ApoE−/− mice.Atherosclerosis (AS) is the major cause of cardiovascular disease, and dyslipidemia is a principal determinant of the initiation and progression of AS. Numerous works have analyzed the lipid signature of blood, but scarce information on the lipidome of vascular tissue is available. This study investigated the lipid profile in the aorta of ApoE−/− mice were randomly divided into two groups: (1) the normal diet (ND) group and (2) the high-fat diet (HFD) group. After feeding for 8 weeks, the plasma low-density lipoprotein (LDL), total cholesterol (TC), and triglyceride (TGs) levels were measured. UHPLC-Q Exactive plus MS was used to assess the lipid profile using both positive and negative ionization modes.LDL and TC levels were significantly increased in HFD mice, and lipid deposition, plaque area and collagen fiber levels were increased in HFD group. In addition, a total of 131 differential lipids were characterized, including 57 lipids with levels that were increased in the HFD group and 74 with levels that were decreased. Further analysis revealed that the levels of several differentially expressed phosphocholines (PCs) and lysophosphocholines (LPCs) were significantly increased. These PCs included PC (38:3), PC (36:4), PC (36:3), PC (36:2), PC (36:1), PC (34:1e), PC (34:1), PC (32:1), PC (18:0/18:1), and PC (38:5), and the LPCs included LPC (18:1), LPC (18:0) and LPC (16:0).Our findings indicate the presence of a comprehensive lipid profile in the vascular tissue of atherosclerotic mice, particularly involving PC and LPC, which exhibited significantly increased levels in AS.The online version contains supplementary material available at 10.1186/s12986-022-00723-y. Cardiovascular and cerebrovascular diseases caused by atherosclerosis (AS) remain the leading cause of morbidity and mortality worldwide , 2. AS iApoE−/− mice, including 11 lysophosphatidylethanolamine (LPE), 12 SM, 15 LPC, 27 cholesteryl esters (CE), 35 ceramides (Cer), 38 diacylglycerol (DAG), 60 phosphatidylethanolamine (PE), 64 PC, and 470 triglyceride (TAG). Therefore, further comprehensive exploration of the lipid characteristics related to atherosclerosis, including in serum and vascular tissue, is conducive to enabling an in-depth understanding of the pathogenesis of atherosclerosis and is also expected to provide new insights for the diagnosis, prevention and cure of atherosclerosis.Lipidomics is a powerful analytical tool for analyzing the lipid composition in biological samples under physiological and pathological conditions to elucidate disease pathogenesis, identify biomarkers or study the effect and mechanism of drugs , 13. Liq. have developed a new Multi-ABLE method that can be used to assess small tissue samples (< 5 mg) and have performed aortic lipidomics in ApoE−/− mice [ApoE−/− mice. Therefore, we used classic LC‒MS/MS lipidomics technology to provide a basis for elucidating the lipid profile of atherosclerotic thoracic aortas in ApoE−/− mice.In summary, omics approaches are used to identify lipid profiles in plasma, serum, and plaques to establish a disease prediction model, elucidate disease pathogenesis, improve diagnostic efficiency and provide drug targets. To date, due to the limited sample size, although Jihan Talib et al−/− mice , there hThe chemicals methanol (No. A452-4), acetonitrile (No. A998-4), formic acid (No. A117-50), and isopropanol (No. A451-4) were purchased from ThermoFisher , ammonium acetate (10001218) was purchased from Sinopharm , Lyso PC17:0 (No. 855676P) was purchased from Avanti , chloroform (No. G75915B) was purchased from Greagent . Distilled water was purchased from Watsons . All aqueous solutions were prepared using purified water at a Milli-Q grade.ApoE−/− mice  were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Animals were kept in a specific pathogen-free (SPF) environment. The feeding conditions included a light–dark cycle for 12 h, temperature 22–25 °C, and a relative humidity of 60%. The mice were divided into two groups: (1) mice were fed a normal diet, and (2) mice were fed a high-fat diet . After feeding for 8 weeks, the mice were fasted overnight and anesthetized with sodium pentobarbital solution  by intraperitoneal injection. The plasma was separated from the blood, which was collected from the abdominal aorta, after standing for 2 h and centrifugation at 8000 rpm for 8 min. The plasma was transferred to an EP tube and preserved at − 80 °C refrigerator before being used for analysis. The thoracic aorta was peeled off and preserved at − 80 °C refrigerator.This study was approved by the Chengdu Women’s and Children’s Central Hospital Ethics Committee. The cryopreserved plasma (100 μL) obtained following fasting was thawed and then centrifuged. The biochemical markers associated with lipid metabolism, including the levels of LDL, TC, and TG, were detected using a commercial kit according to the manufacturer’s instructions .The heart, including the aortic root, was fixed in paraformaldehyde. Then, the tissues were embedded in OCT compound and sliced into sections (7 μm) . Then, HAll images were photographed using a panoramic MIDI scanning microscope  and then quantified using Image-Pro Plus 6.0 software. The plaque area was measured as the total area of plaque. Lipid deposition is presented as the percentage of positive cells in total plaque. The collagen area was measured as the collagen-positive area.As previously described, the expression of CD68 in the aortic root was assessed by immunostaining . BrieflyDue to the limitation of sample weight, the thoracic aorta vessels of every two mice were combined in the analysis. After quickly freezing with liquid nitrogen, the samples were placed in and stored at − 80 °C refrigerator until analysis. Lipids were extracted as follows: (1) 20 μL Lyso PC-17:0  was added to the samples as internal standard to reflect the repeatability of the extraction. And then mixed the samples with methanol: water . Subsequently, the samples were subjected to grinding with two small steel balls  in grinder  and then added 300 μL chloroform for ultrasonication for 10 min, and stored at − 20 °C for 20 min. After centrifugation for 10 min, 200 μL of the chloroform layer was transferred into a new centrifuge tube. (2) The residual samples were added to chloroform:methanol  and then vortexed for 30 s. The samples were ultrasonicated for 10 min, placed at − 20 °C for 20 min, and centrifuged for 10 min, and then taken 150 μL of the chloroform layer into the centrifuge tube. (3) The two chloroform layers were combined and then dried under a nitrogen stream. The samples were re-dissolved in 400 μL isopropanol:methanol , vortexed for 30 s and sonicated for 3 min. Before detection, the solution was filtered through a 0.22 μm organic phase pinhole filter. All sonication was performed in an ice-water bath, and centrifugation was performed at 13,000 rpm at 4 °C. QC samples were prepared by mixing aliquots of all samples, and then were injected at regular intervals (every 6 samples) into the samples throughout the analysis procedure to evaluate the stabilityof the equipment. All chemicals and solvents were analytical or HPLC grade.Lipid profiling was performed using an UPLC system  coupled to a Q-Exactive plus mass spectrometer equipped with a heated electrospray ionization (ESI) source . In detail, the samples were separated using an ACQUITY UPLC BEH C8 column  and detected using ESI positive and ESI negative ion modes. During the analysis, the samples were kept at 4 °C. The gradient elution system consisted of two mobile phases, (A) acetonitrile: water  and (B) isopropanol: acetonitrile . The flow rate was 0.26 mL/min, and the column temperature was 55 °C. The injection volumes were 2 μL and 4 μL in positive and negative modes, respectively. The separation was achieved using the following gradient: 0 min, 32% B; 1.5 min, 32% B; 15.5 min, 85% B; 15.6 min, 97% B; 18.0 min, 97% B; 18.1 min, 32% B; 20.0 min, 32% B.The mass spectrometer was operated in both positive electrospray ionization (ESI+) mode and negative electrospray ionization (ESI−) mode. The acquisition parameters: the spray voltage value of positive ions is 3.5 kv, and 3.0 kv negative ions; Capillary Temp 300 °C; Mass range (m/z): 150–1500; Full ms resolution: 70,000; MS/MS resolution: 17,500.https://hmdb.ca/) [P value) > 2 as the threshold and then defined Z score > 2 and Z score < − 2 as the threshold of significant activation and inhibition, respectively. The consistency scores were calculated by assessing regulatory effects and molecular networks, and the higher the consistency score was, the more accurate the regulatory effect analysis results. In addition, we used a P value of overlap < 0.05 as the inclusion criteria for upstream regulators.Ingenuity pathway analysis (IPA) is a widely used bioinformatics software for predicting the correlations among components, targets, pathways, and diseases , 22. Themdb.ca/) . In canop values < 0.05.The original data obtained from Q Exactive plus (LC‒MS/MS) analysis were processed by Lipid Search software. The molecular structure of lipids and the additive mode of its positive and negative ions were confirmed according to the parent ions and multistage mass spectrometry data of each individual sample. To sort the original data matrix, results were aligned according to a certain retention time range. The peak signals in each sample were normalized, by converting its signal intensity into the relative intensity in the spectrum, and multiplying it by 100,000. Lipids which the relative standard deviation (RSD) of QC greater than 30% were discarded. These highly variable ions should be rejected as unacceptable for the analysis. Following the extraction of data, peaks with missing values (ion intensity = 0) in more than 50% of groups were removed and replaced the zero value with half of the minimum value. Positive and negative ion data were combined into a data matrix. Principal component analysis (PCA) was carried out to monitor the overall sample distribution and the stability of the whole analysis process. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and partial least squares-discriminant analysis (PLS-DA) were used to authenticate the results regarding the differential lipids. Variable importance of projection (VIP) value was used to assess the variable importance. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to analyze metabolic pathway enrichment. The statistical graphs were generated by GraphPad Prism 8 software, and a two-tailed Student’s T test was used for statistical analysis. All data are presented as the means ± standard errors of the means, SEM). Differential lipids were selected with VIP values > 1.0 and ApoE−/− mice fed a high-fat diet to establish an animal model of atherosclerosis and then collected blood vessel tissue to extract lipids for mass spectrometry analysis. The study design is shown in Fig. ApoE−/− mice were fed a high-fat diet (HFD) for 8 weeks to establish an atherosclerosis model, while ApoE−/− mice fed a normal diet (ND) were used as a control. The lipid species in thoracic aorta were assessed via HPLC‒MS/MS. The body weight was not significantly different between the ND group and the HFD group  of positive ions and negative ions in the ND group and HFD group are shown in Additional file To evaluate the stability of the equipment, the lipid intensity distribution of the QC samples is shown in Fig. P < 0.05. A total of 57 lipids exhibited increased levels, and 74 exhibited decreased levels in the HFD group  was used to predict metabolic pathways based on the lipids with increased and decreased levels. As shown in Fig. KEGG pathway enrichment analysis , PE (18:1/20:4), TG (18:1/18:1/18:1), TG (18:1/18:2/18:2), PS (37:1), PE (16:0p/22:6), TG (18:0/18:1/18:1), TG (16:0/18:1/18:1), TG (16:1/16:1/18:2), TG (20:0/18:1/18:1), PC (34:1), TG (18:0/18:0/18:1), PA (24:2/20:4), PC (36:1), LPE (20:4), PA (26:2/20:3), PE (16:0p/20:4), TG (16:1/14:0/18:2), TG (16:0/18:2/18:2), and LPE (22:6) in the AS group.Dyslipidemia is a major risk factor for atherosclerosis and atherosclerosis-related cardiovascular disease . A conseApoE−/− mice. The lipids included DAG (14:0/18:2), PC (18:0/20:1), LPC (18:2), LPC (20:2), PC (18:2/22:6), PE (18:0/18:2), SM (D18:1/16:0), LPE (20:1), TAG48:1-FA12:0, TAG48:1-FA14:0, TAG49:2-FA17:0, TAG52:6-FA16:0, TAG53:3FA16:0, TAG53:3-FA17:0, TAG54:6-FA20:5, TAG54:2-FA16:0, and TAG54:0-FA18:0. Jihan et al. [ApoE−/− mice. The results showed that 52 lipid molecules exhibited increased abundance in lesion-containing tissue and included SM (n = 7), phospholipids (n = 24) and CL (n = 3). The results regarding the lipids identified by lipidomics in thoracic aorta of ApoE−/− mice in this article and those in previous studies are inconsistent, which suggested the data from vascular lipidomics should be further investigated to provide an in-depth understanding of the complex pathogenesis of AS.Recently, omics approaches have been used to improve the understanding of the pathogenesis of AS. For example, Wang et al.  used ultDAG 14:0/:2, PC , PC (36:4), PC (36:3), PC (36:2), PC (36:1), PC (34:1e), PC (34:1), PC (32:1), PC (18:0/18:1), and PC (38:5). Phosphatidylcholine (PC) is a subclass of glycerophospholipids with choline as the head group. The physiological functions of PC include membrane integrity/stability, cell proliferation and survival, fuel and energy storage/source, glycerophospholipid metabolism and cell signaling [In addition, we found that the levels of 10 PCs from HFD-treated ignaling , 39. It ignaling . In contignaling .Lysophosphatidylcholine (LPC) is the main component of oxidized low-density lipoprotein (oxLDL). In cells, saturated LPC (16:0) and PC can be interconverted. Specifically, PC can be cleaved by phospholipase A2 (PLA2) to form LPC , 43, andAlthough we revealed the lipidomic profiling of vascular tissue of atherosclerotic mice by LC‒MS/MS technology and identified some differentially expressed lipid molecules, there are still some limitations. First, due to the continuous development of LC‒MS/MS technology, lipidomics methods are widely used to comprehensively analyze lipids and their interactions with other molecules. However, it is not clear whether all lipids in some biological samples can be comprehensively detected and accurately identified due to the complex composition of lipids. It is also difficult to conduct accurate localization analysis on the detected lipids. Therefore, the development of new instruments and new bioinformatics schemes, such as ion mobility mass spectrometry (IM-MS) , NanoLC‒Our findings indicate that in the comprehensive lipid profile in the vascular tissue of atherosclerotic mice, a total of 131 differential lipids were identified, including 57 lipids with levels that were increased in the HFD group and 74 with levels that were decreased. Further analysis revealed that the identified differentially expressed PCs and LPCs exhibited significantly increased levels. We particularly analyzed the details of PCs and LPCs, which exhibited significantly increased levels in AS. Therefore, targeting PCs, LPCs and lipid metabolism might be a novel and effective therapeutic strategy for atherosclerosis.Additional file 1: Figure S1. The base peak chromatograms (BPCs) of positive ions and negative ions. A The BPCs of positive ions in the ND group. B The BPCs of negative ions in the ND group. C BPCs of positive ions in the HFD group. D The BPCs of negative ions in the HFD group. BPC, base peak chromatogram; ND, normal diet; HFD, high-fat diet.Additional file 2: Figure S2. The multivariate statistical analysis. A PCA analysis. B OPLS-DA. PCA, principal component analysis; OPLS-DA, orthogonal partial least squares discriminant analysis.Additional file 3: Figure S3. IPA network pathway analysis. The IPA network pathway analysis revealed that these differentially expressed lipids were related to NF-κB signaling, PI3K/AKT signaling, and JAK/STAT signaling. IPA, ingenuity pathway analysis; CP, classical signaling pathway.Additional file 4: Figure S4. Chromatogram analysis of various lipid components. A A total of 131 differential lipids, including 53 TG (40.46%), 24 PE (18.32%), 10 PC (7.63%), 10 PS (7.63%), 7 SM (5.34%), 7 PA (5.34%) and 20 other lipids (15.27%), were identified. B TG, C PE, D PC, E PS, F PA, G SM, H Other lipids. TG, triglyceride; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PA, phosphatidic acid; SM, sphingomyelin; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; DG, diglyceride; CL, cardiolipin; PG, phosphatidylglycerol; PEt, phosphatidylethanol; dMePE, dimethyl phosphatidylethanolamine.


Oreochromis niloticus), a primary vertebrate, are investigated. The open reading frame of OnSAP is 645 bp of a nucleotide sequence encoding a polypeptide of 214 amino acids. As a calcium-binding protein, the structure and relative motif of OnSAP is highly similar to those of humans, containing amino acid residues Asn, Glu, Gln and Asp. In healthy fish, OnSAP mRNA is extensively distributed in all eleven tissues examined, with the highest level in spleen. The mRNA expression of OnSAP was significantly up-regulated after being challenged with gram-positive bacterium Streptococcus agalactiae and gram-negative bacterium Aeromonas hydrophila in vivo. In addition, recombinant OnSAP ((r)OnSAP) protein had capacities of binding S. agalactiae or A. hydrophila in the presence of Ca2+. Further, (r)OnSAP helped monocytes/macrophages to efficiently phagocytize bacteria. Moreover, the (r)OnSAP was able to enhance the complement-mediated lysis of the chicken red blood cells. Collectively, the evidence of SAP in tilapia, based on the results including its evolutionary conserved protein structure, bacterial binding and agglutination, opsonophagocytosis of macrophage and hemolysis enhancement, enriches a better understanding of the biological functions of the pentraxin family.Serum amyloid P component (SAP), an ancient short pentraxin of the pentraxin family, plays an essential role in resistance to bacterial infection. In this study, the expression and functional characterization of SAP (OnSAP) in Nile tilapia ( Innate immunity plays an important role in resistance against pathogens, consisting of cellular and humoral arms . SolubleHxCxS/TWxS, x representing any amino acid). As for long-chain pentraxins, for example, pentraxin member 3 (PTX3), has an additional N-terminal domain preceding the PTX domain [Pentraxins, including short-chain pentraxins such as C-reactive protein (CRP/PTX1), serum amyloid P component (SAP/PTX2) and long-chain pentraxins, are PRMs of the immune system, constituting a conservative protein superfamily characterized by a cyclic multimeric structure ,6. ProteX domain , whereasX domain .2+-dependent protein, SAP is mainly secreted from the hepatocytes and macrophages into the circulation [Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes and Neisseria meningitidis) and parasites such as malaria [SAP is a key member of pentraxins with a PTX domain and an intramolecular conserved pentraxin signature, which could form an oligomer by the interaction of five identical units, playing a critical role in innate immune defense . As a Caculation . SAP is  malaria ,11. Besi malaria ,13. More malaria ,11,14,15Oncorhynchus mykiss), Atlantic salmon , half-smooth tongue sole (Cynoglossus semilaevis) and rock bream (Oplegnathus fasciatus) [Escherichia coli LPS and Aeromonas