R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance  modification is well distributed in prokaryotic cells and plays important roles in diverse physiological functions. Here, we found that the Kac modifications in a porin family protein Aha1 of  Edwardsiella tarda were altered under oxytetracycline (OXY) stress in our previous work, whereas only a few proteins were confirmed to relate to OXY resistance  modification is a well-known protein posttranslational modification (PTM), frequent in eukaryotic and prokaryotic cells. Kac modifications in proteins have been reported to play important roles in diverse bacterial biological functions such as metabolism, quorum sensing, chemotaxis, and virulence , 9, 10. fication , 11, 12.himurium . HoweverS) and OXY-resistance (OXYR) strain of pathogenic A. hydrophila in both whole protein and Kac modification peptide levels using quantitative proteomics approaches. We found that an outer membrane protein, Aha1 (gene name AHA_2785), was significantly decreased in both whole protein and Kac peptide levels. The functional validation showed that the deletion of aha1 significantly increased antibiotic resistance against OXY. Moreover, the Kac modifications on this protein's K57, K187, and K197 sites negatively regulated OXY resistance. Finally, we demonstrate that these Kac modifications on Aha1 negatively regulated the entry of OXY into the outer membrane, which was consistent with the OXY resistance data. Moreover, the acetylation modifications in the porin Aha1 of A. hydrophila displayed multidrug resistance features. To the best of our knowledge, this is the first report showing that protein Kac modification directly regulates bacterial antibiotic resistance. Our results indicate a novel antibiotic resistance mechanism mediated by PTMs and provide a potential drug target against antibiotic resistance in the future.To better understand the role of Kac modification in bacterial antibiotic resistance, here, we first compared the differential expression between OXY-sensitive , imipenem, aztreonam, ertapenem (ETP), kanamycin, tobramycin, apramycin, gentamicin, erythromycin, lincomycin, roxithromycin, azithromycin, colistin sulfate (CS), polymyxin sulfate B (PB), trimethoprim, pefloxacin, enrofloxacin, ciprofloxacin, enoxacin, levofloxacin, moxifloxacin, carbenicillin (CAR), penicillin G (PG), ampicillin (AMP), latamoxef, cefazolin, cefamandole, and ceftriaxone.A. hydrophila ATCC 7966 (OXYS) and its antibiotic-induced strain OXYR, Escherichia coli MC1061-λpir, E. coli S17-λpir, plasmids pRE112 and pBBRMCS1. A. hydrophila and E. coli were cultured in LB medium at 30 °C or 37 °C, respectively. The OXYR strain had a final minimum inhibitory concentration (MIC) to OXY 16-fold higher than the MIC of the original OXYS strain. It was induced from A. hydrophila ATCC 7966 by sequential subcultures method as previously described  was added. The cells were broken with ultrasonication (10 repetitions of the cycle 100 W ultrasonication for 10 s and 10 s on ice), centrifuged at 15,000g for 20 min, the supernatant was taken into a new tube, and the protein concentration was determined by Bradford method.After an overnight incubation for 16 h, the g for 10 min to isolate the supernatant. After a 12% SDS-PAGE electrophoresis, the gel stained with Coomassie bright blue, for whole-cell and Kac modified protein digestion, a total of 12 mg of each sample was added to the final concentration of 10 mM DTT at 37 °C for 2.5 h. Next, we added the final concentration of 50 mM iodoacetamide to the samples and kept them in the dark for 30 min. Then, the concentration of UA buffer was diluted to 1.5 M using five times the volume of water, and trypsin (Promega) was added at a ratio of 1:50 to digest at 37 °C for 18 h. Finally, the digested peptides were desalted by SPE C18 column (Waters WAT051910) and lyophilized by CentriVap concentrator (Labconco Inc) for future mass spectrometry analysis  propane sulfonic acid), pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl) was added to the peptide samples for redissolution. Pretreated anti-Kac-antibody beads (PTMScan Acetyl-Lysine Motif (Ac-K) Kit, Cell Signal Technology) were added to the samples. The sample was incubated at 4 °C for 1.5 h and then centrifuged at 2000AGE Tips .2O) and buffer B (98% ACN/0.1% FA in H2O) in a 30 min solvent gradient listed as follows: 5% B, 0 min; 5%–24% B, 20 min; 24%–35% B,5 min; 35%–80% B, 1 min; 80% B, 2 min; 80%–5%,1 min; 5%,1 min. An electrospray voltage of 2.2 kV was applied. The mass spectrometer was programmed to acquire by data-dependent acquisition tandem mass spectra from the top 25 ions in the full scan from 350 to 1500 m/z. Dynamic exclusion was set to 10 s, singly-charged ions were excluded, full mass spectrum (MS) resolution to 30,000 and MS/MS resolution to 15,000. The normalized collision energy was set to 28, automatic gain control to 2e5, max fill of MS to 250 ms, max fill MS/MS to 100 ms.For whole-cell proteomics, the desalted peptides were fractionated to six fractions for each sample using a HPLC system (Thermo DINOEX Ultimate 3000 BioRS) and a Welch C18 . The peptides samples were dried by vacuum centrifuging and analyzed using TripleTOF 5600 plus coupled with the Eksigent micro LC system (SCIEX). Briefly, peptides were separated by a binary mobile phase system of buffer A (2% ACN (acetonitrile)/0.1% FA (formic acid) in H2O) and buffer B (84% ACN/0.1% FA in H2O). Chromatographic column Thermo EASY column SC200 150 μm∗100 mm (RP-C18) balanced with 100% liquid A. Samples were fed to Thermo EASY column SC001 traps 150 μm∗20 mm (RP-C18) (Thermo) by the automatic sampler, separated by chromatographic column at a flow rate of 300 nl/min. The relative liquid gradient is as follows: 0 to 110 min, a linear gradient of liquid B ranges from 0% to 55%; 110 to 118 min, a linear gradient of liquid B from 55% to 100%; 118 to 120 min, liquid B maintained at 100%. The hydrolysates were separated by capillary HPLC and analyzed by Q-Exactive mass spectrometry (Thermo Finnigan). Detection method: positive ion, mother ion scanning range: 350 to 1800 m/z, the mass charge ratio of peptide and peptide fragments was collected according to the following methods: 20 fragment maps  were collected after each full scan. MS1 has a resolution of 70,000 at M/Z 200, and MS2 has a resolution of 17,500 at M/Z 200.For Kac-enriched proteomics, the enriched lysine-acetylated peptides were analyzed by LC-MS/MS. HPLC easy-NLC1000 was used for separation with buffer A (2% ACN/0.1% FA in HA. hydrophila ATCC 7966 database containing 4131 sequences (downloaded from the Uniprot database on 12/23/2019). The common parameter set were as follows: digested by trypsin (specific enzyme digestion of K and R) with two maximum missed cleavage, oxidation (methionine) and acetyl (protein N-term) as the variable modifications, carbamidomethylation as the fixed modification, and the peptide and protein false discovery rate cut-off were set to 0.01. The mass error was set to 20 ppm for the first search, 20 ppm for the main search, and 0.5 Da for fragment ions. For whole-cell proteins, at least two unique peptide matches were required for each protein. For Kac peptide identification, Acetyl(K) as the additional variable modification, max at seven charges, and five modifications per peptide, the best Maxquant Kac peptide score >35 and localization probability >0.75 were used.All LC-MS/MS original files were searched by Maxquant software (version 1.6.17.0) for identification and quantification against R and OXYS strain were both performed in three biological replicates. Each biological sample underwent LC-MS/MS analyses. All raw files were imported into MaxQuant software for database search and data analysis. If the label-free quantitation (LFQ) intensity of identified protein was zero in any of the samples, the protein was excluded from further quantification. The protein ratio between OXYRversus OXYS >1.5 or <0.667 with t test p-value<0.05 was considered to be an altered. Besides these parameters, we optimized the Kac peptide identification and quantification criteria for better calculation as follows: (1) If the peptide which has two or three LFQ intensity was zero in the three biological runs of each group, the zero was replaced by the smallest digit except for zero; (2) If only one of the LFQ intensity of identified peptide was zero in the three biological runs of each group, the peptide was excluded from further quantification; (3) If a total of six LFQ intensity were zero in both groups, the peptide was excluded as well. For the peptides that met these criteria, the average LFQ intensity ratio (OXYRversus OXYS) in three runs >1.5 or <0.667 with t test p-value<0.05 was used as a significant change cut-off.The whole cell and Kac quantitative proteomics between OXYhttps://david.ncifcrf.gov/) (http://bioinformatics.sdstate.edu/go/#), and the top 15 terms with the lowest p-value were visualized (https://plogo.uconn.edu/#)  to predict the AMR protein (E. coli (PDB ID: 6wtz) as the template, and then visualized and analyzed by PyMOL with PyTMs 1.2 and Caver 3.0.3 plugins  . The keysualized . The for TBtools . Amino an.edu/#) . The dat protein . SWISS-M plugins , 24, 25. plugins , 27.cobB, acuC, and aha1 genes were amplified using A. hydrophila ATCC 7966 and as templates respectively using primer pairs listed in E. coli BL21. The positive recombinant strains were inoculated in 150 ml liquid LB, cultured at 37 °C until A600 nm = 0.6, and then incubated at 16 °C for 8 h under 0.6 mM IPTG induction. The cells were collected by centrifugation and washed twice with 1× PBS buffer solution (pH = 7.4) before ultrasonic crushed in binding buffer solution (5 mM imidazole in PBS). The supernatant was gone through a Ni-TNA resin column, washed with 1× PBS buffer, and then eluted with 300 mM imidazole to purify recombinant proteins.The recombinant expression protein was constructed as previously described . Brieflyaha1 gene was amplified from A. hydrophila genomic DNA. A DNA fragment with a 6×His tag sequence was then introduced into the PBBR1-MCS1 plasmid (paha1) at HindIII and BamHI restriction sites and transformed into competent E. coli DH5α cells. Next, PCR and sequencing were performed to verify the correctness of the recovery plasmid. Site-directed mutagenesis was performed using Mut Express II Fast Mutagenesis Kit V2 according to the manufacturer's protocol with the corresponding primers listed in aha1-P5 and P6 and further verification by sequencing. Finally, the plasmids carrying aha1, point mutation aha1, and empty vector pBBR1MCS1 were transferred into Δaha1 competent cells by electrotransformation to construct rescued and point mutations.The complementary and site-directed mutagenesis of the target gene was performed as previously described . Briefly2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, 0.05%(V/V) Tween-20) was used to wash the membrane for 5 min. After that, a blocking solution (5% skim milk to PBST) was added and the membrane was incubated at room temperature for 1 h. Then anti-Kac antibody (PTM BioLab) was used as the primary antibody at 1:1000 and incubated at 4 °C