differing photosynthetic sensitivities in a dicot and a monocot species. Rice ( Furthermore, an unbalanced K/Mg ratio inhibits photosynthesis and N metabolism (Magnesium (Mg) is vital for plant growth and reproductive success as it is an irreplaceable component of cells and tissues , with a tabolism . Therefotabolism . Plants tabolism . The rootabolism , as Mg itabolism .2 assimilation since Mg is not only a chlorophyll structural component but also the activator of numerous photosynthetic enzymes (J)  declined in various plant species under Mg-deficient conditions  . Ribulost stroma . Additiontration , which anditions . Reducedn center .2 increases to generate reactive oxygen species (ROS) such as superoxide radicals (O2·−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·) (Pr) and non-photochemical quenching (NPQ)  . Abioticng (NPQ) ; elevating (NPQ) ; enhancing (NPQ) ; and prong (NPQ) . Howeverng (NPQ) .Pn) in monocots are lower than in dicots . Our primary objectives were to 1) compare the physiological and photosynthetic differences between rice and cucumber under Mg deficiency, 2) reveal the underlying mechanisms of photosynthesis downregulation under Mg deficiency, and (3) investigate the cause(s) of differing sensitiveness between the two plant species. Our results will provide insight into the mechanisms underlying the effects of Mg deficiency on plant growth and photosynthesis and increase our knowledge of specific Mg nutrient management on different crops, which is vital to maintain sustainable agricultural development.Oryza sativa L. cv. Shanyou 63) and cucumber (Cucumis sativus L. cv. Jinchun 4) were grown in a greenhouse. The greenhouse has a stable environment, provided with a 14-h photoperiod, a constant relative humidity of 40%∼60%, a photosynthetic photon flux density (PPFD) of 400 μmol m−2 s−1, and a day/night temperature of 30°C/25°C. Rice seeds were sterilized in 10% H2O2 for 1 h and then germinated in moist gauze. Cucumber seeds were soaked in water for 1 h and germinated in sterile quartz sand. After a preculture for 2 weeks, the uniform seedlings with 3 visible leaves were transferred to 6.5 L containers with a quarter-strength nutrient solution for the first 4 days, then transferred to a half-strength nutrient solution for another 4 days before providing a full-strength nutrient solution. Rice and cucumber seedlings were then supplied with two Mg-deficient treatments  and one Mg-sufficient treatment , and Mg was provided by MgSO4·7H2O. The nutrient composition of the full-strength culture solutions was as follows: 2.86 mM of N provided by equimolar amounts of (NH4)2SO4 and Ca(NO3)2, 1.43 mM of Ca provided by equimolar amounts of Ca(NO3)2 and CaCl2, 0.32 mM of P and 1.03 mM of K provided by KH2PO4 and K2SO4, 35.8 μM of Fe-EDTA, 9.10 μM of MnCl2·4H2O, 0.52 of μM (NH4)6Mo7O24·4H2O, 18.5 μM of H3BO3, 0.15 μM of ZnSO4·7H2O, 0.16 μM of CuSO4·5H2O, and 0.1 μM of Na2SiO3·9H2O. Dicyandiamide was added to each nutrient solution to prevent ammonium oxidation. The nutrient solutions were renewed every 4 days, and the pH was adjusted to 5.5 ± 0.1 every day with HCl (1 M) and NaOH (1 M).Rice , and maximum fluorescence (F′m) were recorded when stability was achieved after equilibration (approximately 20 min after clamping the leaf). The minimal level of fluorescence (Fo) and the maximal fluorescence level (Fm) of the leaves were measured from 2:00 to 3:00 after dark adaption sufficiently.Gas exchange and photochemical measurements were determined on the second fully expanded leaves by a portable photosynthesis system  equipped with an integrated fluorescence leaf chamber. Four leaves of each treatment were selected to measure from 9:00 to 14:00, set with a PPFD of 1,500 µmol mJ), and the excess of photosynthetic linear electron transport not used for carbon assimilation (Jexcess) were calculated as follows , the proportion of thermally dissipated energy through NPQ (ΦNPQ), the fraction of absorbed light energy dissipated by additional quenching mechanism  , the ele follows :αleaf is the leaf absorbance and β reflects the partitioning of the absorbed quanta between PSII and PSI, and Rd is the day respiration rate. Light response curves and ΦPSII were measured in gas (98% N2 and 2% O2) at nine levels of PPFD  by the portable photosynthesis system. The slope of the relationship between ΦPSII and 4ΦCO2 (the quantum efficiency of CO2 uptake) is calculated to be the value of αleaf × β (Where leaf × β .J method proposed by gm) and CO2 concentration in chloroplasts (Cc) as follows:The variable 2 compensation point in the absence of mitochondrial respiration (Γ*) and Rd were measured through the Laisk method, as reported by The COFv/Fm) and the NPQ were computed as follows:The maximum quantum efficiency of photosystem II  at a constant CO2 partial pressure (400 μmol mol−1). For CO2 response curves, measurements were determined by adjusting CO2 concentrations to 11 levels  at a constant PPFD . All the gas exchange parameters of the leaves were recorded when stability was achieved at the corresponding ambient environments. The slope of the linear part of the light response curve  and the CO2 response curve  