salmonicida, the dynamics of trout SAP level were investigated [In teleost fish, the SAP has been reported in a few species, including rainbow trout (sciatus) ,16,17,18stigated ,18. ExisOreochrmis niloticus), a primary vertebrate. The ORF of O. niloticus SAP (OnSAP) gene was identified and analyzed. The mRNA expression of OnSAP in different tissues of healthy or stressed fish has been explored. Further, functions of recombinant OnSAP protein towards bacteria binding, agglutination and promotion of phagocytosis were characterized. Moreover, we also found that the OnSAP could facilitate complement-mediated cell lysis. These findings give important evidence that OnSAP is likely to involve in host defense against pathogen infection in a primary vertebrate.In this study, the functional characterization of SAP was examined and identified in Nile tilapia  was located in the middle of the protein, which is similar to other species. As for the similarities among sequences, OnSAP shares a 56.73%, 47.98%, 47.27%, 36%, 38.12%, 37%, and 37.37% sequence similarity with Oryzias melastigma SAP, Oncorhynchus kisutch SAP, Cynoglossus semilaevis SAP, Chelonia mydas SAP, Mauremys reevesii SAP, human SAP and mouse SAP, respectively, revealing its evolutionary conservation OnSAP) was purified following the protocol of Ni-NTA Agarose Resin . According to SDS-PAGE analysis, (r)OnSAP had a mass weight of about 40 kDa A. Identisaves.mbi.ucla.edu/ (accessed on 20 July 2022)), which contains the information that the proportion of the most favored regions residues is 93%. Three-dimensional model of OnSAP from different angles was shown in 2+, (r)OnSAP had the ability to bind S. agalactiae (A. hydrophila (The binding between (r)OnSAP protein and bacteria was evaluated by the ELISA assay. In the presence of Caalactiae A and A. drophila B in a pr2+ using FITC-labeled S. agalactiae and A. hydrophila. As shown in S. agalactiae and A. hydrophila. However, results of the (r)Trx and TBS group indicated that there was no agglutination capability in the control groups for both bacteria.Agglutinating activity of (r)OnSAP was detected under the microscope in the absence of CaA. hydrophila and S. agalactiae. After being treated by (r)OnSAP, the fluorescent signal (Effect of (r)OnSAP regulation towards phagocytosis of MO/Mø was detected by flow cytometer. An increase in phagocytosis rate occurred while evaluating the MO/Mø phagocytosing (r)OnSAP-bacteria complex. For details, the MO/Mø phagocytosing percentage was almost 0 and almost no fluorescent signal is released without the existence of bacteria F,G towart signal F,G was sTo prove that OnSAP could stimulate complement-mediated hemolysis, (r)OnSAP, (r)Trx, normal tilapia serum  and CRBCs were carried out. The results demonstrated that (r)OnSAP significantly improved the CRBCs lysis compared with the TBS and (r)Trx groups. Moreover, by adding highly concentrated (r)OnSAP at the same time, the hemolysis rate was almost close to the total hemolysis rate H.A. fumigatus, S. pneumoniae and Candida albicans by activating the complement system and promoting phagocytosis [Serum amyloid P component is a soluble pattern recognition molecule and is also known as acute-phase protein, which makes it possible to recognize various pathogenic microorganisms, activate the complement system, join the process of opsonophagocytosis and regulate the inflammatory reaction . In mammocytosis ,11,23. Hocytosis .SICSTWDS), which contains conserved cysteine residues that form intramolecular disulfide bonds OnSIn conclusion, the functional characterization of SAP in Nile tilapia was explored and identified in this study. First, the transcript of OnSAP was found to express widely in many tissues and its expression was up-regulated significantly after in vivo stimulation with bacterial pathogen. Further, recombinant OnSAP possessed the capabilities to bind and agglutinate bacteria, and even promoted phagocytosis bacteria cells by monocytes and macrophages. Moreover, the OnSAP could enhance complement-mediated hemolysis. To sum up, our result enriched the function of SAP in a primary vertebrate, which could help us to better understand the biological functions of the pentraxin family.Nile tilapia weighing 80 ± 10 g were harvested from a Tilapia Cultural Farm in Guangzhou, Guangdong, China. Upon arrival, fish were cultured in the automatic filtering aquaculture system with a stocking rate of 10 g/L under 28 ± 2 °C for three weeks . All anig for 10 min and was stored at −20 °C for further use [To explore the expression of SAP in healthy fish, eleven tissues such as the liver, head kidney, hind kidney, muscle, heart, spleen, skin, gills, intestine, peripheral blood and thymus were harvested after being anesthetized with 0.02% MS-222 . Liquid nitrogen was immediately applied to freeze them and later stored at −80 °C for further use . Fresh sther use .6 CFU/fish, 1 × 107 CFU/mL concentration) live bacteria liquid such as S. agalactiae (ZQ1901) or A. hydrophila (BYK00810) dissolved in PBS , and grouped in stimulation groups, respectively [The fish were immunized with 100 μL . Similarly, the templates of every sample collected before were synthesized using the same protocol and then they were stored at −80 °C for further analysis [Total RNA from the healthy fish liver was extracted using Trizol Reagent  for amplifying the ORF of tilapia SAP. After a successful acquisition of total RNA, the template was synthesized with the HiScriptanalysis .OnSAP gene was cloned according to the predicted sequence of Oreochromis niloticus SAP mRNA (GenBank accession LOC109196457) based on the primer devised by software Primer Premier 5.0 .Complete ORF of mier 5.0 . After bhttp://expasy.org/tools (accessed on 25 May 2021)) were achieved by the DNAMAN software and Clustalw2 program ). Using the neighbor-joining method with 1000 bootstrap replications, the MEGAX was employed to give a phylogenic tree [Multiple amino acid sequence alignments of OnSAP analyzed by ExPASy tools , 2 μL of each primer shown in t method ,34,36,39t method .O. niloticus weight 200 ± 10 g and grinded into cellular homogenate using a 1 mL disposable sterilized syringe in a cell culture dish with 10 mL RPMI 1640 medium. Different concentrations of percoll could be selected to further isolate MO/Mø cells as described. Diluted with 100 mM PBS in a ratio of 9:1, the processed original percoll solution  could be used to further adjust the concentration to 54% and 31% using 10 mM PBS. Then, the separation liquid was prepared using 10 mL 54% percoll covered with 10 mL of 31% percoll. After slightly adding head kidney MO/Mø cell suspensions of Nile tilapia to separation liquid and centrifuging, the MO/Mø fraction could be collected from the 31–54% interface of percoll [Head kidneys were collected from healthy  percoll .6 cells/mL. The above system was incubated and cultured in 96-well plates (100 μL/well)  at 25 °C for 3 days. Removing non-adherent cells, the MO/Mø were washed with 10 mL RPMI 1640 basic medium 3 times and resuspended. Finally, they were cultured in the 96-well plates as mentioned above for further experiment [After being diluted with cell culture (10% FBS + 1% penicillin-streptomycin), the cell concentration was appraised by 0.4% trypan blue exclusion in order to control the density to 5 × 10periment .EcoR I and HindIII as restriction sites, the PCR products were inserted into the pMD-18T vector. Later, recombinant pMD-18T plasmid and pET-32a digested with EcoR I and Hind III were the further form expression plasmid pET-32a-SAP, which was transferred into BL21 (DE3) , and cultured in LB-ampicillin. Induced at 37 °C for 6 h after adding Isopropyl-β-D-thiogalactopyranpside (IPTG) to a final concentration of 1 mM, once the culture medium reached an O.D. 600 of 0.6–0.8, the culture medium was centrifuged at 3300× g for 30 min at 4 °C. PBS was employed to re-suspend the cells. Next, lysozyme  was added to the resuspended cells at a ratio of 1:100 and kept the reaction for 3 h. After sufficient reaction, above cells mixture was disrupted by an Ultrasonic Processor  to release the protein. Then, cell lysate was centrifuged at 10,000 rpm for 30 min at 4 °C, and the precipitate was re-suspended by Lysis Buffer . Ni-NTA His Band Resin columns  were applied to purify following the protocol [2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and 5 mL Elution Buffer  with different concentrations of imidazole  were added in sequence and collected respectively to obtain the recombinant protein. Obtained protein was concentrated by PEG 20,000 and separated by 12% SDS-PAGE gel Electrophoresis.Amplified by the specific primers EOnSAP-F and EOnSAP-R using protocol . The colhttps://www.rcsb.org/ (accessed on 16 December 2021), PDB identity number: 1GYK). Moreover, the structures of OnCRP (Gene symbol LOC109204179) and OnPTX3 (XP_005474584.1) were also predicted by AlphaFold 2.0 for comparison. Further the similarity among OnSAP, Human SAP and CRP was compared by Pymol 2.5.Tridimensional structure of OnSAP was predicted by Alphafold 2.0, which was deployed on the Ubuntu 16.04.7 lts (GNU/linux5.4.0-67-generic x86_64) GPU server equip with Tesla v100 32 GB . The humβ-2-ME processing) were transferred onto nitrocellulose membranes. Washed three times with 1 × TTBS  containing 0.05% (v/v) Tween-20 (PBST), mouse against 6 × His tag IgG monoclonal antibodies  (1:1500 dilution) and mouse polyclonal antibodies (1:500 dilution) were employed as the primary antibody for 1 h incubation at 37 °C. As for mouse polyclonal antibodies against recombinant OnSAP, briefly, six-week-old female BALB/c mice ) were intraperitoneally immunized with (r)OnSAP proteins 50 μg (100 μL) mixed in complete Freund’s