overnight. The next day, the PVDF membrane was washed with PBST for three times at room temperature, and then the secondary antibody was added at 1: 4000. After incubation at room temperature for 1 h, the membrane was washed with PBST buffer five times. Finally, an ECL chemiluminescence reagent (Bio-Rad) was used for exposure and color rendering in the imager.Approximately, 20 μg protein samples from each group were separated by 12% SDS-PAGE. Bio-Rad Trans-Blot Semi-Dry Transfer instrument  was used to transfer the protein samples in the protein glue to PVDF membrane, and then PBST (8 mM Na600nm reached 1.0) were diluted 100 times, and then 2 μl of each sample was spotted on the agar plate containing different concentrations of antibiotics and cultured at 30 °C for 16 h. Each experiment was repeated at least two independent times.As previously described, this study used agar dilution to determine the minimal bacterial concentrations (MBCs) to antibiotics . Briefly600nm = 1.0). The bacterial cells were collected by centrifugation and then suspended in Mg2+ buffer . OXY in Mg2+ buffer was added to the final concentration of 2 mM and the samples were left standing for 2 h. After centrifugation, the supernatant was removed. The pellets were resuspended in Mg2+ buffer and then aliquoted into a 96-well Costar black plate (Corning) at 200 μl) with three well duplicates for each well. The absolute amount of OXY accumulation was measured by a fluorescence spectrophotometer (Molecular Devices) with excitation wavelength at 400 nm and emission wavelength at 520 nm. Each experiment was repeated three independent times.The OXY accumulation assay was measured by fluorescence spectrometry as previously described . The oveSA. hydrophila strain and its induced OXYR strain by Western blotting, which had a MIC to OXY 16-fold higher than that of OXYS. As shown in Figure 1A, when compared with OXYS, the Kac level of OXYR sharply decreased, indicating the important role of Kac in antibiotic resistance. Next, we compared the different expression patterns between OXYR and OXYS from whole-cell protein and Kac modification peptide samples using label-free–based quantitative proteomics technologies (B). The protein ratios of log2(OXYRversus OXYS) were normally distributed, and the median is close to zero , suggesting that the altered proteins were unbiased at the protein level (E). Therefore, in whole-cell proteomics, we identified 1379 proteins, including 92 proteins (account for 6.7%) that were altered between OXYR and OXYS strains (F), of which 54 increased and 38 decreased in abundance (H).To better understand the role of Kac modification on bacterial antibiotic resistance, we first detected the Kac modification levels between an OXYnologies  and S3. nologies B. The prin level E. Theref strains F, of whibundance H.Fig. 1WC). We then identified a total of 4122 Kac peptides with considerable high-quality MS, with an average score of 128.69 and an average mass error close to zero (0.056) (D). Unlike whole-cell proteomics, the distribution of Kac peptide ratios of log2(OXYRversus OXYS) has a little bias to a negative value, which was consistent with the previously detected sharp decreased of the Kac level in the OXYR sample by Western blotting (E). When compared with the OXYS sample, 726 Kac peptides (corresponding to 474 Kac proteins) of a total of 4122 identified Kac peptides (corresponding to 1182 Kac proteins) were altered in the OXYR sample (G). For most of them (628), the levels were decreased, while only 98 Kac peptides showed increased abundance (I).In Kac peptide-enriched proteomics, the correlation factors in both group repeats was relatively low (R > 0.65), which may be explained by the experimental variations of laborious PTM enrichment procedures C. We the (0.056) D. Unlikeblotting E. When cR sample G. For mobundance I.A, 31 proteins overlapped between both comparisons, and 17 of them were downregulated, with three proteins  being both upregulated in whole-cell and Kac-altered proteins (B). Interestingly, there was only one protein (SapD) upregulated in Kac proteins and downregulated in whole-cell protein, and one protein (DeoD-2) upregulated and downregulated in different Kac sites and upregulated in whole-cell protein. In addition, 25 Kac proteins that have several Kac sites increased, whereas some others decreased in the OXYR strain, indicating the different biological functions of these Kac sites in the complex physiological processes.We compared the expression patterns between altered whole-cell proteins and Kac proteins. As shown in Figure 2proteins B. IntereC and p-value were cytoplasm, metal-binding, nucleotide-binding, ATP-binding, and protein biosynthesis  in many motifs, such as EK, DK, KxxK, KxxxK, and KxxxxK (x represents any amino acid residue). In contrast, altered Kac sites have only four significant motifs: DK, EK, EK, KF, and KxxK. This observation indicates that drug-resistant–related sites may prefer these motifs to the other nonspecific Kac sites. Moreover, the DK motif is more frequent in the upregulated Kac sites, whereas the KF, EK, QK, DK, and NxxK motifs are more frequent in the downregulated Kac sites Figure 2F and We further investigated the motif characteristics of Kac proteins. As showed in Figure 2A, the downregulated proteins are mainly related to valine, leucine, and isoleucine degradation pathways, whereas the upregulated proteins show no enrichment. For Kac proteomics, despite the significant differences between upregulated and downregulated Kac protein numbers, both were involved in several central metabolic pathways, such as carbon metabolism, pyruvate metabolism, and biosynthesis of antibiotics  encoding proteins that increased, and four genes  encoding proteins that decreased in the OXYR strain in whole-cell proteomics, were traceless knocked out in A. hydrophila ATCC 7966 (A–F). When compared with the WT strain, the deletion of aha1 significantly increased the MBC of OXY eight times, while other mutants did not show significant drug resistance to OXY, suggesting that aha1 may play an important role in the resistance to OXY antibiotics in A. hydrophila. Furthermore, the aha1 rescued strain restored the OXY susceptibility phenotype, further validating the role of Aha1 in OXY resistance (G).We then asked about the role of altered proteins in OXY resistance. A total of 11 genes, including seven genes  is an outer membrane protein that belongs to the Gram-negative porin family, we speculated that Aha1 might affect the uptake of OXY into the cells. Therefore, fluorescence spectroscopy was used to detect the intracellular accumulation of OXY among Δaha1, aha1 rescued, and their control strains under a high dose of OXY stress. As shown in Figure 4H, the accumulation of intracellular OXY in Δaha1 was significantly reduced, whereas its rescued strain restored the OXY concentration to the level of the WT A. hydrophila. Therefore, our results confirmed that the outer membrane protein aha1 might influence bacterial drug resistance through outer membrane permeability.According to the protein description in Uniport, the Aha1 . These data further confirmed our whole cell protein and Kac quantitative proteomics results.We then further detected the expression of Aha1 in OXYR strain I and J. R strain against CARD database, and a total of 39 AMR genes/proteins that have Kac modifications were detected. The Kac sites of these AMR proteins and their MS intensities in Kac proteomics and their possible resistance mechanisms predicted from CARD were displayed in (A). According to resistance mechanisms, these proteins could be classified into four classes, antibiotic efflux, antibiotic activation, antibiotic target alteration, and reduced permeability to the antibiotic. Of the 39 AMR Kac proteins, Aha1, PtsI-2, Uup, IleS, and A0KLT8 were the top five abundant Kac sites, while many proteins, such as AccC, UvrA, CheY, CueR, and GyrB, have only one altered Kac site. Most altered Kac sites were downregulated, except for K5 on Tuf1, K111 on RlmL, K363 on MetG, K93 on A0KP78, K203 on SapD, and K408 on Uup. Several Kac sites, K408 and K363 on Uup, K363, and K133 on MetG, for example, displayed opposite modification patterns in one protein, indicating the delicate regulation of Kac modifications on bacterial antibiotic resistance.The potential antibiotic resistance roles of Kac proteins were further analyzed by homologous searching altered Kac proteins in OXYlayed in A. AccordS and OXYR showed that Aha1 has eight Kac sties , and seven of them (except for K164) were downregulated in the OXYR stain (B). We then asked the lysine acetyltransferase or deacetylase responding for Kac level of Aha1. Firstly, the deletion of A. hydrophila cobB (NAD+-dependent deacetylase sirtuin family) did affect the bacterial growth in OXY stress, while ΔacuC (zinc-dependent deacetylase Rpd3/Hda1 family) and ΔAHA_0149 (lysine acetyltransferase family) did not, indicating that cobB may involve in OXY resistance (A. hydrophila (C and The current quantitative acetylome comparison between OXYYR stain B. We thesistance . Moreovedrophila C and S3.A–F). We then further investigated the relationship among the three Aha1 mutants by multiple site-directed mutagenesis. As shown in Δaha1 strain, suggesting that the deacetylations of K57, K187, and K197 play an important role in the OXY resistance and the role of these three Kac sites on OXY resistance should be independent. Moreover, the following OXY accumulation assays were consistent with the antibiotic resistance characteristics of these Aha1 derivatives (G). Besides these, we also constructed aha1 deleted, rescued, and site-specific mutagenesis mutants in an A. hydrophila clinical pathogen strain namely LP 2 that was isolated from diseased European eel (Anguilla anguilla) with a considerable OXY resistance characteristic (A and B). When compared to WT A. hydrophila LP-2 strain, the deletion of lpaha1 significantly increased the MBC of OXY four times. The plate MBCs assays showed that K57R, K187R, and K197R of LpAha1 derivatives significantly increased OXY resistance, and their MBCs to OXY were four-fold higher than the corresponding K57Q, K187Q, and K197Q derivatives (C–H). These findings further confirmed that Aha1 plays an important role in bacterial OXY resistance and provides a novel mechanism that Kac modifications may affect the uptake of OXY into the cell by regulating the Kac modification status on specific lysine sites of this protein.Combined with the clues mentioned above for the phenotypes of Aha1 against OXY stress, we were interested in the effect of Kac modifications of this porin protein on OXY resistance. First, the seven Kac sites were mutated to arginine (R) or glutamine (Q) to mimic lysine deacetylation and acetylation modifications, respectively , A–F. Weivatives G. Besideteristic , A and Bivatives , C–H. ThA. hydrophila Aha1 using MSA and 3D structure prediction. As a member of the porin family, Aha1 shares a considerably high identity with E. coli OmpF, PhoE, and OmpC. Those 3D structures as homotrimers were well documented, and their roles as antibiotic uptake channels were well characterized. According