was calculated as apparent quantum yield (α) and carboxylation efficiency (CE). The maximum rate of RuBP carboxylation (Vcmax) and the maximum rate of RuBP regeneration (Jmax) were evaluated by the CO2 response curve fitting model provided by After the above measurements of photosynthetic parameters, three rice and three cucumber leaves were selected to conduct light and CO2 concentration in the chloroplasts at which the transition from Rubisco to RuBP regeneration limitation occurs (Ctrans) was calculated as follows:COKc = 27.24 Pa, Ko = 16.58 kPa, and O = 21 kPa.Rubisco kinetics parameters were according to 2 and 2% O2; FFPD, CO2 concentration, relative humidity, and leaf temperature were maintained as described in the measurement of Pn, and the net photosynthetic rate in 2%O2 (Pn2%O2) was recorded when stability was achieved after equilibration.Photorespiration rate measurements were also determined on the second fully expanded leaves by a portable photosynthesis system, which was connected to a gas containing 98% NPr) was computed as follows:The photorespiration rate . Four plants in each treatment were also dried under 80°C to constant weight to determine biomass.After the measurements of photosynthetic parameters, 0.2 g of fresh second fully expanded leaves were cut into little pieces and extracted with 20 ml of alcohol . After a 24-h extraction in a dark place, the absorbance of the extract was measured by a spectrophotometer at 665, 649, and 470 nm, and then the chlorophyll and carotenoid contents were calculated from those three absorbances. Another part of these leaves was used to determine leaf mass per area (LMA) and Mg contents. Leaves were shot for leaf area estimation, and LMA was calculated by dividing leaf dry matter by leaf area concentrated. Fresh leaves were dried under 80°C to a constant weight, then weighed and digested in mixed HNOg for 15 min at 4°C, and then the supernatant was collected for enzymatic activity analysis. SOD, POD, and CAT activities were determined by the methods described by Fresh second fully expanded leaf (0.2 g) was precooled by liquid nitrogen and ground with 2 ml of cold phosphate buffer  containing insoluble polyvinylpyrrolidone (1%) and ethylenediaminetetraacetic acid (0.2 mM). The homogenate was centrifuged at 10,000ANOVA with Duncan’s multiple range test was conducted on all measured parameters to identify the difference between Mg treatments using SPSS 18.0 software. Graphics and regression analysis were conducted using OriginPro 8.5 software and Adobe Illustrator 2020.Rice and cucumber plants under 0.01 mM of Mg were seriously inhibited physiologically  were also limited with increasing Mg deficiency severity, while for Ci, no significant difference was observed in 0.01 mM, compared with 1 mM Mg treatments  significantly decreased under Mg deficiency and were consistent among the two plant species. Cucumber was more sensitive to Mg deficiency than rice, with wider disparities in Jmax/Vcmax and Ctrans between 0.1 and 1 mM Mg treatments.J and ΦPSII significantly declined with Mg deficiency regardless of plant species  increased by 14.0% and decreased by 37.8%, respectively. There was no difference in rice αleaf × β between Mg-deficient and Mg-sufficient leaves, while cucumber αleaf × β decreased by 18.4% in 0.01 mM Mg treatment. For ΦNPQ, rice and cucumber showed different responses to Mg deficiency, while cucumber Φf,D increased by 40.4% under 0.01 mM Mg treatment. Jexcess declined significantly only in rice under Mg deficiency, and rice and cucumber plant Jexcess/J under 0.01 mM of Mg increased by 27.9% and 39.9%, respectively.Pr) significantly increased in 0.01 mM Mg treatment, by 42.9% compared with Mg-sufficient treatment. However, the highest cucumber Pr was found in 0.1 mM Mg treatment, where it increased by 14.3% compared with Mg-sufficient treatment. Compared with 1 mM Mg treatment, rice and cucumber Pr/Pn  ratios all increased as the Mg supply was reduced, by 71.8% and 56.8%, respectively, at 0.01 mM of Mg  decreased rapidly and continued to decline until the end of the measurements. As Pn declined, Fv/Fm of all rice treatments was stable and unchanged . Furthermore, the declining Jmax/Vcmax and Ctrans suggest a greater limitation by RuBP regeneration than by carboxylation, which implies that under Mg deficiency, the limited energy generated by the electron transport was unable to meet the normal demands for CO2 fixation  and  reduced Table 4, carnosa . Strengt reduced .Fv/Fm significantly decreased under Mg deficiency  ratio rates were similar between rice and cucumber under Mg deficiency treatments  increased by 39.9%, while in rice, it was only 27.9% under the same conditions. It was reported that high rates of electron transfer to some additional/alternative electron transport pathways, such as PTOX-mediated electron transport to oxygen and Mehler reaction, may dissipate excess electrons , which induced energy captured by light harvesting to exceed utilization by carbon assimilation; ii) Mg deficiency leads to a surplus of energy generated by photophosphorylation, with an increased ATP and ADP ratio, as well as more H+ accumulated in the thylakoid