adjuvant . The immunized mice were bled from the tail vein on the fifth day and boosted every ten days using incomplete Freund’s adjuvant  mixed OnSAP proteins 25 μg (100 μL). The serum containing polyclonal antibodies was collected from mice blood and stored at −80 °C [Being resolved in 12% SDS-PAGE gel electrophoresis, recombinant OnSAP proteins , a volume of 100 μL/well (1 × 107 CFU/mL) S. agalactiae or A. hydrophila was coated in the 96-well plates  at 37 °C for 1 h. In every step, the plates were washed three times using TBS/Ca2+ (2 mmol/L) and the temperature of ELISA was controlled at 37 °C. The (r)Trx and recombinant OnSAP was binded to biotin and diluted to different concentrations . Each well of plates was blocked at 200 μL. As a control group, the well was filled with 200 μL 1 × TBS/Ca2+. After being washed, biotinylated antibodies were employed in combination with streptavidin-HRP . ABTS (0.4 mg/mL)  in substrate buffer with 0.03% H2O2 was used to develop plates and the optical density rate (O.D.) was measured using a Microplate Reader  at 405 nm [Diluted with coating buffer  were washed for four times and heated at 65 °C for 15 min. Afterwards, being labeled with 200 μL (1 mg/mL) fluorescein isothiocyanate , the bacteria were lucifugal incubated for 30 min at 25 °C. After being washed four times and re-suspended in TBS/Ca2+ buffer, labeled bacteria were mixed with (r)OnSAP (40 μg/mL or 80 μg/mL), (r)Trx (40 μg/mL) as well as TBS buffer, respectively. After incubating for 1 h at 25 °C, treated samples were observed and photographed through a fluorescence microscope  [After being re-suspended in TBS/Cazerland) .S. agalactiae (2 × 107 CFU/mL) or A. hydrophila (2 × 107 CFU/mL) was mixed with 100 μL (r)OnSAP (50 μg/mL) in the presence of Ca2+ the in dark at 25 °C for 1 h. Then, the above mixture was incubated with 1 × 105 cells of the MO/Mø suspension. Incubated at 25 °C for 1 h, mixture tubes were shaken every 10 min. As for the control, bacteria were similarly treated by TBS or (r)Trx instead of (r)OnSAP. Mixtures after incubation were centrifuged under 100× g for 10 min to remove the non-ingested bacteria from the macrophages, washed using 1 × PBS and re-suspended in TBS (pH 7.4). After adding 1 μL of trypan blue (0.4%) 10,000 individual cells of each sample were analyzed in flow cytometer FACS Aria III . Defined as the percentage of the macrophages with one or more engulfed bacteria within the total cell population, the phagocytosis rate was analyzed by Flowjo V10 software [To explore the influence of (r)OnSAP on phagocytosis, flow cytometric analysis was employed ,45. Briesoftware ,36.2, 142 mM NaCl, 5 mM sodium barbital, 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.5), the buffer for the assay, was applied for serum dilution. First, for the activation of the complement system, 2 μg mannose was added to 1 mL serum (1:10 dilution) and the above-mentioned mixture was incubated at 25 °C for 1 h. Second, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μL serum (1:10 dilution) were added to the tube and then DVB buffer was added to achieve a total of 150 μL of volume. Afterward, the tubes after adding 100 μL CRBCs were incubated at 25 °C for 1 h. Finally, cells were centrifuged at 500× g for 5 min at 4 °C. The resultant supernatant of 100 μL was moved to a 96-well microporous plate. Absorbance at O.D. 405 nm was detected by a microplate reader  [Fresh tilapia serum and 2% chicken red blood cells (CRBCs)  were employed for the CH50 assay. DVB  .2+. Then, the protein and control group TBS were added to activated tilapia serum for 1 h at 25 °C. After incubation, 100 μL CRBCs and DVB were added to make a total volume of 250 μL. The above mixture was also incubated for 1 h at 25 °C and then centrifuged using 500× g for 5 min at 4 °C. Similarly, 100 μL of the resultant supernatant was employed for testing O.D. 405 nm absorbance. The calculation of lysis percentage was analyzed as described [The (r)OnSAP and (r)Trx were diluted to different concentrations  using 1 × TBS/Caescribed . All the


The narrow substrate scope limits the wide industrial application of enzymes. Here, we successfully broadened the substrate scope of a nitrile hydratase (NHase) through mutation of two tunnel entrance residues based on rational tunnel calculation. Two variants, with increased specific activity, especially toward bulky substrates, were obtained. Crystal structure analysis revealed that the mutations led to the expansion of the tunnel entrance, which might be conducive to substrate entry. More importantly, molecular dynamics simulations illustrated that the mutations introduced anti-correlated movements to the regions around the substrate tunnel and the active site, which would promote substrate access during the dynamic process of catalysis. Additionally, mutations on the corresponding tunnel entrance residues on other NHases also enhanced their activity toward bulky substrates. These results not only revealed that residues located at the enzyme surface were a key factor in enzyme catalytic performance, but also provided dynamic evidence for insight into enzyme substrate scope broadening. Mutations of two gating residues at the substrate access tunnel entrance direct the substrate scope of NHases. To extend the application of certain enzymes, it is particularly important to broaden the substrate scope. Efforts have been made to identify enzymes with a broad substrate scope using screening methods such as gene mining,2,3 but the obtained enzymes cannot always meet the laboratory or industry requirements. A second option for expanding the substrate scope is to modify the existing enzymes.4For centuries, traditional chemical catalysis has dominated the synthesis of valuable compounds. However, with biocatalysis being a promising and environmentally friendly alternative over the past few decades, traditional chemical catalysis is becoming overwhelmed by its green counterpart.5 For example, through elucidation of the gating characteristics and constitution of the substrate access tunnel, it was determined that threonine deaminase increased catalytic efficiency towards bulky substrates 90.6-fold.6 Mutations on the substrate access tunnel forming residues, V333 and T334, of prodigiosin ligase PigC, which involve the translocation of small pyrroles, enhanced the catalytic efficiency more than 45-fold.7 On mutating two bottleneck residues in the cytochrome P450BsβHI substrate access tunnel, its decarboxylation activity increased by 15.2-fold.8 Therefore, applying the existing knowledge and strategy to modify the substrate-binding pocket and access tunnels is promising for refining the substrate scope of enzymes.Traditional substrate scope engineering of a particular enzyme involves modifying the substrate-binding pocket and substrate access tunnel.9 It has been widely applied in the industrial production of acrylamide and nicotinamide and shows great potential in the future production of high-value amide products.10 However, the activities of NHases towards bulky nitriles such as thiacloprid and toyocamycin are very low.11,12 Therefore, further industrial applications for producing various valuable amides using NHase are strictly hindered by their narrow substrate scope, and academia and industry require impressive tailor-made NHases. The NHase active site contains either a non-heme iron or non-corrin cobalt,13 buried in a deep internal pocket,14 similar to most enzymes in nature. Hence nitrile substrates should pass through a tunnel to access the active site, and rational tunnel engineering might be a plausible choice for NHase substrate scope modification. Modifying the bottleneck region of the substrate access tunnel of NHases has been reported to result in the well-tailored regio- and stereo-selectivity of NHases.15,16 Therefore, the catalytic performance of NHase toward bulky nitrile substrates can also be improved through the tunnel engineering strategy. However, the current tunnel engineering strategies mainly focus on the internal regions inside the tunnel space, and the influence of the entrance region at the substrate access tunnel surface on the catalytic performance has not yet drawn enough attention.Nitrile hydratase , comprising α- and β-subunits, is a metalloenzyme that catalyzes nitriles into amides.Pseudonocardia thermophila JCM 3095 (PtNHase), based on substrate access tunnel calculations. Two successful mutants, PtNHase-βM46R and PtNHase-βA129R, which exhibited high catalytic activity toward bulky substrates were obtained. Structural analysis revealed that the mutation of the gating residues introduced anti-correlated movements to the substrate access tunnel, resulting in a “breathing mode” of the tunnel entrance. These results not only revealed that residues located at the enzyme surface were a key factor in enzyme catalytic performance, but also provided dynamic evidence for insight into enzyme substrate scope broadening.In this study, we discovered that two gating residues at the substrate access tunnel entrance play a key role in expanding the substrate scope of NHases, especially for bulky nitrile substrates. We rationally refined the tunnel entrance region of NHase from 13 In the present study, five NHases from different strains that were applied or have the potential to be applied in industrial amide production were tested using two regular nitrile substrates (acrylonitrile 1a and 3-cyanopyridine 1f) and two relatively bulky nitrile substrates (pentanenitrile 1c and cinnamonitrile 1h). All the tested NHases exhibited high or moderate activity toward 3-cyanopyridine and acrylonitrile  was chosen for further study.In general, NHases exhibit high activity toward regular nitrile substrates such