to the X-ray crystallographic analysis of 3D structure, E. coli OmpF is a homotrimer. The traditional prion monomer has a series of 16 antiparallel β-strands connected by eight external loops (L) and eight periplasmic turns (T) (A) . Except for K164, all other Kac sites of Aha1 were downregulated in the OXY resistance strain. Since the important role of Kac/lysine deacetylation modifications on K57, K187, and K197 sites of Aha1 in OXY resistance, the Kac modifications of Aha1 on these three sites were highlighted in its 3D predicted model in monomer and homotrimer (C and D). The K57, K187, and K197 sites were all located in the extracellular loops. Moreover, The PyMOL plugin of Caver 3.0.3 was employed to predict the effect of Kac states on the channel of this porin. As showed in We then asked about the OXY resistance mechanism mediated by Kac sites on urns (T) . Loop2 purns (T) A 32). W. WA. hydin loop4 B. Exceptmotrimer , C and DA. hydrophila against OXY stress, we then asked whether this pattern works only for OXY antibiotic or multidrugs resistance. Thus, the antibiotic susceptibility of the aforementioned seven Aha1 derivatives were further measured against a total of 31 antibiotics in this study  to the same MBC folds as Δaha1. Although it belongs to β-lactams as well, it is a surprise that the K57R mutant did not affect the MBCs to penicillins, including AMP, PG, and CAR and that the K187R mutant did not affect the MBCs to CAR. Also surprising was the finding that K166Q decreased the MBCs to polypeptides (CS and PB) by two-fold, whereas other Aha1 mutants, including Δaha1, did not affect polypeptides susceptibility, indicating the complicated Kac regulation of multidrugs resistance. Moreover, the multiple site-directed mutagenesis generated K57, K187, and K197 mutants displayed the same resistance behaviors as their corresponding single site-directed mutagenesis mutants. Therefore, the multiple site-directed mutagenesis mutants did not reveal a superposition effect for antibiotic resistance.Moreover, the K57R, K187R, and K197R mutants of tet(X) from the environmental microbiota can effectively inactivate TETs by enzymatic alteration mechanism, which confer high-level resistance to all TETs , A0KJP9 (AHA_1969), and A0KQZ1 (AHA_4275) did not affect the antibiotic susceptibilities of A. hydrophila against TETs, including the OXY antibiotic. However, the deletion of aha1 (AHA_2785) did significantly increased the MBC of A. hydrophila four to eight fold to OXY and TET  were downregulated in the OXYlic flux , 48. Theogenesis . Meanwhiogenesis , 51. TheR strain, suggesting that the Kac modification on AMR proteins may affect their roles in antibiotic resistance. We were then interested in the impact of Aha1 Kac modification on OXY resistance because Aha1 has seven downregulated Kac sites in the OXYR strain, and the deletion of the aha1 gene significantly increased the antibiotic resistance against OXY. Interestingly, the K57R, K187R, and K197R mutants significantly increased their MBCs to OXY eight fold, similar to the MBC of Δaha1 to OXY. These results indicate that Aha1 controlled the porin permeability for OXY uptake mediated by Kac or deacetylation modification, and the OXY accumulation assay confirmed this hypothesis.Further analysis showed that at least 71 Kac peptides from 39 proteins belonging to different resistance mechanism classifications in the CARD database were altered in the OXYA. hydrophila. Therefore, we further measured the MBCs of these derivatives to a total of seven types of antibiotics on a large scale. To our surprise, the Δaha1 and its K57R, K187R, and K197R mutants increased their MBCs up to 128-fold to many kinds of antibiotics, including tetracyclines and β-Lactam antibiotics; notably, for the resistance to oxacephem (RAO) and ETP, the MBCs were 64- and 128-fold, respectively. These results further confirmed that Aha1 and its Kac states were involved in bacterial multidrug resistance. Interestingly, the effects of the corresponding Aha1 derivatives  were the same as Δaha1, whether as single point or multipoint mutants, indicating that there are no synergistic or superposition effects among them. Moreover, we also observed that Δaha1 increased resistance to quinolone antibiotics but showed no differences among the series of Aha1 site-directed mutagenesis–generated mutants, suggesting the existence of other active sites in Aha1 besides the K57, K187, and K197 sites that are involved in the uptake of some specific antibiotics.Based on the fact that the Aha1 homolog in the CARD database is classified as a general bacterial porin with reduced permeability to β-lactams, we hypothesized that the Aha1 and its Kac states might affect bacterial multidrug resistance in E. coli OmpF, OmpC, PhoE, and A. hydrophila Aha1, the K57, K187, and K197 residues of Aha1 were not evolutionarily conservative residues, probably because the extracellular part of the protein undergoes more rapid mutational alterations to adapt to environments , ampicillin NH3+ and OmpF Y32 residue (corresponding to the nearby of K57 in Aha1), that is helpful for ampicillin to bind to the extracellular pore vestibule of OmpF, oriented perpendicular to the pore axis. Moreover, the different charge features of β-Lactam antibiotics  display different binding patterns with OmpF for antibiotics uptake , which is a reminder that some AMR proteins could regulate bacterial antibiotic resistance by PTMs alone that escape the popular gene sequencing technologies used in the clinic.Overall, our current data provided evidence to better understand how the Kac modification states on the extracellular loops of Aha1 porin affect outer membrane permeability and then trigger bacterial antibiotic resistance and may be helpful in the development of new antibiotic therapy strategies. In addition, although K to R (mimicking lack of Kac) and K to Q (mimicking presence of Kac) are useful models that have been well-used in many recent researches, the mutants remain imperfect since they are not real corresponding lysine modifications. That may partial explain some negative results obtained with these mutants.via the PRIDE partner repository with the dataset identifier PXD028447.Raw mass spectrometry proteomics data for whole cell proteins and Kac peptides have been deposited to the ProteomeXchange Consortium This article contains supplemental data.The authors declare no competing interests.


Cyclotella sp. HB162 had the highest dry biomass of 0.46 g/l and relatively high triacylglycerol (TAG) content of 237.4 mg/g dry biomass. The highest TAG content of 246.4 mg/g dry biomass was obtained in Fistulifera sp. HB236, while Nitzschia palea HB170 had high dry biomass (0.33 g/l) but relatively low TAG content (105.9 mg/g dry biomass). N. palea HB170 and Fistulifera sp. HB236 presented relatively stable growth rates and lipid yields under fluctuating temperatures ranging from 28 to 35°C, while Cyclotella HB162 maintained high lipid yield at temperatures below 25°C. The percentage of saturated fatty acids and monounsaturated fatty acids in all the 6 strains was 84-91% in total lipids and 90-94% in TAGs, which makes them the ideal feedstock for biodiesel.Screening suitable strains with high temperature adaptability is of great importance for reducing the cost of temperature control in microalgae cultivation, especially in summer. To obtain high temperature-tolerant diatoms, water samples were collected in summer from 7 different regions of China across the Northeast, North and East. A total of 731 water samples was collected and from them 131 diatom strains were isolated and identified based on the 18S rRNA sequences. Forty-nine strains out of the 131 diatoms could survive at 30°C, and 6 strains with relatively high biomass and lipid content at high temperature were selected and were found to be able to grow at 35°C.  With the exhaustion of fossil fuels and the aggravation of environmental problems from fossil fuels usage, renewable and environmentally friendly energy sources are being explored . BiodiesNannochloropsis spp., Chlorella species including Chlorella protothecoides and Chlorella vulgaris, diatoms Cyclotella cryptica and Phaeodactylum tricornutum, and so on [Screening high quality microalgae and determining the microalgae oil production efficiency are the basis and core of microalgae energy development . Until nnd so on -13. Diatnd so on . They arnd so on . Diatomsnd so on .Growth rate, lipid productivity and fatty acid composition of diatoms are influenced tremendously by temperatures, thus temperature adaptation is of great importance for screening strains to ensure high lipid productivity . DiatomsCyclotella sp. HB162, Fistulifera sp. HB236 and Nitzschia palea HB170, showed good adaptation to high or fluctuating temperatures and could serve as potential feedstock for outdoor biodiesel production.In this study, diatom strains isolated from freshwater in seven provinces of China were screened by thin-layer chromatography (TLC) for a rough identification of high lipid productive strains on the basis of the relative TAG content. High oleaginous diatoms with high temperature tolerance were cultivated at different temperatures. Their growth, total lipid and TAG contents, and fatty acid compositions were evaluated to obtain good diatom strains with inherent tolerance to high or fluctuating temperatures. Three diatom strains, 2 s) continuous illumination before use.Water samples were collected in summer (mainly from June to September) from reservoirs, rivers, lakes and ponds in the provinces of Liaoning (LN), Jilin (JL), Zhejiang (ZJ), Heilongjiang (HLJ), Hebei (HB), Shanxi (SX) and the Inner Mongolia Autonomous Region (NM) of China. The location, pH, temperature and electrical conductivity of the water bodies were recorded . Algal cUlothrix zonata (Z47999) and Gloeotilopsis planctonica (Z28970) as the outgroup.All isolated microalgae strains were initially examined by microscopy and those diatom strains were then identified by 18S rRNA sequence analysis. Total genomic DNA of the diatom strains was extracted according to the manufacturer’s instructions for a glass milk DNA isolation kit . The 18S rRNA gene amplification was carried out, using the general primers 18S-1 (5’-tggttgatcctgccagtagtc-3’) and 18S-2 (5’- tgatccttctgcaggttcacc-3’), by polymerase chain reaction (PCR) as previously described . Agarose5 cells/ml) at 22°C under continuous illumination of 50 μmol photons/(m2 s) for 15 d, then total lipids were extracted from the cell pellets and analyzed using TLC for the comparison of the TAG content. TLC was performed as described by Reiser and Somerville [All diatom strains were grown in 50 ml flasks containing 20 ml CSi liquid medium  at 28°C for 10 days. Cell density and size were determined under microscope using a Malassez chamber (0.2 mm). For comparison, relative cell biomass was obtained based on cell density and cell area, and the relative lipid yield was calculated from relative cell biomass and the total lipid content was measured as above.For preliminary screening of high temperature-tolerant diatom, all strains were grown at 30°C with the starting density of 2× 