stroma inducing NPQ increment; and iii) in parallel, declining Jmax and ΦPSII exhibited a RuBP regeneration limitation, and Mg deficiency also increased the proportion of electron transport to photorespiration (Jo), further competing with Rubisco carboxylation (Jc). Mg deficiency significantly decreased NPQ and increased electron transport rates to other oxygen-dependent pathways in cucumber; meanwhile, rice possessed a stronger photoprotection capacity than cucumber, was in a better condition, and remained green.We summarized the mechanisms underlying these responses Figure 5Jmax, Vcmax, and gm. Reduced photosynthetic rate provoked an imbalanced energy between light capture and utilization by carbon assimilation, inducing NPQ increment. In parallel, more electron transport to photorespiration further competed with Rubisco carboxylation. Greater cucumber sensitivity under Mg deficiency was attributed to lower NPQ and higher electron transport rates to alternative pathways and subsequently increased oxidative stress. Overall, these results revealed the inherent mechanism of suppressed photosynthesis and suggested the crucial role of photoprotection capacity under Mg deficiency.Under magnesium deficiency stress, rice and cucumber growth and photosynthesis were inhibited. Suppressed photosynthesis was attributed to integrated limitations of The original contributions presented in the study are included in the article/SG, XM and SB conceived and designed the experiment. XM, SW and KC performed the experiments. XM, SB, YP and KX analyzed the data and were responsible for the table and figures. XM wrote the paper. SG, YP and MW proofread and polished the manuscript. All authors contributed to the article and approved the submitted version.


The exposure of ionizing radiation during early gestation often leads to deleterious and even lethal effects; however, few extensive studies have been conducted on late gestational exposures. This research examined the behavior al effects of C57Bl/6J mouse offspring exposed to low dose ionizing gamma irradiation during the equivalent third trimester. Pregnant dams were randomly assigned to sham or exposed groups to either low dose or sublethal dose radiation  at gestational day 15. Adult offspring underwent a behavioral and genetic analysis after being raised under normal murine housing conditions. Our results indicate very little change in the behavioral tasks measuring general anxiety, social anxiety, and stress-management in animals exposed prenatally across the low dose radiation conditions. Quantitative real-time polymerase chain reactions were conducted on the cerebral cortex, hippocampus, and cerebellum of each animal; results indicate some dysregulation in markers of DNA damage, synaptic activity, reactive oxygen species (ROS) regulation, and methylation pathways in the offspring. Together, our results provide evidence in the C57Bl/6J strain, that exposure to sublethal dose radiation (<1000 mGy) during the last period of gestation leads to no observable changes in behaviour when assessed as adults, although some changes in gene expression were observed for specific brain regions. These results indicate that the level of oxidative stress occurring during late gestation for this mouse strain is not sufficient for a change in the assessed behavioral phenotype, but results in some modest dysregulation of the genetic profile of the brain. Ionizing radiation, defined as sufficient incident energy to remove an electron from an atom, can create a cascade of physiological effects at the molecular level, leading to organ and system dysfunction or failure at sufficient dosage exposures . CatastrDuring the third trimester in humans, neural development is characterized by increased neuronal organization, migration, dendritogenesis, and synaptogenesis; all pertinent to healthy cognition and behavioral processes ,10. MediFetal programming describes the impact of the in-utero environment on the phenotype of the offspring, whereby adverse environments lead to predispositions for adult diseases ,17,18. DAdverse behaviors, such as depressive and anxious tendencies, have been associated with late gestational prenatal stress. Critical periods of neural sensitivity to radiation lie between gestational days 11 and 17 in mice, where neurons are still growing and differentiating . The hipThe current study examined the impact of low dose radiation during the late gestation on adult behavioural outcomes and environmental experiences (such as perception and learning), with a focus on relevant regions of the brain . Gene analysis examined the effects of prenatal radiation exposure on DNA damage, ROS regulation, and synaptic activity to investigate different neural regions and potential genetic modifications.p < 0.0001. No other behavioral measures were significant for this task and no effects of prenatal exposure to ionizing radiation were found. Observational data for the Open Field Task measured exploratory activity as an indicator for anxiety See . A main p = 0.032, η2 = 0.13. In contrast, the 1000 mGy offspring (41.25 ± 2.57 rears) were more active than Sham offspring (28.67 ± 2.78 rears). There