as 3-cyanopyridine and acrylonitrile whereas their activity toward bulky substrates is very low.onitrile ; howeverPtNHase, structural information is necessary. The crystal structure of PtNHase (PDB ID: 1IRE) is available in the Protein Data Bank (PDB), and its substrate access tunnel has been identified.19,20 For a bulky substrate to be catalyzed by an enzyme, the substrate must firstly migrate through the access tunnel and then be accommodated inside the catalytic cavity. Static PDB structure analysis using the CAVER tool showed that the volume of the WT active site cavity was 159.1 Å3, which was larger than that of bulky substrates such as cinnamonitrile  and 1-naphthonitrile   . TherefoPtNHase. The length of the calculated tunnel was 12.8 Å, and the entrance region at the enzyme surface comprised ten amino acid residues which were mainly distributed on an “upper entrance loop” and a “lower entrance loop”: βPhe41, βMet46, βGly47, βLeu48, βLeu127, βPro128, βAla129, βArg131, βIle177, and βGlu188  with conservation grades lower than seven were chosen as hotspots for mutant library construction . Interestingly, when bulky sangivamycin was used as a substrate, the WT enzyme activity was not detected, whereas both mutants exhibited detectable catalytic activity. These results indicated that the substrate scope of PtNHase was successfully broadened.The two mutants were then purified Fig. S4 and charKm values of βM46R and βA129R were 2.2- and 1.7-fold lower, respectively, than that of their parent enzyme. The kcat values of the corresponding mutants increased by 3.1- and 3.8-fold, respectively. Accordingly, the kcat/Km values of these two variants increased by 6.8- and 6.3-fold, respectively. When using 1c or 1h as a substrate, both mutants βM46R and βA129R had slightly lower Km values compared to that of the wild-type, and their kcat values improved to varying degrees, among which βM46R showed 2.8- and 3.6-fold higher kcat values for 1c and 1h while βA129R showed 1.2-fold and 3.5-fold higher kcat values for 1c and 1h, respectively.The kinetic parameters of mutants βM46R and βA129R toward substrates 1c, 1f and 1h were determined , the side-chain orientations of the β46 site differed tremendously. The hydrophobic side chain of βMet46 in WT was buried and oriented towards the interior of the protein, whereas the bulky side chain of βArg46 in PtNHase-βM46R formed a hydrogen bond with a water molecule, showing an obvious outward offset to the solvent  simulations of the WT and the two variants were performed based on the crystal structures. Root mean square deviations (RMSD) from 200 ns simulations were calculated for each dimer of the NHase tetramer Fig. S7. The RMS27,28 Compared with the weak correlations and anti-correlation observed in the WT  information analyses were performed to check the amino acid residue movement correlations based on MD simulations.n the WT , strong n the WT . The strcavities . The act30 However, it is inscrutable that the RMSF values of the β181 to β187 region in the two mutants were much lower than those of the WT, while the RMSF values in other regions were very similar. In addition, stability and activity are generally found to be in a trade-off relationship,25 where activity improvement usually leads to stability reduction. Gibbs free energy (ΔΔG) calculation based on DynaMut2, and assessing changes in stability and flexibility upon single and multiple point missense mutations,31 indicated that compared with the WT, the ΔΔG of the two variants were −0.95 and −0.62 kcal mol−1, respectively, a stability reducing result  also has great potential in amide production because of its excellent stability.34,35 Tailoring the amino acid forming the substrate access tunnel entrance of these NHases might further improve their catalytic performance. Therefore, based on amino acid sequence alignment . Kanamycin, dithiothreitol, and isopropyl β-d-thiogalactoside (IPTG) were purchased from Sangon Biotech . Desthiobiotin was purchased from Sigma-Aldrich . Other commercial reagents like standards and solvents were purchased from Aladdin Reagents , Macklin , Takara , and Sinopharm Chemical Reagent Co., Ltd, respectively.Substrates 1a (acrylonitrile), 1b (isobutyronitrile), 1c (pentanenitrile), 1d (hexanenitrile), 1e (2-cyanopyrazine), 1f (3-cyanopyridine), 1g (benzonitrile), 1h (cinnamonitrile), 1i (1-naphthonitrile), 1j (thiacloprid), 1k (toyocamycin) and the corresponding amides were purchased from Macklin Co., Ltd . Tryptone and yeast extract were purchased from Thermo Fisher Scientific . NaCl, KCl, NaPtNHase were created by site-directed mutagenesis using a whole-plasmid approach.36,37 The primers are listed in ESI Table 2.E. coli BL21(DE3) cells. Single colonies were picked and grown in 5 mL LB medium  containing 50 μg mL−1 kanamycin at 37 °C. Next, CoCl2·6H2O and IPTG were added to a final concentration of 0.1 g L−1 and 0.4 mM when the OD600 value reached 0.6. After induction at 24 °C for 16 h, the cells were harvested by centrifugation at 10 000g for 5 min, and the pellets were washed and resuspended 2–3 times with 10 mM kalium phosphate buffer (KPB) . To screen efficient variants initially, enzyme activity assays were firstly performed using a certain number of whole cells for 1c and 1h at 25 °C for 10 and 5 min in a metal bath thermostat, respectively , and the enzyme was isolated with an AKTA purifier using an isocratic elution of 2.5 mM desthiobiotin in PBS buffer . The target enzyme samples were pooled, and the concentration was adjusted to 0.5 mg mL−1 with the Bradford assay. Purity was checked using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Purified enzymes were stored in aliquots at −80 °C in PBS buffer at pH 7.4.After incubation in an ice bath, the suspensions of the selected variants were sonicated using an ultrasonic cell disruptor for 30 min . Subsequently, the supernatant was collected by centrifugation at 12 000PtNHase activity was assayed using procedures performed previously.38 Enzyme activity was assayed by measuring the amount of amide product generated from the nitrile substrate. The reaction mixture of a total volume of 0.5 mL contained 10 mM KPB (pH 7.4), a certain substrate concentration, and 10 μL of appropriately purified enzyme. The reaction was performed at 25 °C for an appropriate time and terminated by adding acetonitrile (0.5 mL). The amount of product formed in the reaction mixture was determined using an HPLC (Hitachi) equipped with a C18 reverse-phase column. Acetonitrile : water (1 : 2) was used as the mobile phase. The liquid phase detection conditions, including the flow rate, collection wavelength, and time, are listed in ESI Table 3.Km, kcat) of PtNHase and mutants were determined by the enzyme activity detection method mentioned above with a minor modification: the reaction time was changed to 2 min. The substrates were set to 5–7 gradient concentrations from 1 to 100 mM (1c), 2 to 200 mM (1f) and 0.05 to 5 mM (1h), respectively. Statistical analysis was performed by using the Graphpad Prism 7 software package  via a Michaelis–Menten kinetic derivation.The kinetic parameters . The enzyme solution was loaded onto a 6 mL Resource Q column  and separated with a linear gradient of 0.15 to 0.3 M NaCl in a buffer.The crude enzyme samples of PtNHase variants M46R and A129R were further purified by size exclusion chromatography using a Superdex 200 pg HiLoad 16/600 column  equilibrated with 10 mM KPB. The purified enzymes, M46R and A129R, were concentrated to 10 and 6 mg mL−1, respectively, using a 30 K Millipore Amicon Ultra tube. Sessile drop vapor diffusion crystallization experiments were set up in 192-well sessile drop culture plates  by mixing 0.7 μL of the protein solution with 0.7 μL of the reservoir solution containing 1.0–1.4 M trisodium citrate, 0.1 M HEPES, pH 7–7.8, similar to the above previous crystallization of the wild-type PtNHase. Both PtNHase M46R and A129R crystals appeared after two days. Before data collection, crystals were soaked in a cryoprotectant solution containing 1.0 M trisodium citrate, 0.1 M HEPES, pH 7.5, and 50% glycerol. X-ray diffraction measurements for M46R and A129R were performed on a Bruker D8 VENTURE diffractometer.39 The diffraction data for M46R and A129R were indexed, integrated, and merged using the Proteum2 software suite. The crystals of M46R and A129R belonged to the same space group as that of wild-type PtNHase, with nearly identical unit cell dimensions, allowing the wild-type crystal structure (PDB entry 1IRE) to be used directly for initial refinement and electron-density map calculations. Subsequently, the structures were adjusted using model-building to replace the side chains of the mutated residues. Model adjustment and refinement were conducted using Refmac5 and Coot.40 A summary of the data collection and model refinement statistics is provided in Table S5.https://pymol.sourceforge.net/).PtNHase simulation, biological assembly 1 of 1IRE containing an αββα tetrameric crystal structure was used.19 βM46R and βA129R crystal structures with PDB codes 7W8L and 7W8M were used to simulate mutational variants. To obtain tetramers, structural alignment with 1IRE was performed. Before the simulation, protonation states were determined using the PROPKA tool installed in the PDB2PQR server.41–43 Next, the structures were solvated with at least 10 Å in each direction and 0.15 mol L−1 NaCl ions, and a neutralization option was added. The initial simulation box was 93 × 111 × 82 Å. For each variant, we performed a 200 ns Langevin molecular dynamics (MD) simulation at 300 K with a 1 fs time step under atmospheric pressure, with long-range electrostatic interactions calculated using particle mesh Ewald summation. Before the main simulation, 1 ns of water and ion equilibration, 1000 steps of energy minimization, and gradual heating up to 300 K for 60 ps were performed. The NAMD 2.14 code with the CHARMM27 force field was used.44–47 Parameters for the nonstandard active site with cobalt and posttranslationally oxidized cysteines were obtained based on extensive DFT/B3LYP/6-31G and HF/6-31G* quantum calculations published in Peplowski's PhD thesis, used for the first time in SMD simulation for PtNHase in 2008.20 Caver Analyst 2.0 BETA with a probe size of 2.0 Å was used in the analysis of cavities.29 DCCMs have been calculated in R with a bio3D plugin.48For the WT All data included in this study are available upon request by contact with the corresponding author.Dong Ma, Zhongyi Cheng and Zhemin Zhou conceived the project and wrote the paper. Lukasz Peplowski wrote the part about MD simulations. Dong Ma, Zhongyi Cheng, Lukasz Peplowski, Laichuang Han, Yuanyuan Xia, Xiaodong Hou, Junling Guo, Dejing Yin, and Yijian Rao designed and performed all the experiments. Dong Ma, Zhongyi Cheng and Lukasz Peplowski analyzed the results.The authors declare no competing interests.SC-013-D2SC02319A-s001