10Cyclotella sp. HB162, Fistulifera sp. HB236, Nitzschia palea HB170, Nitzschia sp. ZJ183, Nitzschia sp. ZJ184 and Nitzschia sp. HLJ48) were selected and cultivated in glass culture tubes containing 180 ml of CSi medium with aeration by sterile air under continuous illumination of 70 μmol photons/(m2 s) at different temperatures for 10 days. Cell density was determined by cell counts using the Malassez chamber every 2 days and specific growth rates (μ) were calculated according to μ = (lnXt − lnX0)/t, where X0 is the initial cell density and Xt is the cell density after t days. Algal dry biomass was determined by the weight method at the end of the experiment.Six diatom strains  was used as an internal standard.Total lipids of 6 representative diatom strains from 25°C cultures were obtained as described above. TAGs were separated by TLC and the band of TAGs on the plates was scraped out and then extracted with chloroform. Total lipids and TAGs were methylated with sulfuric acid-methanol as described before . Gas chrAchnanthidium, 1 from Craticula, 7 from Cyclotella, 23 from Fistulifera, 1 from Gomphonema, 2 from Lemnicola, 65 from Nitzschia, 3 from Pinnularia, 10 from Sellaphora and 1 from Synedra based on the morphological characteristics and 18S rRNA gene sequence analysis.Diatoms are widely distributed in freshwater, and are remarkably dominant species in the cold season during winter and early spring . TherefoNitzschia was divided into two monophyletic groups, which is consistent with the previous report of Lundholm, et al. [Nitzschia sigma, N. thermalis, N. palea and isolated strains HLJ48, ZJ183, ZJ184, HB170, and etc. , 51 from the North  and 41 from the East (Zhejiang Province). The ecological habitat information on all the strains except 7 diatoms was shown in Although 246 and 84 water samples were collected from Zhejiang and Jilin, 41 and 7 diatom strains were obtained from these samples respectively. Only 24 from the former and 19 from the latter were collected at temperatures lower than 25°C with the highest record of 38 and 28°C, respectively. However, 20, 12, and 11 diatom strains were obtained from 58, 50, and 51 water samples collected from Liaoning, Heilongjiang and Inner Mongolia, whose average temperatures were around 20°C. In particular, the lowest temperature was 11°C in Liaoning, 19°C in Inner Mongolia, and 20°C in Heilongjiang. It is indicated that diatoms have a preference for low temperatures and thus thrive in cold waters , 31. In Cyclotella strains and 5 Nitzschia strains, were isolated from acidic waters. This indicates that most of the diatoms possess efficient CO2 concentrating mechanisms (CCMs), and are able to assimilate bicarbonate directly or via extracellular carbonic anhydrases [Nitzschia accounted for a significant proportion (over 50%) in the isolated diatoms, indicating a wide distribution in freshwater habitats. Among them, 19 strains belong to N. palea, which was present in water samples of all the 7 regions. N. palea has been found to be a widely distributed freshwater diatom in various lotic and lentic habitats [Nitzschia strains inhabited water with a temperature range from 25 to 30°C and pH range from 7.5 to 8.5.Most of the isolated diatoms (114 strains) prefer alkaline waters, and only 7 strains, including 2 hydrases . Strainshabitats . About hNitzschia and Fistulifera strains amounted to over 40%, which makes our screening for high oleaginous diatoms possible.More than 40% of the isolated diatom strains boasted a total lipid content of over 30%, suggesting diatoms are a good candidate for lipid production. Diatoms mainly store carbon and energy in the form of lipids, especially under stress . The lipFistulifera sp. SX5, Fistulifera sp. SX100, Nitzschia sp. SX21, Cyclotella sp. SX119 and Achnanthidium sp. SX85) from Shanxi, 4 strains  from Hebei, 2 strains (Fistulifera sp. NM19 and Fistulifera sp. NM82) from Inner Mongolia, one strain (Nitzschia sp. HLJ64) from Heilongjiang, 4 strains  from Zhejiang, 2 strains (Nitzschia sp. JL39 and Cyclotella sp. JL92) from Jilin, 4 strains  from Liaoning showed relatively higher TAG contents. Especially, strains HB235, HB 236, NM19, ZJ69 and LN106 had rather high TAG contents.TAGs, which can be converted via a transesterification process, are believed to be superior to other lipids for biodiesel production . In thisNitzschia, 9 Fistulifera, 6 Sellaphora, 3 Achnanthidium, 1 Cyclotella and 3 unknown species. Twenty-nine strains out of the 49 high temperature-tolerant ones were isolated from waters at above 25°C, and among them 7 were from waters at above 30°C. Although diatoms prefer low temperatures, screening diatom strains with high temperature tolerance in hot seasons is also feasible. For example, Nitzschia sp. ZJ183 and Nitzschia sp. ZJ184 were isolated at around 30 °C, and Cyclotella sp. HB162, N. palea HB170 and Fistulifera sp. HB236 were from waters of 23-26°C. However, Nitzschia sp. HLJ48 was isolated from waters below 20°C, and it could also survive at 30°C.After preliminary experiments, a total of 49 isolated diatom strains could survive at 30°C, and they were designated as high temperature-tolerant diatoms. The 49 strains included 26 Nitzschia  showed higher relative cell biomass, while Fistulifera sp. HB236 and Nitzschia sp. JL83 had the highest total lipid content in all the 49 diatoms except for the two unidentified strains LN105 and LN106. Genera Achnanthidium, Craticula and Sellaphora showed much lower lipid yields compared with the others. In contrast, thirteen Nitzschia strains showed much higher relative lipid yields, and strain HB170 had the highest relative lipid yield. HB236 had the highest lipid yield in all 9 high temperature-tolerant Fistulifera strains, and HB162 was the only high temperature-tolerant strain of genus Cyclotella. Nitzschia sp. ZJ184 had a relative low lipid yield among all the high temperature-tolerant Nitzschia strains, and was used as a control in this study. Therefore, Fistulifera sp. HB236, Cyclotella sp. HB162 and four Nitzschia strains  were selected for further detailed research.Growth and lipid yields of the 49 high temperature-tolerant diatoms were compared at 28°C . StrainsFistulifera sp. HB236 and 4 Nitzschia strains not only grew best but also showed the highest lipid content at 25°C, however the optimum growth temperature of Cyclotella sp. HB162 was 28°C, though its lipid content was sharply decreased at that temperature  but the best lipid production occurred in winter (2-17°C). Fistulifera solaris was well adapted to high-temperature environments, and even in summer when average daily ambient temperature exceeded 30°C and temporarily reached 42°C, steady biomass (0.31-0.57 g/l) and lipid content (18%) of the microalgae were observed under outdoor mass cultivation [N. palea HB170 and Fistulifera sp. HB236 presented a small variation of specific growth rates within the scope of 28~35°C, thus having excellent temperature adaptability.Six selected diatoms were cultivated at temperatures ranging from 22 to 40°C to observe their response to temperatures . All strperature . Cyclote at 30°C . Althougg et al.  revealedtivation . In thisCyclotella sp. HB162 had the highest dry biomass of 0.46 g/l and relatively high TAG content of 237.4 mg/g dry biomass. The highest TAG content of 246.4 mg/g dry biomass was obtained in Fistulifera sp. HB236. The cell sizes (areas) of HB162 were the biggest (235 ± 18 μm2) among all high temperature-tolerant diatoms, and were as much as 7 times bigger than that in strain ZJ183 (28 ± 2 μm2). Cell sizes (areas) of strains HLJ48, HB236 and ZJ184 were 34 ± 3, 36 ± 6, and 39 ± 3 μm2, and that of strain HB170 was 84 ± 8 μm2. Thus, the dry biomass at day 10 was much lower in strains ZJ183 (0.12 g/l), ZJ184 (0.17 g/l), and HLJ48 (0.19 g/l). Since the highest cell density was observed in strain HB236 at 25°C, the relatively high dry biomass (0.28 g/l) was also obtained in this strain. N. palea HB170 had high dry biomass (0.33 g/l) but relatively low TAG content (105.9 mg/g dry biomass). TAG contents in strains ZJ183, ZJ184 and HLJ48 were 70.7, 95.5, and 135.9 mg/g dry biomass. Therefore, the highest TAG yield of 109.4 mg/l was obtained in strain HB162 and the lowest of 8.3 mg/l in strain ZJ183.Amphora, whose fatty acid profile is different from that of other diatoms, reported that the PUFAs were higher at the higher temperature [Besides high lipid content, an adequate fatty acid profile is indispensable in microalgae as a renewable feedstock for biodiesel production , 19. Theperature , 47. AltN. palea HB170 and Fistulifera sp. HB236 presented relatively stable growth rate and TAG yield under fluctuating temperatures ranging from 28 to 35°C, while Cyclotella HB162 can be used as an oil source at temperatures below 25°C. Our results demonstrate that strains HB170 and HB236 are highly desirable candidates for outdoor cultivation, while strain HB162 may be a better choice under a relatively low temperature.In conclusion, diatoms store excess photosynthate as lipids instead of carbohydrates, and are recognized as very promising microalgae for biofuel production. However, diatoms have a preference for low temperatures and usually could not survive at high temperatures, making them unsuitable for outdoor cultivation. In this study, 131 diatom strains were isolated from China in summer, and among them, 49 strains could survive at 30°C. Six screened diatoms with relatively high biomass and lipid content out of the 49 strains could grow at 35°C, but showed different responses in growth and TAG yield to high temperatures. http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Vascular endothelial cells are crucial mediators of inflammation during infectious diseases, due to their ability to produce lipid-based inflammatory mediators and facilitate leukocyte migration and translocation to infected tissues. Mastitis is the costliest infectious disease in North America, with over two billion dollars in annual costs due to loss of milk production, medical treatment, and potential loss of the animal. Infections caused by coliform bacteria are particularly deleterious, causing a negative impact on cow well-being and a high mortality rate. Dysfunction and breakdown of the endothelial barrier is a key part of the pathology of coliform mastitis. The endocannabinoid system (ECS), shown to modulate inflammatory responses of vascular endothelial cells in humans and rodents, may be a novel target for inflammatory modulation in dairy cows. The endocannabinoid (EC) arachidonoylethanolamide (AEA) is a potent anti- or pro-inflammatory mediator in endothelial cells, depending on location, timing, and concentration. We hypothesized that elevated AEA during LPS challenge will impair endothelial barrier integrity via increased production of reactive oxygen species (ROS) and activation of apoptotic pathways. Challenge of bovine aortic endothelial cells (BAEC) with 25 ng/mL lipopolysaccharide (LPS) for 8 h induced AEA synthesis, increased expression of cannabinoid receptor 1 and 2 (CB1/2) and the AEA synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), while decreasing gene expression of the AEA degradation enzyme