were no other measurable behavioral differences in either the Social Interaction Task or the Porsolt Swim Task.In the Social Interaction Task, animals were observed for anxiety in relation to a stranger mouse See . There wΔΔCQ + S.E.M.; Few dysregulated genes see  were disIn the cerebral cortices, the 50 mGy and 300 mGy conditions had 6 and 5 dysregulated genes respectively, whereas the 1000 mGy condition had 3. SOD1 was upregulated in 1000 mGy females (1.23 ± 0.08), whereas SOD2 was downregulated in both males (0.50 ± 0.19) and females (0.46 ± 0.06) in the 300 mGy condition. For SOD3, males in the 50 mGy condition were also downregulated (0.86 ± 0.04). Similar to the cerebella, the cortices also displayed dysregulation in DNA methylation, circadian rhythm, synaptic activity, microglia, and apoptotic genes; however, this was predominantly in the 50 mGy and 300 mGy conditions. In the hippocampi, the 50 mGy condition had 6 dysregulated genes compared to 4 and 3 in the 300 mGy and 1000 mGy conditions, respectively. Here, there were no expression changes in the DNA methylation genes, or microglia gene TMEM119. However, SOD1 males (1.47 ± 0.21) in the 50 mGy, males (1.70 ± 0.28) and females (1.52 ± 0.25) in the 300 mGy conditions displayed increased expression. SOD3 50 mGy females were decreased compared to sham controls (0.54 ± 0.15), similarly in p53 (0.40 ± 0.15). p53 in 300 mGy females was also decreased (0.81 ± 0.06). In the 1000 mGy condition, PER2 (0.88 ± 0.05), PSD95 (1.36 ± 0.22), and NeuN (0.72 ± 0.04) were the only significant genetic expression changes.During the late gestation developmental period, animals are susceptible to fetal programming under stressful or adverse in-utero conditions ,18. LateC57Bl/6J mice are a common rodent model for a broad spectrum of research; however, previous literature has implicated they are resistant to radiation exposures ,42,43. IThere was a significant sex difference in gene dysregulation across the brain regions, which is a normal trend for brain region, stress type, and various mouse breeds ,50. The Synaptic activity genes, post-synaptic density 95, synaptophysin, NeuroD, BDNF, NOS3, and NeuN were all differentially expressed in the different brain regions, especially in the 300 mGy cerebella. PSD95 is a synaptic anchoring protein in excitatory glutaminergic synapses, associated with synaptic plasticity. Disruption of the balance of excitatory versus inhibitory synapses, as indicated by the upregulation in female cerebral cortices, can increase AMPA receptors, changing synaptic strength by inhibiting long-term potentiation and enhancing long-term depression ,54. A liEndothelial nitric oxide synthase (NOS3) can be dysregulated in the presence of ROS and promote nitric oxide neurotransmission and vascular changes . PER2 isAll experimental protocols were approved by the Animal Care Committee of McMaster University (AUP-15-11-26) and are in accordance with the Canadian Council on Animal Care guidelines. The study was designed to have 8 males and 8 females per treatment condition, for a total of 4 treatment conditions. Male and female C57Bl/6J mice (8–12 weeks) were obtained from Jackson Laboratories  and bred at McMaster University . Two females were placed in a cage overnight with a single male, for a total of 14 dams per treatment condition. Pregnancies were confirmed with the presence of a vaginal plug in the morning, which was considered gestational day (GD) 0. Pregnant females were then singly housed for the duration of the pregnancy. Normal rodent chow and water was provided ad-libitum ; and animals were maintained on a 12:12 h (7 am–7 pm) light-dark cycle. Animals were housed in standard, non-ventilated cages.137Cs γ-radiation Taylor Radiobiology source at McMaster University. Animals were transported in a temperature-controlled vehicle from the housing facility to the irradiation building in their home cages. Upon arrival, the cages were placed under shielding for acclimatization to the new location for 1 h prior to exposures. Cages were placed equidistant from the source and they received 10 mGy per minute dose on gestational day 15; fetal doses were measured at 8.9 mGy per minute [Pregnant C57Bl/6 mice were exposed to ionizing radiation with a 662 keV, r minute . Animalsad-libitum until the age of 17–18 weeks. Pups then underwent behavioral testing and were euthanized by cervical dislocation within 24 h of the last task. Relevant tissues were harvested, frozen immediately with liquid nitrogen, and then stored at −80 °C until genetic analysis.A maximum of pups from each dam (n = 8 per sex) were utilized for behavioral and genetic testing. Pups were raised under normal conditions; 2–3 per cage and food and water provisioned All behavior was recorded utilizing video-cameras. Experimenters and observers were blind to treatment groups. Behavioral tasks were conducted in random order to prevent ordering effects and were completed over the course of 1.5 days ,68. AnimAs a measure of general anxiety, four open field boxes (40 × 40 × 30 cm) were placed on the floor . Mice weTo measure social anxiety, animals were placed in a clean, normal housing cage for a total