Chlamydomonas sp. MACC-216 was used to investigate total nitrate removal in TAP medium with sodium nitrate as the sole nitrogen source under several light conditions made up of permuted combinations of three light colors  and three light intensities . It was observed that nitrate removal efficiency is influenced by light color as well as light intensity. Additionally, Chlamydomonas sp. MACC-216 was cultivated in synthetic wastewater under four light conditions, namely, Blue 250, Blue 125 + Red 125, Red 250, and White 250, where it showed the highest nitrate removal efficiency and nitrate reductase activity under the Blue 125 + Red 125 light condition. To observe the impact of light color on the nitrate removal capacity of Chlamydomonas sp. MACC-216, the expression of five genes participating in nitrate transport and reduction  was also analyzed; these genes showed the highest expression under the Blue 125 + Red 125 light condition. Based on the above-mentioned findings, the blue + red light combination emerged as a promising light combination for nitrate removal. Hence, our study suggests the importance of the blue + red light combination together with high light intensity, as the optimal light condition for nitrate removal from synthetic wastewater in comparison to other monochromatic lights with high light intensity.In the present study,  With the increasing global population, there is a surge in industrialization which is responsible for releasing various gaseous, solid, and liquid wastes into the environment. Rising levels of nitrogen and phosphorus in water bodies have been associated with major environmental problems in multiple countries. Nitrate, nitrite, and ammonia represent the main forms of inorganic nitrogen. Nitrite and ammonia are unstable, while nitrate is highly stable making nitrate one of the most common chemical contaminants in water bodies . NitrateNannochloropsis sp., Scenedesmus sp., and Chlamydomonas reinhardtii [Scenedesmus sp. [The ability of microalgae to grow and propagate in wastewater can be influenced by multiple factors including nutrient concentration, light conditions, pH, temperature, salinity, etc. Light has an essential role in the life cycle of cyanobacteria, algae, and higher plants; the color or wavelength of the light strongly influences their growth ,9. Lightnhardtii ,12,13. Bnhardtii ,14,15, wnhardtii ,17. In o−2 s−1 as the maximum growth rate has been observed for different species of microalgae in this light intensity range [Chlamydomonas reinhardtii cc124 and Chlorella ohadii can resist photoinhibition and were shown to grow at light intensities of 3000 µmol m−2 s−1 and 3500 µmol m−2 s−1, respectively [Chlorella vulgaris showed an increasing trend in total nitrogen and total phosphorus removal efficiency when the light intensity was increased from 400 µmol m−2 s−1 to 2000 µmol m−2 s−1 [Similar to light color, light intensity is another factor that has been shown to play a role in the life cycle of microalgae. Microalgae grown under various light intensities showed differences in growth rate and nutrient removal rate ,19,20,21ectively ,23. In tChlamydomonas sp. MACC-216. The specific goal was to determine optimal light color and intensity for efficient nitrate removal by Chlamydomonas sp. MACC-216 as this microalga proved in our previous study to be a promising candidate for nitrate removal [Chlamydomonas sp. MACC-216 grown in tris-acetate-phosphate medium with sodium nitrate as the sole nitrogen source (TAP-N) and synthetic wastewater (SWW). Various light conditions made up of combinations of three light colors  and three light intensities  were used to cultivate Chlamydomonas sp. MACC-216. Chlamydomonas sp. MACC-216 was first grown under each light condition to check for growth and nitrate removal efficiency in TAP-N and then the most efficient light conditions showing maximum growth and nitrate removal capacity were selected for further experiments in SWW. In SWW, Chlamydomonas sp. MACC-216 was grown under four different light conditions, namely, Blue 250, Blue 125 + Red 125, Red 250, and White 250, where the numbers 250 and 125 define light intensity in µmol m−2 s−1. Under the aforementioned four light conditions, various parameters such as growth, nitrate removal efficiency, nitrate reductase activity and expression of genes involved in nitrate transport and reduction were investigated in Chlamydomonas sp. MACC-216.The aim behind the present study was to observe whether various light colors and intensities would affect the nitrate removal efficiency of  removal . The preChlamydomonas sp. MACC-216 was mainly influenced by the light intensity in TAP-N5 (TAP with 5 mM nitrate) and TAP-N10 media (TAP with 10 mM nitrate)  . At 50 µChlamydomonas sp. MACC-216 grown under TAP-N5 medium showed the highest nitrate removal efficiency under Blue 25 + Red 25 and Red 50 light conditions at 50 µmol m−2 s−1 light intensity . An increase in the light intensity from 50 µmol m−2 s−1 to 250 µmol m−2 s−1 led to significantly higher nitrate removal efficiency. Thus, Chlamydomonas sp. MACC-216 grown under Blue 250 removed more nitrate than Chlamydomonas sp. MACC-216 grown under Blue 50 and so on. A clear role of light intensity was observed in total nitrate removal as higher nitrate removal efficiency was observed at higher light intensity. Moreover, Chlamydomonas sp. MACC-216 cultivated in TAP-N10 medium under Blue 125 + Red 125 showed the highest significant nitrate removal efficiency in comparison to any other light condition. Chlamydomonas sp. MACC-216 removed 9.64 mM nitrate when grown under Blue 125 + Red 125 light condition .For experiments to be carried out in SWW, 250 µmol mnditions a. Howeveh in SWW a in comph in SWW  at 250 µondition b. Chlamyectively . ChlamydChlamydomonas sp. MACC-216 was quantified to investigate the influence of various light conditions on this enzyme activity. We observed that Chlamydomonas sp. MACC-216 grown under Blue 125 + Red 125 light had significantly the highest nitrate reductase activity in comparison to any other light conditions used in the study (Chlamydomonas sp. MACC-216 grown under Blue + Red 125 light condition produced 31.09 µmol g−1 of FW (fresh weight) min−1 NO2−, whereas Chlamydomonas sp. MACC-216 grown under Blue 250, Red 250, and White 250 light conditions produced 18.47, 28.41 and 25.4 µmol g−1 of FW min−1 NO2−, respectively  carrier protein. Expression of NRT1, NRT2.1, NRT2.2, NIA, and MCP was significantly higher in Chlamydomonas MACC-216 grown under the Blue 125 + Red 125 light condition  on the nitrate removal capacity of Chlamydomonas sp. MACC-216. Since the light spectrum has been known to affect microalgae, we decided to check its effect on the total nitrate removal by microalgae. Additionally, we also wanted to study whether the combination of blue and red light will enhance the growth and nitrate removal efficiency of Chlamydomonas sp. MACC-216 as absorption bands of photosynthetic pigments lie in the blue and red range of visible light. So far, numerous studies have shown the effects of different light colors or wavelengths and intensities on the growth of various microalgae [Chlamydomonas sp. MACC-216 only seemed to be influenced by light intensity as it increased with increasing light intensity, where the maximum light intensity used was 250 µmol m−2 s−1  lighting showed nitrate removal efficiency of 19% and 9%, respectively [Chlamydomonas reinhardtii, Scenedesmus rubescens, and Chlorella vulgaris) was observed under continuous illumination in contrast to 12 h:12 h (Light:Dark) lighting [The present investigation was undertaken to elucidate the effects of combinations of various light colors  and intensities . NRT1, NRT2.1, and NRT2.2 code for nitrate and/or nitrite transporters, NIA codes for nitrate reductase, and MCP codes for Moco (Molybdenum cofactor) carrier protein. Three families of transporters namely, NRT1, NRT2, and NAR1 have been shown to play a role in the nitrate and/or nitrite transport in Chlamydomonas [NRT1 gene in Chlamydomonas belongs to the nitrate peptide transporter family (NPF) [Arabidopsis as a dual affinity nitrate transporter, but there is a lack of information about this transporter in Chlamydomonas [NRT2.1-6) code for the transporters in the NRT2 transporters family in Chlamydomonas [NRT1, NRT2.1 and NRT2.2 genes from nitrate/nitrite transporter families for expression study as these three seem to play the role of the main nitrate transporters. We observed the highest expression of all these three genes under the Blue 125 + Red 125 light condition in comparison to other light conditions  . NRT1 trydomonas ,35. NRT2ydomonas . Both NRydomonas ,37. Otheydomonas ,39. NAR1nditions . This hiNIA gene plays a role in the reduction of nitrate to nitrite in the cytoplasm. Chlamydomonas algae have a single gene encoding nitrate reductase in their genome [MCP gene is known to play a role in the biosynthesis and transfer of the molybdenum cofactor to the nitrate reductase which is necessary for the enzyme to function [NIA and MCP was also found to be light color dependent as both genes were highly expressed under the Blue 125 + Red 125 light condition in comparison to other light conditions  and was initially maintained in TAP medium consisting of 2.42 g L−1 Tris base, 0.374 g L−1 NH4Cl, 0.204 g L−1 MgSO4·7H2O, 0.066 g L−1 CaCl2·2H2O, 0.287 g L−1 K2HPO4, 0.142 g L−1 KH2PO4, 0.049 g L−1 Na2EDTA·2H2O, 0.039 g L−1 ZnSO4·7H2O, 0.011 g L−1 H3BO3, 0.007 g L−1 MnCl2·4H2O, 0.008 g L−1 FeSO4·7H2O, 0.002 g L−1 CoCl2·6H2O, 0.002 g L−1 CuSO4·5H2O, 0.001 g L−1 (NH4)6Mo7O24·4H2O, and 1 mL L−1 CH3COOH with pH maintained at 7. The final concentration of CH3COOH in TAP medium was 16.8 mM. The microalga was maintained at 25 °C under white light with a light intensity of 50 µmol m−2 s−1 with continuous shaking at 180 rpm in a regime of 16:8 light-dark periods.