fatty acid amide hydrolase (FAAH). Trans endothelial resistance (TER), measured through electrical resistance across the monolayer, increased 2 h after 0.5 µM AEA treatment and decreased with 5 µM AEA, compared to LPS alone. Addition of AEA to BAEC challenged with LPS induced mitochondrial dysfunction via increased ROS production, cytochrome-C release, and activation of caspase 3/7. Antagonism of CB1 by 1 µM AM251 ameliorated AEA induced ROS production and cytochrome-C release. Addition of AM251 also eliminated 2 h TER increase and improved TER following 5 µM AEA. Doses of 0.5, 1, and 5 µM AEA delayed endothelial barrier recovery, which was eliminated by the addition of AM251. Mitochondrial dysfunction and activation of apoptotic pathways in response to AEA treatment during LPS challenge of BAEC may act to delay inflammatory resolution and contribute to endothelial dysfunction. Infection of the mammary gland, or mastitis, is one of the diseases with the greatest impact on animal welfare and dairy cow health in North America ,2. InfecBreakdown of the epi- and endothelial monolayers is part of the pathology of coliform mastitis in dairy cows . StimulaClosely tied to oxylipids, are a class of potent lipid-based inflammatory mediators called endocannabinoids (EC). As part of the endocannabinoid system (ECS), EC and dedicated cannabinoid receptors-1 and -2 (CB1/2), are involved in several physiological processes, such as energy homeostasis, cellular metabolism, pain, and inflammation . FluctuaTherefore, we set out to characterize the effects of AEA on BAEC function and barrier integrity, during LPS challenge. We demonstrate that the ECS is not only present in BAEC, but that expression of key genes is altered by LPS challenge. We also demonstrate that CB1 activation by AEA concentrations as low as 0.5 µM, are detrimental to BAEC function and monolayer barrier integrity, during LPS challenge. Lastly, we demonstrate that AEA induces ROS production and the likelihood of oxidative stress in BAEC without LPS challenge, while apparent mitochondrial dysfunction results from AEA treatment of BAEC during LPS challenge, leading to activation of apoptotic pathways.High performance liquid chromatography (HPLC) grade acetonitrile, HPLC-grade methanol, formic acid, glycerol, transferrin, insulin, heparin, sodium selenite, Hanks buffered salt solution (HBSS) powder, collagenase, lipopolysaccharide (LPS) (O111:B4), 156 EDTA, and tri-phenylphosphine were purchased from Sigma-Aldrich . Deuterated and nondeuterated isoprostane standards, indomethacin, 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), arachidonoylethanolamide (AEA) and AM251 were purchased from Cayman Chemical . Butylated hydroxy toluene (BHT) was purchased from Acros . Fetal bovine serum was purchased from Hyclone Laboratories, Inc.  and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and dimethyl sulfoxide (DMSO) were purchased from Corning Inc. . HAM’s F-12k was purchased from Irvine Scientific . Antibiotics/antimycotics, trypsin-EDTA, bovine collagen, and ProLong-Gold antifade were from Life Technologies . Predesigned and custom-made bovine TaqMan primers were purchased from Applied Biosystems . 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Molecular Probes , and von Willebrand’s factor was purchased from Agilent Technologies .g for 10 min at 15 °C. Resulting cellular pellets were plated in T25 cell culture flasks until confluent in Ham’s F12K medium containing 10% fetal bovine serum, 10 mM HEPES buffer, 0.25% sodium bicarbonate, 2 mM L-glutamine, 1% 1:1 antibiotic:antimycotic, 100 μg/mL heparin, 10 μg/mL insulin, 5 μg/mL transferrin, and 40 ng/mL sodium selenite. Cells were detached via incubation with 0.05% trypsin, diluted serially, and plated in 96-well plates. To exclude fibroblasts and other cell types, wells containing only colonies derived from a single cell were selected for propagation. Wells were selected by typical endothelial cobblestone morphology for further propagation and confirmed by von Willebrand factor staining. Cells were frozen at passage 3, grown to passage 6 for RNA isolation, representing the earliest passage at which cells could realistically be used for assays, and discarded after passage 9.Endothelial barrier function was assessed using primary bovine aortic endothelial cells collected as described previously . Briefly5 cells/well in 96 well plates, and 1 × 106 cell/well in 6 well plates, and grown to confluency in 5% FBS Ham’s F12k media containing 20 mM HEPES, antibiotics and antimycotics , heparin (100 µg/mL), insulin (10 µg/mL), transferrin (5 µg/mL), and sodium selenite (10 ng/mL), for 18 h before media was changed to serum free media for 2 h  was added for cell lysis. Buffer RLT was collected and stored at −20 °C for no longer than one month before processing. Extraction of RNA occurred utilizing a Promega Maxwell RSC Instrument following the manufacturer’s protocol. RNA quantity and quality were evaluated using a NanoDrop ND-1000 spectrophotometer .For cDNA generation, RNA was diluted with nuclease-free water to standardize all samples. A master mix of 10× reverse-transcription buffer, 25× dNTP, 10× random primers, Multiscribe reverse transcriptase, RNase inhibitor, and RNase nuclease-free water from a high-capacity cDNA reverse-transcription kit with RNase inhibitor , was added at an equal volume to diluted RNA. Samples were placed in a PTC-200 Peltier Thermo Cycler , which ran as follows: stage 1, 25 °C for 10 min; stage 2, 37 °C for 2 h; stage 3, 85 °C for 5 min; stage 4, hold at 4 °C.−ΔΔCT. Efficiency (E) of PCR was calculated based on the slope of a standard curve of tissue samples, serially diluted. The slope of the standard curve was used to calculate E, using the equation E = (10−1/slope − 1) × 100.Real-time PCR was carried out with predesigned TaqMan primers and FAM-MGB probes . All primers and probes were used in a 7500 Fast Real-time PCR system (Applied Biosystems). Genes were evaluated in triplicate with 2× TaqMan Gene Expression Master Mix (Applied Biosystems), 20× Custom TaqMan Gene Expression Assay Mix (Applied Biosystems), sample cDNA , and nuclease free water for a total of 10 μL per reaction well. A 20× pre-designed TaqMan Gene Expression Assay for RPS9 was used as endogenous control (Applied Biosystems). Thermal cycling conditions for the Fast 2-step PCR system were as follows: stage 1, 95 °C for 20 s; stage 2, 95 °C for 3 s; stage 3, 60 °C for 30 s, with 40 cycles of stages 2 and 3. Data were recorded and compiled using ExpressionSuite Software version 1.0.4 (Applied Biosystems) and analyzed using DataAssist Software version 3.01 . Data from in vitro cell samples, having no standard cell type for comparison, are expressed as 2Quantification of ROS production was carried out using the commercially available OxiSelect fluorometric ROS assay . Briefly, BAEC were cultured in 96-well, black walled and clear bottom plates following the experimental design and the assay was carried out following manufacturer’s recommendations. Cells were preloaded with DCF-DA at 100 μM in serum-free media for 15 min before treatments were added. The assay utilizes a non-fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), which is cleaved by cellular deacetylases and is oxidized by ROS to yield the fluorescent and stable 2′,7′-Dichlorodihydrofluorescein (DCF) product. Temporal ROS production was determined by measuring fluorescence using a BioTek Synergy H1 plate reader  at 480 nm excitation and 530 nm emission wavelengths. Results were analyzed as the fold change in fluorescence signal over untreated controls.Evaluation of AEA effects on viability and cytotoxicity of BAEC was carried out using commercially available assay kits from Promega . Briefly, for multiplex evaluation of viability and cytotoxicity, BAEC were cultured for 18 h in 96-well white wall, flat bottom plates and treated following the experimental design. Viability was evaluated using the Promega CellTiter-Glo Assay, following the manufacturer’s recommendations. The assay is based on the principle that the amount of ATP generated is proportional to the number of viable cells. Luminescence was read on a BioTek Synergy H1 plate reader . A complimentary cytotoxicity assay from Promega was employed as a multiplex assay on the same plate and cells as the CellTiter-Glo assay.Quantification of cytochrome-C released in response to AEA treatment in BAEC challenged with LPS was carried out using the Promega Cytochrome-C Assay . Briefly, BAEC were cultured in white 96-well plates following the experimental design and the assay was carried out following manufacturer’s recommendations. Luminescence was read on a BioTek Synergy H1 plate reader .Effect of AEA on apoptosis via caspase 3/7 of BAEC challenged with LPS was determined via a commercially available Promega Caspase-Glo 3/7 kit . Briefly, BAEC were plated in a white flat bottom 96-well plate and treated according to the experimental design, and the assay was carried out according to the manufacturer’s recommendations. Caspase activation of the substrate releases aminoluciferin and subsequent interaction of the free aminoluciferin with luciferase results in a luminescent signal proportional to caspase 3/7 activity. Luminescence was read on a BioTek Synergy H1 plate reader .g for 45 min at 4 °C and the supernatant was extracted in Phenomenex 8B S100 FCH extraction cartridges. After determining that cell pellet hydrolysis did not change detection of target metabolites, the hydrolysis step was not included in processing samples from subsequent experiments. After thawing, samples were centrifuged , and combined with HPLC-grade water and formic acid to achieve 20% methanol and maintain 0.1% formic acidification, respectively. Samples were extracted with SPE cartridges  preconditioned with methanol followed by HPLC-grade water, washed with 20% methanol solution and cartridges were dried for 15 min under full vacuum. A 7.5 mL 1:1 mixture of methanol and acetonitrile was used to elute extracted samples from the cartridges into glass tubes pre-coated with 10 μL of 20% glycerol in methanol followed by a 30 s drying under full vacuum. Samples were dried in a Savant SpeedVac  with an initial heating phase at 45 °C, residues suspended in 150 μL of a 4:1 (v:v) methanol and water mixture, and centrifuged . The supernatant (120 μL) was transferred to an autosampler vial with a low volume insert and stored at −80 °C before LC–MS/MS analyses.Cell pellet samples collected from cell culture experiments, previously stored at −80 °C, were thawed while on ice and protected from light. Initially, cell pellet samples were hydrolyzed by incubating with a 3 M potassium hydroxide solution at 45 °C for 45 min, allowed to cool to room temperature before being acidified with hydrogen chloride to a pH range of 2–3. The cell pellet samples were centrifuged at 4816× Details of LC/MS/MS analysis of isoprostanes are described in Mavangira et al., 2016 . In shorThe details of LC/MS/MS analysis for endocannabinoids are described in Williams et al., 2007 . In shorFor assessment of endothelial barrier integrity, BMEC were plated on bovine collagen-coated 96-well plates