of 5 min with a stranger mouse . The strOriginally designed as a measure of despondency and learned helplessness, animals were placed in buckets of water, at 32 °C and were observed for a total of 5 min . The watBrain regions were determined utilizing Paxinos and Franklin’s mouse brain atlas . The prePrimers were designed using Primer Bank, Primer3, Primer3Plus, or Gemi; sequences and accession numbers are listed in g for 20 min at 4 °C. The top aqueous phase was removed and added into a fresh tube with 500 µL of isopropanol per 1 mL of TRI Reagent. Samples were then vortexed and incubated at room temperature for 10 min. After incubation, samples were again centrifuged, at 12,000× g for 8 min at 4 °C and pellets were washed with 1 mL of 70% ethanol per 1 mL of TRI Reagent. A final centrifugation at 7500× g for 5 min was conducted and RNA pellets were air dried prior to their resuspension in 20 µL of diethylpyrocarbonate (DEPC) treated nuclease-free water. Samples were then placed on the Thermomixer R  for 10 min at 37 °C. Total RNA concentrations were measured using the spectrophotometric measurement of the absorbance at 260 nm  [Neural tissue was weighed and then mechanically homogenized with 1 mL of TRI Reagent  per 50 mg of tissue in a TissueLyser  for two 2-min cycles at 30 Hz. Supernatant was transferred to a fresh tube and 200 µL of chloroform per 1 mL of TRI reagent was added and vortexed ,79. Afte2 µg of total RNA was treated with DNAse I (Sigma-Aldrich) according to manufacturer guidelines prior to complimentary DNA (cDNA) synthesis. For cDNA synthesis, random primers  were added to the DNAse-treated RNA. Samples were vortexed, spun down, and then heated at 70 °C for 5 min in a thermal cycler  and then placed on ice. According to manufacturer’s guidelines, 2 U/µL Mu-MLV reverse transcriptase , mixed dNTPs (200 µM), M-MuLV 5× Reaction Buffer (Promega), and DEPC water were added to each sample. A negative control was prepared with no reverse transcriptase. Samples were mixed, spun down, and incubated for 60 min at 37 °C. The final concentration of cDNA samples was 0.04 µg/µL.Utilizing the QuantStudio5 Real-Time PCR System , genes were analyzed comparing samples from the Sham, 50, 300, and 1000 mGy animals (n = 3 to 8). 12 µL reaction volumes were used with 0.3 ng/µL of cDNA of each sample, DEPC water, 2 ng/µL of forward and reverse primers, and SYBR green master-mix .p > 0.05. Inter-rater reliability was calculated using Pearson’s Correlation, r = 0.993. General linear model analysis of variance (ANOVA) was conducted, otherwise Welch’s test was utilized with alpha levels set to p = 0.05. All results are presented in mean ± standard error of the mean (S.E.M). Post-hoc analyses were conducted where appropriate, using Tukey’s Honestly Significant Difference (HSD).All statistical analyses for behavioral and genetic comparisons were carried out using IBM SPSS 20.0. All conditions were an n = 8 per sex. Datasets were tested for normality and homogeneity of variance using Shapiro-Wilk and Levene’s test, According to our behavioral results, the pattern of genetic disruption in both male and female offspring lead us to believe that the threshold for fetal programming of adverse coping behavior and anxious behavior is just beyond the doses we have utilized here. These animals are displaying a neural response to prenatal stress exposures that either do not elicit behavioral changes at this level of disruption, or display changes that we have not investigated, such as late-cognitive decline or schizophrenic-behavior as associated with the gene dysregulation patterns in other studies ,48. AlthThe authors recognize that different behavioral tasks targeting other cognitive processes may show significant changes in behavioral phenotypes that are not present in the tasks we have chosen. We also acknowledge that future work should include larger sample sizes to ensure confidence.


Subsequent serial euthanasia occurred at 1-, 3-, 7-, 14-, and 28-day post-exposure. Flow cytometry of right hemispheres revealed two endothelial populations of CD31Hi and CD31Med expressors, with wood smoke inhalation causing an increased proportion of CD31Hi. These populations of CD31Hi and CD31Med were associated with an anti-inflammatory and pro-inflammatory response, respectively, and their inflammatory profiles were largely resolved by the 28-day mark. However, activated microglial populations (CD11b+/CD45low) remained higher in wood smoke-exposed mice than controls at day 28. Infiltrating neutrophil populations decreased to levels below controls by day 28. However, the MHC-II expression of the peripheral immune infiltrate remained high, and the population of neutrophils retained an increased expression of CD45, Ly6C, and MHC-II. Utilizing an unbiased approach examining the metabolomic alterations, we observed notable hippocampal perturbations in neurotransmitter and signaling molecules, such as glutamate, quinolinic acid, and 5-α-dihydroprogesterone. Utilizing a targeted panel designed to explore the aging-associated NAD+ metabolic pathway, wood smoke exposure drove fluctuations and compensations across the 28-day time course, ending with decreased hippocampal NAD+ abundance on day 28. Summarily, these results indicate a highly dynamic neuroinflammatory environment, with potential resolution extending past 28 days, the implications of which may include long-term behavioral changes, systemic and neurological sequalae directly associated with wildfire smoke exposure.Smoke from wildland fires has been shown to produce neuroinflammation in preclinical models, characterized by neural infiltrations of neutrophils and monocytes, as well as altered neurovascular endothelial phenotypes. To address the longevity of such outcomes, the present study examined the temporal dynamics of neuroinflammation and metabolomics after inhalation exposures from biomass-derived smoke. 