−1), and 10 mM (849.94 mg L−1), respectively, of sodium nitrate. In addition, 0.001 g L−1 (NH4)6Mo7O24·4H2O was replaced with 0.006 g L−1 of Na2MoO4·2H2O in TAP-N medium so that there is no other nitrogen source other than sodium nitrate.For further experiments, a modified TAP medium (TAP-N) was prepared by substituting different concentrations of sodium nitrate as the nitrogen source instead of ammonium chloride. TAP-N5 and TAP-N10 media were prepared by adding 5 mM (424.97 mg L3 (5 mM), 0.07 g NaCl, 0.04 g CaCl2·2H2O, 0.02 g MgSO4·7H2O, and 0.28 g K2HPO4 were added, and the pH was set at 7.5.Synthetic wastewater (SWW) used in the present study was prepared according to the procedure mentioned in “OECD guidelines for testing chemicals” after slight modifications . To 1 L Chlamydomonas sp. MACC-216 was grown in TAP-N5 and TAP-N10 media under various combinations of light colors (or wavelengths) and light intensities. In total, 12 light conditions made up of combinations of three light colors  and three light intensities  were selected to cultivate Chlamydomonas sp. MACC-216 (Chlamydomonas sp. MACC-216 was grown under each light condition in Multi-Cultivator MC-1000-OD (Photon Systems Instruments) with a continuous light source for 70 h. The absorbance was automatically measured at 720 nm at a gap of 10 h each.MACC-216 . ChlamydChlorella sp. MACC-38 and Chlorella sp. MACC-360 were also grown under the above-mentioned conditions to examine their growth and nitrate removal capacity in SWW.For SWW, only four light conditions were selected namely, Blue 250, Blue 125 + Red 125, Red 250, and White 250  and the w/v) salicylic acid in concentrated H2SO4 was added. Samples were kept at room temperature for 30 min and then 950 µL of 8% (w/v) NaOH in water was added followed by cooling down of samples to room temperature. The absorbance was determined at 410 nm using a Hidex microplate reader.Nitrate estimation was performed at 0 h and 70 h using the salicylic acid method . BrieflyR is the nitrate removal efficiency (%), Ci and Cf are the initial and final concentrations of nitrate (mM) in the growth medium.The nitrate removal efficiency was calculated using the following equation:Chlamydomonas sp. MACC-216 was grown for 48 h under Blue 250, Blue 125 + Red 125, Red, and White 250 light conditions in SWW. After 48 h, 10 mL of culture was collected from each light condition and centrifuged at 4000 rpm for 5 min at 4 °C. Afterwards, supernatants were discarded, and pellets were dissolved in ice-cold 0.1 M phosphate buffer (pH 7.0) followed by sonication at 0.8 cycle, 90% amplitude for 30 s on ice. After sonication, centrifugation was performed at 17,000 rpm for 10 min at 4 °C and supernatants were collected. In a fresh microcentrifuge, to 400 µL of supernatant, 500 µL of 10 mM KNO3, 40 µL of 5mM β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate  and 60 µL of Milli-Q water were added followed by incubation at 25 °C for 30 min. Afterwards, 500 µL of 10 mM zinc acetate was added to stop the reaction followed by centrifugation at 10,000 rpm for 5 min. The supernatant was collected for nitrite estimation by Griess reagent assay as described by Giovannoni et al. [w/v) sulphanilamide  in 5% (v/v) phosphoric acid and 0.1% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride (NED)  in water. Both solutions are stable for several months at 4 °C in darkness. To 100 µL of supernatant, first, 100 µL sulphanilamide solution was added followed by incubation at room temperature for 10 min. Then, 100 µL of NED solution was added followed by further incubation at room temperature for 10 min. After incubation, absorbance was measured at 540 nm in a Hidex microplate reader.For nitrate reductase activity, i et al. . For theChlamydomonas sp. MACC-216 was grown for 70 h under Blue 250, Blue 125 + Red 125, Red 250, and White 250 light conditions in SWW. 1 mL of microalgae sample was collected from each light condition for RNA isolation. RNA isolation was performed using TRIzolTM reagent  by following manual instructions. cDNA was prepared from isolated RNA using RevertAid First Strand cDNA Synthesis Kit  by following manual instructions.For RNA isolation, https://www.ncbi.nlm.nih.gov/). RT-qPCR was performed with 50 ng of cDNA, 500 nM each of forward and reverse primer, 5 µL of SYBR green PCR master mix  and nuclease-free water to a final concentration of 10 µL.To perform quantitative reverse transcription polymerase chain reaction (RT-qPCR), first primers were designed for two reference genes (GAPDH and beta 1 tubulin) and five genes involved in nitrate transportation and reduction . AccessiAnalysis of the results obtained by RT-qPCR was undertaken using Pfaffl method . For thip < 0.05 was considered a significant difference. All experiments were carried out in replicates of 4.Rstudio version 1.2.5019 was used to perform the statistical analyses. All statistical analyses in the current study were performed using one-way analysis of variance (ANOVA) followed by Tukey’s test which was used to detect significant differences among various light conditions, where Chlamydomonas sp. MACC-216 was used to study the effects of various combinations of three light colors  and three light intensities  on its nitrate removal efficiency in TAP-N medium. A role of light intensity as well as light color was observed on the nitrate removal efficiency of the studied microalgae. Furthermore, Chlamydomonas sp. MACC-216 was cultivated under four light conditions namely, Blue 250, Blue 125 + Red 125, Red 250, and White 250 in SWW, where it showed the highest nitrate removal efficiency and nitrate reductase activity under the Blue 125 + Red 125 light condition in comparison to any other light conditions. Relative gene expression values of five genes involved in nitrate transport and reduction were also observed to be the highest under the Blue 125 + Red 125 light condition. Therefore, it is to be concluded that blue + red light combination together with high light intensity represents optimal conditions for the applied green microalgae to efficiently remove nitrate from wastewater.


A 74‐year‐old man was referred to the university hospital for fatigue, weight loss, and thrombocytopenia. Initial examination revealed maculopapular lesions on his body.9/L), and 3% blast cells among leukocytes (14.17 × 109/L). Coagulation assessment found disseminated intravascular coagulation (DIC). Biopsy of cutaneous maculopapular lesions found a perivascular infiltration by blast cells in the dermis, diagnosing leukemia cutis.Subsequent blood tests showed mild normocytic anemia (12.1 g/dL), thrombocytopenia , CD14+ , and CD34−.Flow cytometry immunophenotyping revealed an immature cell population accounting for 50% of bone marrow nucleated cells and harboring the following profile: CD4*12) with 44% VAF and a rare JAK2 point mutation in exon 19, JAK2:c.2437T > G (p.Tyr813Asp) with 50% VAF.Cytogenetic investigations found an isolated loss of the Y chromosome. Next‐Generation Sequencing identified three pathogenic variants: FLT3:c.2503G > T (p.Asp835Tyr) with 13% variant allele frequency (VAF), TET2:c.1304del (p.His435ProfsA diagnosis of acute monocytic/monoblastic leukemia (corresponding to French–American–British M5 of acute myeloid leukemia (AML)) was established according to WHO 2016 classification. Multidisciplinary consultation meeting agreed on chemotherapy treatment using Cytarabin and Venetoclax. Unfortunately, the patient developed a blood transfusion dependency, an aggravation of the cutaneous lesions, and a massive DIC at the start of his treatment and died 19 days later.Haemophagocytic blasts are described in AML and frequently associated with anomalies of chromosome band 8p11 including t KAT6A::CREBBP, inv(8)(p11q13) KAT6A::NCOA2, t KAT6A::EP300, or t, or to t with FUS::ERG fusion gene. Our report provides new cytogenetic and molecular insights into this rare entity. Some authors have underlined the recurrence of association of haemophagocytosis by blasts with DIC with worse clinical outcomes, based on analysis of the few reported cases and series. Up to now, this cytological feature is not included in the disease prognostic criteria, but this question should be seriously considered, given the recurrence of disappointing outcomes in clinical cases showing haemophagocytosis on bone marrow smears.The authors have declared no conflict of interest.