with gold electrodes and cultured for 18 h. Electric currents passing through the monolayer were continuously measured by the Electric Cell-Substrate Impedance Sensing Z-theta system . Approximately 2 h prior to treatment addition, media were changed to serum free media. Trans endothelial resistance (TER) across the monolayer was monitored up to 32 h after LPS addition. TER was normalized to the time point immediately prior to LPS addition.t-test using proc “t-test” procedure and one- or two-way ANOVAs were performed using the proc mixed procedure, with or without repeated measures where appropriate using the SAS9.4 software . A Tukey adjustment for multiple comparisons was performed with a p ≤ 0.05 considered significant when all-pairwise comparisons were performed; otherwise, a Dunnett’s post-hoc correction was performed for comparison of different treatments to the untreated control. For statistical analyses, undetected measures of variables in a treatment class were presented as undetected and assigned a zero value for statistical analyses.For statistical analyses all data sets were tested for normality and variance, before p = 0.0016, p = 0.0003, and p = 0.0010, respectively), whereas expression of FAAH decreased with LPS challenge compared to the media control (p = 0.0010). Addition of 0.5 µM AEA did not alter gene expression of CB1/2, NAPE-PLD, or FAAH, with and without 25 ng/mL LPS. . Extra- and intracellular AEA concentrations did not change with addition of 0.5 µM AEA without LPS compared to the media only treatment. In the presence of 25 ng/mL LPS, 0.5 µM AEA does not alter extra- or intracellular AEA concentrations compared to the LPS control.Analysis by RT-qPCR revealed that gene expression of CB1/2 and NAPE-PLD was elevated with LPS challenge (/mL LPS. a–d. Expop = 0.0457). Treatment concentrations of 1 and 5 µM AEA were detrimental to BAEC TER. Addition of 1 µM AEA resulted in decreased TER at 28, 29, 30, 31, and 32 h compared to the LPS control , and with 5 µM AEA, TER was decreased at 10, 11, and 12 h , and again at 25, 26, 27, 28, 29, 30, 31, and 32 h  compared to the LPS control.Assessment of the trans-endothelial resistance (TER) via ECIS showed that without LPS pre-treatment, 0.5, 1, and 5 µM AEA did not alter barrier integrity compared to the media control . In BAECp < 0.05). Addition of 1 µM AM251 to 0.5 µM AEA increased barrier integrity over the LPS control at 20 h in BAEC pre-treated with LPS (p = 0.0460).Treatment with 1 µM of the CB1 antagonist AM251 eliminated the 2 h TER increase associated with 0.5 µM AEA treatment in BAEC challenged with LPS a. Startip < 0.05). Additionally, TER increased with 1 µM AEA and AM251 over the LPS control at 13 h (p = 0.0429) and again from 17 to 29 h (p < 0.05). Addition of 1 µM AM251 to 5 µM AEA increased TER of LPS pre-treated BAEC over 5 µM AEA only, starting at 9 h through to the 32 h time point (p < 0.05). Lastly, treatment with 5 µM AEA and 1 µM AM251 showed greater TER compared to the LPS control from 12 to 29 h (p < 0.05). By itself, 1 µM AM251 did not alter TER, with and without LPS , while VCAM-1 gene expression did not change with any AEA treatment concentration compared to the LPS control. However, in BAEC pre-treated with LPS gene expression of ICAM-1 was elevated following 4 h of 5 µM AEA treatment compared to 0.5 µM AEA (p < 0.0001). Similarly, expression of VCAM-1 in response to 4 h of 5 µM AEA treatment was greater than that of 0.5 and 1 µM AEA treatments . Expression of ICAM-1 decreased with the addition of 1 µM of the CB1 antagonist AM251 to 0.5 µM AEA, compared to the 0.5 µM AEA only treatment (p = 0.0495). Expression of VCAM-1 was not altered by 0.5 µM AEA and 1 µM AM251. The remaining time points for ICAM-1 and VCAM-1 gene expression can be found in the RT-qPCR analysis of gene expression of ICAM-1 and VCAM-1 in BAEC not challenged with LPS was not altered by 0.5, 1, and 5 µM AEA treatment doses at 4 h, compared to the media control a,b. In Bp = 0.0055) (p = 0.0108) and addition of 1 µM AM251 increased ATP production compared to the 5 µM AEA only treatment (p = 0.0029).AEA concentrations of 0.5 µM elevated ATP production in BAEC challenged with 25 ng/mL LPS compared to LPS alone ( 0.0055) a. Additip = 0.0156) (p = 0.0045).Cytotoxicity was not affected by 0.5 and 1 µM AEA, but increased with 5 µM AEA compared to the LPS control ( 0.0156) b. Additip = 0.0185, p < 0.0001, and p = 0.0001, respectively) (p = 0.0115) and addition of 1 µM AM251 to 0.5, 1, and 5 µM AEA reduced cytochrome-C release compared to the corresponding AEA only treatments .Cytochrome-C release from the mitochondria increased with all AEA doses used  compared to the LPS control (ctively) c. The CBp = 0.0004, p < 0.0001, and p < 0.0001, respectively) .Activation of caspase 3/7 increased with the addition of all three concentrations of AEA  compared to the LPS control (ctively) d. Additip < 0.0001) (p = 0.0480). Interestingly, addition of 1 and 5 µM AEA increased ROS production without LPS compared to the untreated control   , whereas IsoP production of 0.5 µM AEA was unchanged. The remaining time points for ATP-production, cytotoxicity, cytochrome-C release, caspase 3/7 activation, ROS, and IsoP production can be found in the Production of ROS is increased with LPS antagonism compared to the untreated control ( 0.0001) e. Increactively) f. Additip < 0.0001) (p < 0.0001). Production of ROS was dosage dependent and significantly elevated at 0.5 µM, 1 and 5 µM AEA compared to the media control  . Total IsoP concentrations were also elevated over media control with 0.5 µM AEA addition (p = 0.0002) (p = 0.0002). Total IsoP production is also dosage dependent, with 0.5 µM, 1 and 5 µM of AEA inducing IsoP production  (p = 0.0002), but only reduces total IsoP of 1 and 5 µM AEA .Concentrations as low as 0.5 µM AEA induce ROS production in BAEC ( 0.0001) a. Additictively) b. Additi 0.0002) c. Additictively) d. AdditiIn this study, we demonstrate that coliform mastitis endothelial barrier damage may include ECS activation as a mechanistic component. The activation of the endothelial endocannabinoid receptor CB1 mediates effects of AEA on endothelial barrier integrity and mitochondrial function. Addition of AEA altered gene expression of adhesion molecules, induced ROS production, increased cytochrome-C release, and activation of apoptotic pathways via caspase 3/7. However, treatment of endothelial cells with AEA without LPS challenge did increase ROS production and the likelihood of oxidative stress, but did not alter barrier function or activation of apoptotic pathways, indicating that inflammatory state and timing of AEA increase are crucial.Basal expression of CB1 and 2 in this study indicates the involvement of the ECS in non-inflammatory functions of BAEC. Non-inflammatory models utilizing AEA in non-bovine species support the involvement of the ECS in a variety of processes in vascular endothelial cells, such as regulation of vascular tone  and angiTreatment of BAEC with up to 5 µM AEA without LPS challenge did not alter TER, whereas in the presence of LPS even nanomolar concentrations altered TER. Lack of change in gene expression of ICAM and VCAM-1 with 0.5 and 1 µM AEA addition stands in contrast to decreased expression of the adhesion genes in human blood-brain barrier treated with AEA in an ischemia/reperfusion model . SimultaEven though ROS production was not significantly different with addition of AEA in this study, we did observe a numerical increase that was dose dependent, and addition of AM251 significantly reduced ROS production of 1 and 5 µM AEA, indicating that activation of the CB1 receptor by AEA does contribute to overall ROS production and possible mitochondrial dysfunction. Furthermore, addition of 1 and 5 µM AEA to LPS challenged BAEC increased IsoP production compared to the LPS control, which was ameliorated by the addition of AM251. Elevated IsoP production is an indicator for oxidative stress, or damage to healthy tissue by free radicals, such as ROS. Even though the effect of IsoP on bovine vascular endothelial cells remains elusive , increasThe compounding of increased AEA concentrations and CB1 expression may therefore lead to detrimental effects on barrier integrity and endothelial cell function following any additional AEA increase, for example by NSAIDs. Elevated AEA following NSAID treatment has been recorded in several models, in vivo and in vitro, and has been associated with analgesic and anti-inflammatory effects in several cell types. However, treatment of bovine mammary epithelial cells with the NSAID flunixin meglumine (FM), the only FDA approved NSAID for endotoxemia associated with acute coliform mastitis, significantly reduced TER for up to 10 h post treatment . ElevateInterestingly, 0.5 µM AEA treatment increased TER at 2 h but did not exhibit any changes in ICAM/VCAM expression or other parameters that were evaluated see . The incFuture studies should focus on the involvement of CB2 and non-CB receptors, such as TRPV1. Non-published data from this study shows that antagonism of CB2 alters BAEC barrier function independently of AEA addition, and the data was therefore not included in this publication. However, AEA is capable of activating CB2 and modifying the effects of CB2 activation by other agonists via allosteric modulation, particularly at supraphysiological concentrations greater than 5 µM . FurtherOverall, addition of AEA had detrimental effects on BAEC barrier integrity, elevated ROS production, leading to mitochondrial dysfunction highlighted by cytochrome-C release, and activation of apoptotic pathways, namely caspase 3/7. Reports on anti- and pro-inflammatory effects of AEA are numerous, and indication of pro-inflammatory effects in BAEC pre-treated with LPS are shown here. Nonetheless, further studies on timing and concentration dependent effects of AEA on BAEC function are needed, as activation of CB1 by AEA can reduce or increase ROS production, depending on cell type, timing of administration, and concentration.There are limitations to our study design and functional assays. Utilization of the ECIS as a functional assay of endothelial barrier function allows for real-time determination of barrier integrity and advanced modeling thanks to multiple frequency electrical resistance and capacitance measurements. However, the embedded electrodes impede imaging of the cells after completion of the ECIS assay. Therefore, the mechanism causing increases in TER at 2 h post 0.5 µM AEA could not be determined with the data presented in this study. Future studies should determine the morphological changes associated with CB1 activation by AEA.Increased AEA concentrations during coliform mastitis further deteriorates endothelial barrier integrity. Treatment of endothelial cells with AEA at physiological concentrations (0.5 µM) results in ROS production, increasing likelihood of oxidative stress and activation of apoptotic pathways. However, dynamic concentrations of cannabinoids and expression of related genes emphasizes the importance of timing of endocannabinoid treatment. Additional studies pertaining to the timing of concentration ranges of endocannabinoids and endocannabinoid related drugs are necessary to determine the true effect of AEA and other endocannabinoids on endothelial barrier function. Improved endothelial barrier function is a crucial facet of improving welfare and reducing mortality and cost associated with coliform mastitis.