2-month-old female C57BL/6 J mice were exposed to wood smoke every other day for 2 weeks at an average exposure concentration of 0.5 mg/mThe online version contains supplementary material available at 10.1186/s12974-023-02874-y. These wildfire exposures have been linked to cardiovascular [Wildland fire acres burned per year have roughly doubled since 1985 [vascular , pulmonavascular , 3, oculvascular , 5, nasavascular , 7.Hi (anti-inflammatory) cells exhibiting a reduced level of CCL2, inducible nitric oxide synthase, and tumor necrosis factor-α (TNFα) following particle inhalation [Med expressing endothelial cells, however, exhibited higher expression of those same inflammatory proteins, implying that the inflammatory response was mixed and exhibiting a switchover from induction to resolution.Neuroinflammation following acute and subchronic exposures to wildfire smoke, along with other pollutants such as ozone and diesel emissions have been reported –11. The halation . CD31MedPeripheral immune cells can express leukocyte function-associated antigen-1 (LFA-1) and interact with CCL2, which both act as transmigration signals for peripheral invasion into the neurological milieu. However, myeloid cells can also express CCL2 in response to stimuli . In addiPrevious studies have shown that wildfire smoke particulate matter exposure caused only modest pulmonary inflammation yet initiated numerous neuroinflammatory sequelae, including microglial activation, peripheral immune infiltration, and decreased neuroprotective metabolites , 7. To bn = 6 per exposure per timepoint  at 8 weeks of age were housed in AAALAC-approved facilities, on a 12 h light:dark cycle and provided a standard chow diet and water ad libitum, in quarantine for 1 week of acclimation prior to exposure. The exclusive use of female C57BL/6J mice in our study was strategically chosen due to evidence of sex-based differences in inflammatory and metabolic responses, particularly relevant to neuroinflammation , 22. ThiTo obtain bronchoalveolar lavage fluid, the whole lung was flushed with 800 µl of Dulbecco's phosphate buffered saline (PBS). Post-lavage, the sample was centrifuged to separate cells and liquid supernatant. The supernatant was immediately frozen in liquid nitrogen at − 80 °C for later analysis. The cell pellet was resuspended and used to prepare cytospin slides. These slides were fixed and stained using a modified Wright–Giemsa protocol. Stained slides were then examined by hemocytometer for quantitative differential counting of cell populations.Exposure concentrations were measured in real time from a sampling tube positioned centrally in the exposure chamber with a DustTrak II  and gravimetrically using 47 mm quartz filter collected for the duration of each daily exposure and weighed on a microbalance  in a temperature-controlled laboratory. Particle size distribution was quantified for the overall system prior to mouse exposures , again sampling directly from the center of the exposure chamber. Size distribution was measured over a single 2-h run  perfusion. Hippocampus samples were dissected from the left hemisphere for metabolomics analysis (below), and all portions of the left hemisphere were flash-frozen in liquid Nescribed , 26. Bri6 cells were transferred in fluorescence activated cell sorting (FACs) tubes, stained with viability dye eFlour 450  for 30 min. After a wash with FACs buffer  and incubation with a saturating solution of Fc block , cells were stained for surface antigens for 25 min in the dark on ice. Antibodies against mouse CD11b, CD45, MHC-II, and CD31 were all purchased from Thermo Fisher Scientific, MA, USA and used as 0.125–0.5 μg/106 cells, as recommended by the manufacturer. Cells were examined for intracellular levels of various proinflammatory factors: TNFα, CCL2 and inducible nitric oxide synthase (iNOS). For intracellular staining, cells were fixed with fixation buffer and then permeabilized using intracellular fixation and permeabilization buffer . Cells were then stained with fluorochrome conjugated-antibodies for the intracellular immune factors for another 1 h at room temperature in the dark. After another wash with 1 × permeabilization buffer, cells were resuspended in 250–300 μl FACs buffer and immediately quantified in the flow cytometer. At least 50,000 live cell events were collected for each sample. Single-stained controls and isotype controls were used for laser compensation