However, little is understood about global changes in carbohydrate and energy-related metabolites during the transition from mature pollen grain to hydrated pollen, a prerequisite to pollen germination, in most plants, including Arabidopsis. In this study, we investigated differential metabolic pathway enrichment among mature, hydrated, and germinated pollen using an untargeted metabolomic approach. Integration of publicly available transcriptome data with metabolomic data generated as a part of this study revealed starch and sucrose metabolism increased significantly during pollen hydration and germination. We analyzed in detail alterations in central metabolism, focusing on soluble carbohydrates, non-esterified fatty acids, glycerophospholipids, and glycerolipids. We found that several metabolites, including palmitic acid, oleic acid, linolenic acid, quercetin, luteolin/kaempferol, and γ-aminobutyric acid (GABA), were elevated in hydrated pollen, suggesting a potential role in activating pollen tube emergence. The metabolite levels of mature, hydrated, and germinated pollen, presented in this work provide insights on the molecular basis of pollen germination.Pollen germination is an essential process for pollen tube growth, pollination, and therefore seed production in flowering plants, and it requires energy either from remobilization of stored carbon sources, such as lipids and starches, or from secreted exudates from the stigma. Transcriptome analysis from  Successful pollination in flowering plants is essential to fertilization and seed formation and is a key determinant of seed yield . Mature Arabidopsis  right after collection followed by centrifuging at 15,000 g for 1 min (4°C). For hydrated pollen and germinated pollen samples, in brief, pollen pellets were washed with 1 ml of liquid Pollen Germination Medium  before they were resuspended in 30 μl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). A 70 μm mesh was used to cover the pollen droplet to create a thin layer for optimal germination for each Petri dish. The Petri dishes were covered and placed in the dark for 45 min or 4 h and collected as hydrated pollen or germinated pollen, respectively. All pollen samples were washed by 1 ml ice-cold ddH2O three times before being stored in a -80°C freezer.For mature pollen samples, collected pollen grains were resuspended in 2 ml of ice-cold Pollen Isolation Buffer  kept in the dark for 10 min and washed twice using 1 ml ddH2O was added. The samples were then centrifuged at 14,000 rpm for 10 min to achieve phase separation and the upper aqueous phase, as well as the lower organic phase, were collected separately. The aqueous phases containing polar and non-polar metabolites were split into two equal parts and dried using a speed vacuum centrifuge . The two dried parts were re-suspended in 50 μL 80% (v/v) methanol, and 30% (v/v) methanol for metabolomics analyses using hydrophilic interaction chromatography (HILIC) and reverse phase chromatography, respectively. The organic phase was also dried using a speed vacuum centrifuge and re-suspended in 50 μL of 49:49:2 (v/v/v) mixture of acetonitrile:methanol:chloroform. All samples were filtered using a 0.8 μM PES membrane centrifuge filter  and transferred to a glass vial for injection into an LC-MS/MS system.Total metabolites from pollen were extracted using a phase separation method previously described  with sli2O (solvent A) and 10 mM ammonium bicarbonate in 95:5 (v/v) Acetonitrile: ddH2O (solvent B) and a flow rate of 15 μL/min. The following gradient was used for HILIC; 0-2 min at 100% B, 3 min at 85% B, 16 min at 50% B, 17 min at 30% B, 18 min at 30% B, 20 min back to 100% B and equilibration up to 30 min. Reverse-phase chromatography was performed using a Targa C18 (100 × 0.3 × 5 μm) column with the mobile phases, 0.1% formic acid in ddH2O (Solvent A) and 0.1% formic acid in Acetonitrile (Solvent B), and a flow rate of 15 μL/min. The gradient conditions used for the C18 method are as follows; 0-3 min at 2% B, 13 min at 100% B, 16 min at 100% B, 19 min at 2% B, and equilibration up to 30 min. For lipidomics, a custom-made C8 column (100 × 0.5 × 1.7 μm) from Higgins Analytical Inc.  was used with the mobile phases 1% 1 M ammonium acetate, 0.1% acetic acid in ddH2O (solvent A) and 1% 1 M ammonium acetate, 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol (solvent B), and a flow rate of 40 μL/min. The following gradient, 0–1 min at 55% B, 3 min at 75% B, 8 min at 89% B, 10 min at 99% B, 11 min at 99% B and 12 min at 55% B followed by equilibration up to 18 min, was used, which was modified from Three different chromatographic methods, including a HILIC, a reverse phased C18 and a reverse phased C8 columns, were used to attain a wide coverage of compound groups. The aqueous fraction of the extraction, which is expected to contain core and specialized metabolic intermediates, was used for HILIC and C18 chromatography, while the organic fraction was used for C8 based chromatography for the separation of lipids. An Eksigent Ekspert microLC 200-chromatography system  and a CTC Analytics Leap HTS PAL liquid handler hooked to a benchtop Q-Exactive Orbitrap MS  were used for all untargeted LC-MS analysis. HILIC separation was achieved using a custom made zic-pHILIC (100 × 0.5 × 3 μm) column obtained from Higgins Analytical Inc.  with the mobile phases, 10 mM ammonium bicarbonate in ddH5 and 100 ms, respectively. The heated electrospray ionization (HESI) source was operated with sheath gas, 15 arbitrary units; auxiliary gas, 5 arbitrary units; capillary temperature, 250°C; auxiliary gas heater temperature, 50°C; and S-lens RF level, 50. The spray voltage was 4.2 and 3.9 kV in positive and negative modes, respectively. One sample in each group was also used for the top 12 data-dependent acquisition experiments in both ionization modes to generate MS/MS datasets for compound identification. These experiments involved a full MS scan at 70,000 resolution, AGC target 5 × 105, maximum IT 100 ms and MS/MS scans at 17,500 resolution, AGC target 5 × 104, maximum IT 50, 2.0 m/z isolation window, stepped collision energy of 15, 25, and 35 eV, intensity threshold 1 × 104 and 15-s dynamic exclusion.Data for untargeted metabolomics using all three chromatographic methods were acquired for mass ranges of 70–1,000 m/z by full MS at 70,000 resolution in both positive and negative ionization modes. The automatic gain control (AGC) and maximum injection time (IT) were 5 × 10t-test was carried out to determine the significantly changed metabolites (P < 0.05) in pollen from the hydration or germination stage when compared with the mature pollen stage. The resultant subset of features that showed significant differences were annotated using “Functional Analysis” module of MetaboAnalyst v5.0 ” was set as the integration method.For data analyses, the raw data files in Thermo.RAW format obtained in the profile mode were first centroided by conversion into .mzML format using ProteoWizard  with peayst v5.0 . Notablyyst v5.0 , the conThe Shapiro-Wilk test was used to test the normality of data. For the data that passed the normal distribution test, one-way ANOVA followed by multiple comparison tests (Fisher’s LSD method) was used for the difference comparisons among multiple subjects. For the data of metabolites  non-normally distributed, the Kruskal-Wallis ANOVA followed by multiple comparison tests (Dunn’s test) was used. All statistical analysis was performed using OriginPro 2021b software .Pollen germination rates were initially evaluated using a light microscope. Consistent with previous observations , no pollP < 0.05) between mature and hydrated pollen, and between mature and germinated pollen, we noted the significantly increased or decreased metabolites in each pair, with verified identities presented in After determining the significant changes that occurred in metabolite levels  and AtSUC9 (At5g06170), which encode plasma-membrane localized sucrose transporters, had increased expression in germinated pollen. AtSIP2 (At3g57520), which encodes a raffinose synthase, had elevated expression in germinated pollen. The gene expression levels were consistent with increased sucrose and raffinose that accumulated in germinated pollen (Arabidopsis pollen grains after staining with iodine containing solution (AtUGE1 (At1g12780), which encodes a UDP-glucose epimerase, was expressed to a higher degree in germinated pollen, and AtUGE3 (At1g63180) was also elevated in both hydrated and germinated pollen. Plant UGEs are important to the regulation of cell wall carbohydrate biosynthesis , which encodes the key diacylglycerol acyltransferase for TAG biosynthesis , which accumulate a high level of lipids instead of starch , which when absent result in a compromised pollen germination phenotype in mutants without affecting fatty acid content  that hydrolyze sucrose into fructose and glucose, are highly expressed in pollen  website, the Metabolomics Workbench, JW and L-QC conceived and designed the experiments. JW and SK conducted the experiments and performed the metabolomics analysis. DA supervised the metabolomics analysis and edited the manuscript. JW, SK, and L-QC wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intriguingly, artesunate inhibited the upregulation of spinal chemokine CCL21, triggering receptor expressed on myeloid cells 2 (TREM2) and DNAX-activating protein of 12 kDa (DAP12) expressions and microglia activation in fracture mice. Furthermore, spinal CCL21 neutralization attenuated the severity of fracture-associated post-surgical pain. Exogenous CCL21-induced acute inflammatory pain was impaired by artesunate therapy. Additionally, the pharmacological blockage of TREM2 reduced recombinant CCL21-elicited behavioral hypernociception. The present findings demonstrate that artesunate therapy reduces the initiation and maintenance of fracture-associated chronic postoperative pain by inhibiting CCL21-dependent TREM2/DAP12 inflammatory signaling and microglia activation, thus suggesting that artesunate could emerge as a therapeutic strategy for fracture pain management.Chronic pain after bone fracture and orthopedic surgery is often refractory to most analgesics currently in use, thus emphasizing the urgent need for improved therapeutic medications. Chemokine-dependent neuroinflammation is critical for excitatory synaptic plasticity and central nociception sensitization. Recent studies have focused on the inhibition of inflammatory responses by artesunate, the first anti-malaria drug extracted from artemisinin. The present study investigated the analgesic effects and potential targets of artesunate in a mouse model of chronic pain induced by tibial fracture and orthopedic surgery. Three injections of artesunate were intrathecally administered on a daily basis from days 4 to 6 after fracture. We reported that repetitive exposure to artesunate (10 and 100 μg but not 1 μg) dose-dependently prevented fracture-induced mechanical and cold allodynia. Moreover, single intrathecal injection of artesunate (100 μg) alleviated the established chronic pain on day 14 after fracture surgery. Intraperitoneal artesunate (10 and 50 mg kg With the rapid development of industry, construction, and transportation and the aggravation of population aging, the occurrence of fracture following industrial accidents, construction injuries, traffic injuries, and osteoporosis in the elderly also increases . ChronicChemokine-mediated neuroinflammation involves microglia activation and neuronal plasticity in pain neurocircuits that, subsequently, maintain the pain phenotype . ChemokiGiven that current analgesics including opioid agents and non-steroidal anti-inflammatory drugs have several side effects and may interfere with bone healing , alternaIn this study, we characterized the potential role of intrathecal (i.t.) artesunate in chronic postoperative pain using a mouse model of tibial fracture with intramedullary pinning. Spinal expressions of CCL21, TREM2, and DAP12 were measured to verify the nociceptive pathogenesis and anti-nociceptive targets of artesunate in our orthopedic model. Our findings identified that the inhibition of neuroinflammation by artesunate may offer a novel therapeutic strategy for pain control after orthopedic surgery.ad libitum. All animals were purchased from the experimental animal center of the Chinese Academy of Military Medical Science. All experimental studies and protocols were conducted in strict accordance with the International Association for the Study of Pain directives and approved by the Animal Ethical and Welfare Committee of Tianjin Medical University .Adult male C57BL/6J mice, 8–10 weeks old, were raised in an artificially regulated 12-h light–dark environment with food and water 5 and L6 using a 30-G needle  was dissolved in 10% dimethyl sulfoxide  for i.t. injection. Recombinant CCL21 , a neutralizing antibody against CCL21  and a neutralizing antibody against TREM2 , was dissolved in normal saline or 10% DMSO for i.t.; delivery and the doses of these reagents were chosen based on previous reports . The intG needle ; 5 μl ofThe tibial fracture-associated postoperative pain model was established according to the previously described procedures . BrieflyAll tests were conducted between 10:00 a.m. and 3:00 p.m. on that day in a temperature-controlled room at 24°C. The baseline threshold was tested 1 day before the treatment, and the mice were habituated 2 h per day in the testing apparatus for 3 days prior to the baseline threshold test.In the von Frey test, the mice were placed on a test platform with a grid spacing of 1.5 mm, covered with a plexiglass box of 49 cm × 33 cm × 40 cm, and allowed to acclimatize for 2 h. The paw withdrawal threshold (PWT) of the mice was measured with the von Frey filaments  between 0.16 and 2 g using the up-and-down method. Starting with 0.16 g to stimulate the left hind paw, licking or withdrawal during the 5 s stimulus was considered as a positive response. The force was reduced in the case of a positive reaction; otherwise, the force was increased, and finally the PWT was calculated . With reFor measurement of cold allodynia, two acetone applications (20 μl each) were gently applied to the left hind paw bottom using a pipette and the responses to acetone were scored: 0, no response; 1, quick withdrawal, paw stamping, or flicking; 2, prolonged withdrawal or repeated flicking of the paw; and 3, repeated paw flicking and licking .The same investigator blinded to the treatments collected the behavioral data.4-5 segments of spinal cord. The spinal cord tissues were collected before and after tibial fracture, as well as at hour 12 after exogenous CCL21 administration. The spinal cord tissues were homogenized in a lysis buffer containing protease and phosphatase inhibitors. The tissue samples were centrifuged at 12,500 ×g for 10 min, and the supernatant was collected. The BCA protein assay (Pierce) was employed to determine protein concentrations. For each reaction in a 96-well plate, 100 μg of proteins of samples were used. All ELISA experiments followed the manufacturer’s protocol. The optical densities of samples were measured using an ELISA plate reader (Bio-Rad) at a wavelength of 450 nm, and the levels of CCL21, TREM2, and DAP12 were calculated using the standard curves and normalized to the total protein levels.An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of CCL21 , TREM2 , and DAP12  in the L4–L5 spinal cord was dissected and dehydrated in 30% sucrose for 2 days. The tissues were then frozen in OCT and cut into 5-µm frozen sections using a cryostat . The sections were blocked with 0.3% Triton X-100 for 10 min and 5% goat serum for 1 h. They were then incubated with primary antibodies overnight at 4°C. The following primary antibody was used: anti-Iba-1 . After rinsing three times with PBS, the sections were incubated with a fluorescence-labeled secondary antibody for 1 h. Images were collected using a fluorescence microscope , and the analysis was performed using Image J software.The mice were deeply anesthetized and transcardially perfused with pre-cooled PBS following 4% paraformaldehyde. The whole spinal cord was blown out using the hydraulic pressure method. The Lpost hoc comparisons. The results of biochemical experiments were analyzed using one-way ANOVA with Bonferroni post hoc comparisons. A p value <0.05 was considered statistically significant.All statistical analyses were performed with SPSS 18.0 software . All animals were randomly assigned to experimental conditions. All data were expressed as mean ± standard error of mean (SEM). The sample size was calculated as previously described . The Shap > 0.05, n = 8; p > 0.05, n = 8; p > 0.05, n = 8; p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; First, we did not observe any significant differences in basal mechanical and cold sensitivities between two groups  was injected in naïve animals. We found that as compared to baseline, artesunate treatment did not impair peripheral mechanical and cold sensitivity in naïve mice  reduced the spinal up-modulation of CCL21, TREM2, and DAP12 expressions after tibial fracture with pin insertion  on 14 days after fracture (in the late phase) produced a rapid and transient attenuation of the established mechanical allodynia for 5 h, as reflected by the increase in paw withdrawal threshold [F  = 86.87, p < 0.001; n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; p = 0.008, n = 8, two-way ANOVA; To further examine whether the CCL21 pathway is central for the development of chronic fracture pain, repetitive anti-CCL21  was delivered on a daily basis from days 4 to 6 (in the early phase) after tibial fracture with pin insertion. Strikingly, pretreatment with anti-CCL21 at 10 μg but not 0.1 and 1 μg generated a significant alleviation of mechanical and cold allodynia, which sustained for more than 4 days after three injections, as demonstrated by the increase of paw withdrawal threshold [F  = 192.9, p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; p < 0.05, n = 5; Then, we further tested the hypothesis that artesunate would control the spinal CCL21-dependent nociception sensitization. We previously reported that acute exposure to recombinant CCL21  directly initiated an acute allodynia . Herein,p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; Next, we evaluated the effect of TREM2 signaling on pathological pain development. Intriguingly, a neutralizing antibody against TREM2  ameliorated the established mechanical allodynia [F  = 37.75, via intraperitoneal injection for the treatment of several diseases in rodents (−1) following intraperitoneal injection on 14 days after orthopedic surgery. Interestingly, the systemic delivery of artesunate at 10 and 50 mg kg−1 relieved the fracture-induced mechanical allodynia and cold allodynia, as manifested by the elevation in paw withdrawal mechanical threshold [F  = 31.23, p < 0.001, n = 8, two-way ANOVA; p < 0.001, n = 8, two-way ANOVA; Given that artesunate is often administered  rodents , we finaIn this current study, the major findings are as follows: first, tibial fracture originates from the persistent mechanical allodynia and cold allodynia with the spinal up-modulation of CCL21 and TREM2/DAP12 expressions following orthopedic surgery. Second, the intrathecal delivery of artesunate prevents fracture-associated allodynia in a dose-dependent manner and reduces fracture-caused CCL21 over-expression, as well as the TREM2/DAP12 accumulation in the spinal dorsal horn. Third, the postoperative therapy of both intrathecal and intraperitoneal artesunate attenuates the established fracture allodynia. Fourth, spinal CCL21 neutralization is sufficient to impair the generation and maintenance of fracture-associated pain. Fifth, the pre-administration of artesunate impairs exogenous CCL21-evoked acute inflammatory pain by reducing the spinal TREM2/DAP12 overload. Sixth, the pharmacologic inhibition of TREM2/DAP12 also ameliorates fracture-associated chronic pain and CCL21-elicited acute pain. These results therefore elucidate a previously undescribed role of artesunate as an alleviator of chronic pain following tibial fracture and orthopedic surgery by the inhibition of CCL21-dependent TREM2/DAP12 neuroinflammatory signaling.Neuroinflammation driven by chemokines is a cardinal feature of chronic pain following peripheral tissue damage, nerve trauma, chemotherapy, and cancer . We prevIt is noteworthy that the involvement of TREM2/DAP12 in neuron–microglia interactions is indispensable for neuroinflammation-associated pain perception in several rodent models . Especia−1) is sufficient and effective in abrogating fracture-associated chronic pain. Taken together, these results emphasize that artesunate therapy protects against the generation and maintenance of fracture-associated chronic pain through inhibiting spinal CCL21-dependent TREM2/DAP12 inflammatory signaling and microglia activation. Indeed, microgliosis is capable of raising excitatory neuronal responsiveness by promoting the secretion of tumor necrosis factor-α (TNF-α)  . Thus, i2O2)-induced retinal neuronal dysfunction, sodium nitroprusside-induced cortical neurotoxicity, and neurodegenerative disease (via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activation in dorsal horn neurons of fractured animals (Apart from its anti-inflammatory properties, artemisinin and its derivatives can activate the antioxidant system to exert potent neuroprotective effects on hydrogen peroxide (H disease . Oxidati disease . Accordi animals . HoweverIn summary, the current findings recapitulate an unconventional pharmacological role of artesunate in the prevention and alleviation of fracture-associated postoperative pain by inhibiting spinal CCL21-dependent TREM2/DAP12 inflammatory processes. These results suggest that artesunate administration and CCL21 neutralization may generate innovative therapeutic concepts for a targeted neurotherapeutic strategy for fracture pain in clinics.