and data analysis. Data were acquired using the BD LSR Fortessa cell analyzer  and analyzed using Flow Jo software v10.7.1.For flow cytometry analysis, live cells were counted on a hemocytometer using trypan blue staining exclusion criteria. Between 0.2 and 1.0 × 10Hi expression (CD45Hi CD11b +). Activated microglia were distinguished from non-activated microglia based on combined expression levels of CD45 and CD11b [Hi Ly6C +). Median or geometric mean fluorescent intensities were plotted for activation markers or cytokine expression on these different immune and endothelial cell subsets.The gating strategy for determining different cell subsets in the brain tissues is similar to that described in our prior reports , 26. Brind CD11b . In addi13C3-lactate and 142 μM 13C5-glutamic acid) in an Eppendorf tube using a Bullet Blender homogenizer . Then, 800 μL MeOH:PBS  was added and, after vortexing for 10 s, the samples were stored at – 20 °C for 30 min. The samples were then sonicated in an ice bath for 30 min. The samples were centrifuged at 14,000 RPM for 10 min (4 °C), and 800 μL of supernatant was transferred to a new Eppendorf tube. The samples were then dried under vacuum using a CentriVap Concentrator . Prior to mass spectrometry analysis, the obtained residue was reconstituted in 150 μL 40% PBS/60% acetonitrile. A quality control sample was pooled from all the study samples.Briefly, each hippocampus sample  was homogenized in 200 μL MeOH:PBS . Briefly, LC–MS/MS experiments were performed on an Agilent 1290 UPLC-6490 QQQ–MS  system. Each hippocampus sample was injected twice: first a 10 μL volume for analysis using negative ionization mode and second a 4 μL volume for analysis using positive ionization mode. Both chromatographic separations were performed in hydrophilic interaction chromatography mode on a Waters XBridge BEH Amide column . The flow rate was 0.3 mL/min, auto-sampler temperature was kept at 4ºC, and the column compartment was set at 40 °C. The mobile phase was composed of Solvents A  and B . After the initial 1 min isocratic elution of 90% B, the percentage of Solvent B decreased to 40% at t = 11 min. The composition of Solvent B maintained at 40% for 4 min (t = 15 min), and then the percentage of B gradually went back to 90%, to prepare for the next injection. The mass spectrometer is equipped with an electrospray ionization source. Targeted data acquisition was performed in multiple-reaction-monitoring mode. The whole LC–MS system was controlled by Agilent Masshunter Workstation software . The extracted MRM peaks were integrated using Agilent MassHunter Quantitative Data Analysis . Resultant data were normalized by tissue weight before subsequent normalization steps.Targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique was used to measure NADThe untargeted LC–MS metabolomics method used here was modeled after that developed and used in a growing number of studies –31. Briet tests were used. For non-normally distributed data, t tests were employed on log2 transformed data based on right-tailed distribution. Tests were either conducted in GraphPad Prism v9.1.1, Excel (Version 2211 Build 16.0.15831.20098) or RStudio v1.4.1564, R v4.3.4. Venn diagram was generated using an online multiple list comparison tool [t tests were performed on the data sets and uncorrected as these data were not individually examined, but qualitatively explored as overlapping matrices. These values were input into Excel, RStudio, or downloaded directly for figure generation. Volcano plots were generated using a raw p < 0.05  or FDR corrected p < 0.1 (untargeted panel), both with fold-change > 1.5. Heatmapping was performed using limma-based linear regression[+) with metabolite number explicitly stated in the figure legends.Quality control samples were inserted at 10-sample intervals during mass spectrometry measurement and were utilized as a pooled sample group to compensate for temporal variability on the machine. Data were analyzed using the R package MetaboAnalyst 5.0 , 33. Norson tool , with Jaegression  were below the USEPA standards  post-exposure until day 14 . D1CD31Hi: 1.64/3.05, D1CD31Med: 17.73/8.30, D3CD31Hi: 2.28/3.99, D3CD31Med: 3.09/1.58, D7CD31Hi: 0.41/0.86, D7CD31Med: 3.88/2.4525, D14CD31Hi: 1.45/2.47, D14CD31Med: 4.60/3.99, D28CD31Hi: 2.16/2.65, D28CD31Med: 14.53/15.63. The population of endothelial cells expressing CD31Hi has been suggested as an anti-inflammatory reaction to stimuli [Med was depleted until D14, which then returned to normal levels relative to control  abundances were increased, with decreased abundance seen in glutamate, indole, niacin, and N’-Formylkynurenine. Although no metabolites appeared significantly altered by woodsmoke between D3–D14, D28 metabolites of NAD+ and NADH showed decreased abundance. After removing outliers, we plotted graphs on the Preiss–Handler and salvage pathways to examine specific points of interest for future investigation  [Summarily, these data indicate that the neuroinflammatory and neurometabolomic alterations following woodsmoke exposure can persist up to and possibly longer than 28 days. The neuroinflammation driven by woodsmoke was consistent with that observed after a real-world exposure to PM in New Mexico that was largely derived from California wildfires . In the 3) . The obs3) , as well3) –46.Hi endothelial cells that exhibited reduced relative expression of inflammatory markers . We observed a reduced population of CD31Med endothelial cells associated with promoting the neuroinflammatory response to woodsmoke, but these cells exhibited increased expression of VCAM-1, ICAM-1, CCL2, TNFα, and iNOS to varying degrees even 14 days after the exposures had ended. Both pro- and anti-inflammatory endothelial cell responses had largely subsided by day 28. Current understanding points to a reduced endothelial expression of CD31 in Alzheimer’s disease compared to healthy individuals and decreased expression in arterial beds relative to venous and capillary [Neurovascular endothelial cells exhibited a response to woodsmoke that included both inflammatory and anti-inflammatory components. Following 2 weeks of intermittent exposure, mice showed a robust increase in the CD31apillary . AccelerInterestingly, the activated microglial response to woodsmoke increased on D7, continuing through D28. Activated microglia can release quinolinic acid, and our metabolomic results show a gradual increasing concentration of quinolinic acid until statistical significance is achieved on D28. If this response lasts, neurological damage could occur due to quinolinic acid having the potential for excitotoxic effects. Here, we reported a population of activated microglia expressing Ly6C on D14. However, there is evidence that these could be microglial precursors being differentiated and recruited to fight ongoing infections/stimuli . If trueWe also observed a D14 neutrophil and non-specific peripheral immune invasion. On this point, there are two current schools of thought, with one postulate that the inhaled particles are able to escape the lungs and enter the circulation , 49. Upo+, and the decreased ratio of NAD+/NADH. 5α-DHP is a neuroprotective [A receptor that is synthesized from progesterone and reductions have been linked to social isolation and depression [Hippocampal metabolite changes may reaffirm recent observations of reduced learning and attention after wildfire exposure, using an app-based experiment performed in humans . This cootective  agonist pression . 5α-DHP pression . 3-MT ispression , while gpression . Broadly+ panel benzenepropanoic acid. We are yet unclear as to the meaning, which warrants further investigation. In addition, we observed a decrease to the dipeptide arginine–glutamine (Arg–Gln), which is formed from L-arginine and L-glutamine residues. On D3, we observed a potential compensation mechanism for reduced Arg–Gln through an increased oxoproline. This metabolite has been shown to facilitate amino acid transport across the blood brain barrier . Finally+ panel, the decreased abundance at D28 may imply increased demand from NAD+ consuming enzymes or pathways. Although we were unable to determine which enzyme or pathway was consuming the NAD+, these data do corroborate previous studies that show a decrease in NAD+ abundance after wildfire smoke exposure [+ consuming enzymes within the brain parenchyma, including sirtuins and poly(ADP-ribose) polymerases. Without these molecules—and coupled with the highly responsive neutrophil data on D28—there exists increased potential for damage and inflammatory persistence. Furthermore, if the model of fragmented peptides reaching the BBB is correct, then these neutrophils are potentially reacting to self. This has much broader implications, given that wildfire acres burned yearly have roughly doubled since 1985 and the steadily increasing incidence of autoimmunity based on epidemiological evidence [Regarding the NADexposure . There aevidence .3 for 13 consecutive days [Caveats to this study include a limited study design related to fuel source, timing of exposures, mouse model (age and sex), duration of examined timepoints, and observational outcomes. There are a multitude of organic and non-organic fuel sources for wildfires, including brush, grasses, leaves, cars, paint, asbestos, plastics, and others. In addition, our exposure paradigm emphasized replicability at the partial expense of real-world parameters. Our exposure levels were quite modest compared to some wildfires, including reported exposures in Washington State from 2020, where it was estimated that 7.1 million people were exposed to average daily particulate matter concentrations ranging from 30 to 190 µg/mive days . We chosSummarily, the major findings of this study include the duration of resolution, impacts on inflammatory and metabolite profiles, and corroboration with recent human exposure data. The duration of endothelial neuroimmune activity and resolution appeared to be resolved within 7–14 days. However, microglial activation, peripheral immune infiltration, and neutrophil invasion began during endothelial response and lasted until our final timepoint examined (Additional file Additional file 1: Table S1. List of all metabolites affected from limma-based linear modelling. Fig. S1. Neuroinflammatory and Metabolomic Temporal Dynamics.