Neuropathic pain is a condition affecting the quality of life of a substantial part of the population, but biomarkers and treatment options are still limited. While this type of pain is caused by nerve damage, in which lipids play key roles, lipidome alterations related to nerve injury remain poorly studied. Here, we assessed blood lipidome alterations in a common animal model, the rat sciatic nerve crush injury. We analyzed alterations in blood lipid abundances between seven rats with nerve injury (NI) and eight control (CL) rats in a time-course experiment. For these rats, abundances of 377 blood lipid species were assessed at three distinct time points: immediately after, two weeks, and five weeks post injury. Although we did not detect significant differences between NI and CL at the first two time points, 106 lipids were significantly altered in NI five weeks post injury. At this time point, we found increased levels of triglycerides (TGs) and lipids containing esterified palmitic acid (16:0) in the blood plasma of NI animals. Lipids containing arachidonic acid (20:4), by contrast, were significantly decreased after injury, aligning with the crucial role of arachidonic acid reported for NI. Taken together, these results indicate delayed systematic alterations in fatty acid metabolism after nerve injury, potentially reflecting nerve tissue restoration dynamics. Neuropathic pain, defined as pain caused by nerve damage, is a condition affecting 6–10% of the population, substantially impacting their quality of life . For resNerve injury is inherently associated with a disruption in lipid composition, since they are the main constituent of important parts of the nervous system, such as the myelin sheath, and are central for proper function . While tWhile all contemporary lipidomics analytical methods are based on mass spectrometry, each approach has specific advantages, such as direct-infusion methods producing robust measurements and straightforward absolute quantification and reverse-phase liquid chromatography coupled to mass spectrometry (LC-MS) providing a wide coverage of lipid compounds . MoreoveWe assessed blood plasma lipidome alterations after sciatic nerve crush injury in a time-course experiment involving seven rats with nerve injury (NI) and eight control rats (CL). The time course included three blood collection points: (i) immediately after the surgery, (ii) two weeks after the surgery, and (iii) five weeks after the surgery  fatty acid residues. Of the six decreased NI-associated lipids annotated at the fatty acid composition level, five had arachidonic acid in their structure , and diglycerides (DG) for palmitic acid a, and PCc = 0.57, p = 7.1 × 10−13). Accordingly, lipids with the strongest statistical differences between NI and CL contained multiple arachidonic acid residues, multiple palmitic acid residues, or a mixture of palmitic acid and the other three “increased” fatty acid residues, i.e., 16:1, 18:1, and 20:3. For instance, the lipid with the strongest decrease between NI and CL was found to be PC 20:4/20:4 , whereas the lipid with the strongest increase between NI and CL was found to be PC 16.0_20.3  among all classes and TG 16.0/16.0/16.0  among TGs  and arachidonic acid (20:4). At the marginal level of statistical significance, we also noted alterations of other fatty acids: 16:1, 18:1, and 20:3. The study reporting alterations in the blood plasma following nerve injury , which iIn nervous tissue, the most consistently reported lipidomic change following peripheral nerve injury is an increase in arachidonic-acid-containing phospholipids ,7,8,9. IThe changes in blood lipid profile were most substantial for storage lipid classes, such as TGs and CEs, which are used to transport fatty acids to tissues . NeverthTaken together, these results suggest systematic alterations in fatty acid metabolism following peripheral nerve injury, possibly reflecting the nerve regeneration processes that occur in the nervous tissue. While the relevance of these results with respect to human studies is exemplified by reports of the association between fatty acid levels and peripheral nerve function , additioThis study was performed using male Wistar rats weighing 150–200 g . All animals were housed in groups of up to 8 per cage (size: 60 cm × 50 cm × 60 cm) under an artificial 12 h light–dark cycle (8:00 a.m.–8:00 p.m.) at a controlled temperature (21 ± 1 °C) and humidity (50–60%). Food and water were available ad libitum. Animal care and handling throughout the experimental procedures were in accordance with the guidelines established in Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.All surgical procedures were performed under anesthesia with combined injection of Zoletil 50  and XylaVET  administered intramuscularly to the animals.Sciatic nerve crush was applied according to the method described by Angelis . BrieflySurgical procedures and blood collection were carried out in two separate temporal batches including both control animals (CL) and rats with nerve injury (NI) . For each batch, blood samples were collected immediately after the surgery, as well as two weeks (14 days) and five weeks (35 days) post surgery. Blood samples collected for analysis of plasma composition were taken from the right ventricle of the heart. Blood was sampled in sterile test tubes . Tubes were stored for 10 min at 3 °C, then centrifuged for 15 min at 2000 rpm at 4 °C. Plasma samples (250–500 µL) were transferred into sterile chilled vials , frozen immediately at −30 °C, with a temperature of −78 °C for long-term storage.2O = 3:10:2.5 was added to the residue for re-extraction. The sample was shaken again for 1 min at 4 °C and centrifuged for 5 min at 13,000 rpm at 4 °C. Another 300 µL of the top layer was separated and pooled, affording a 1300 μL non-polar fraction. The resulting lipid solution was evaporated to dryness in a SpeedVac centrifuge vacuum concentrator at 25 °C.Extraction was performed using the protocol described in . Lipid ev/v)). The sample was shaken for 10 min, incubated in an ice-cold ultrasonic bath for 10 min, and centrifuged for 5 min at 13,000 rpm at 4 °C. Prior to UPLC/MS analysis, samples were diluted 1:5 and 1:2 with ACN:IPA solution (7:3 (v/v)) for positive and negative ionization measurements, respectively.Dry lipid samples were stored at −78 °C for a several days before being re-dissolved and analyzed by UPLC-MS. Dry residue was re-dissolved in 200 µL of a cool (0 °C) mixture of ACN:IPA solution (7:3 (Ultraperformance liquid chromatography mass spectrometry (UPLC-MS) analyses were performed in randomized order. Analyses were performed on an QExactive mass spectrometer equipped with a heated electrospray ionization source  interfaced with a Waters Acquity UPLC chromatographic system . Liquid chromatography (LC) separation was performed using an ACQUITY UPLC BEH C8 reverse-phase column  with a Vanguard pre-column with the same sorbent maintained at 60 °C. Mobile phase A consisted of 10 mM ammonium acetate in water with 0.1% formic acid, and mobile phase B consisted of 10 mM ammonium acetate in ACN:IPA (7:3) with 0.1% formic acid. For the negative detection mode, formic acid in the composition of the mobile phase was replaced by acetic acid. The flow rate was set to 0.4 mL/min in gradient elution mode according to the following program: 1 min 55% B, 3 min linear gradient from 55% to 80% B, 8 min linear gradient from 80% B to 85% B, and 3 min linear gradient from 85% B to 100% B. The phase composition was maintained at 100% B for 4.5 min, after which the column was set to initial conditions (55% B) for 4.5 min. The injection volume was 3 μL.m/z 100–1500 with a mass resolution of 70,000 (FWHM for m/z 200). HESI source conditions were set as follows: ion transfer tube temperature, 350 °C; vaporizer temperature, 250 °C; spray voltage, 4.5 kV; S-lens RF level, 70; AGC target value, 1 × 106; sheath gas (N2) flow rate, 45 arbitrary units (a.u.); auxiliary gas (N2) flow rate, 20 a.u.; sweep gas (N2) flow rate, 4 a.u. External calibration was applied using Pierce LTQ Velos ESI positive ion calibration solution and Pierce ESI negative ion calibration solution before sample analysis to confirm mass accuracy.MS detection was carried out in positive and negative ionization modes in the mass range of m/z values for each polarity and then repeated with an exclusion list containing the same m/z values. Parameters for full MS scan mode were set for both ionization modes as follows. Resolution: 70,000 at m/z 200; AGC target: 5 × 105; IT: 50 ms; mass range: 200−1800. All ions from the inclusion list within the 10 ppm range were subjected to fragmentation with following parameters. Resolution: 17,500; AGC target: 5 × 104; IT: 100 ms. The intensity threshold was maintained at 8 × 103, and the isolation width was set to 1.2 Da. Stepped normalized collision energy was set to 15, 20, and 25%. The dynamic exclusion parameter was set to 12 s. Data was acquired in profile mode, the peptide match option was off, and isotope exclusion was set to on.To achieve a wide coverage of lipid species annotation, iterated data-dependent acquisition (DDA) approach was used. Briefly, an analytical MS/MS protocol was performed with inclusion lists containing approximately 400 Technical samples were analyzed together with the plasma samples. Empty tubes without plasma (extractions blanks) were placed at the end of the experiment and subjected to the same analysis steps as plasma samples. Quality control samples (QC) were incorporated every 12th position to account for sample preparation and measurement variability and were also used for MS/MS analysis. QC samples consisted of aliquots of plasma, mixed after extraction.UPLC-MS spectra were converted from raw to ABF format using an ABF converter. Then, files were processed using MS-DIAL software (version 4.70) . Parametp-value < 0.05 and absolute difference of mean log2 abundances in NI and CL (absolute log2 fold-change) > 0.3 were considered significant. The false-discovery rate (FDR) for significant lipids and overall statistical effect were assessed by permutation procedures. Specifically, for each time point, sample labels were randomized 10,000 times. Then, the mean number of significant lipids for the 10,000 permutations was used as the FDR estimate. To calculate the permutation p-value, we assessed the proportion of permutations for which there was more or an equal number of significant lipids than the true number of significant lipids.Statistical analyses and data visualization were performed in R ,36. For p-values for lipid classes and fatty acid residues were corrected for multiple testing using Benjamini–Hochberg correction, separately for NI-associated lipids decreased and increased in nerve injury. Fatty acids and lipid classes with a corrected p-value < 0.05 were considered significantly enriched, whereas those with a nominal p-value < 0.05 were considered marginally significant. For lipid classes, enrichment scores were calculated as follows: Ntotal is the total number lipids in the analysis, Nsign is the number of NI-associated lipids with increased or decreased abundances, Nclass is the number of lipids in the lipid class, and N is the number of lipids in the intersection of the latter two groups. For fatty acid residues, enrichment scores were calculated similarly by counting fatty acid residues instead of lipids.To study the over-representation of lipid classes and fatty acid residues among NI-associated lipids, we used a hypergeometric test. The test was applied separately for NI-associated lipids with increased and decreased abundances in NI compared to CL, defined according to their difference of mean log2 abundances (log2 fold change) between groups. For lipid classes, the number of lipid species from the corresponding lipid class was considered in the enrichment analysis. For fatty acid residues, the number of times the fatty acid was found in the structure of a lipid, including multiple occurrences of the same fatty acid in a compound, was considered in the enrichment analysis, discarding lipids without annotation at the fatty acid composition level. Sphingoid bases, such as 18:1;O2 for Cer 18:1;O2/18:1, and ether-linked fatty acids, such as O-18:1 for PC O-18:1/16:0, were disregarded in this analysis. The hypergeometric test In this work, we assessed changes in the rat plasma lipidome after sciatic nerve crush injury, an often-used model of neuropathic pain. Blood lipids were measured at three time points: immediately after, as well as two and five weeks post injury. Of the three time points, only the latter showed significant blood plasma lipid alterations between rats with nerve injury and controls. These alterations involved lipids with particular chemical properties: triglycerides and lipids containing specific fatty acid residues, i.e., 20:4, 16:0, 16:1, 18:1, and 20:3, of which the former two fatty acid residues showed strongest statistical effects. These results indicate delayed systematic changes in lipid metabolism potentially associated with post-injury nerve tissue recovery.


Tartary buckwheat “Chuanqiao-2” under 150 mmol·L−1 salt (NaCl) stress was treated with 5 or 10 mmol·L−1 H2O2, and seedling growth, physiology and biochemistry, and related gene expression were studied. Treatment with 5 mmol·L−1 H2O2 significantly increased plant height (PH), fresh and dry weights of shoots (SFWs/SDWs) and roots (RFWs/RDWs), leaf length (LL) and area (LA), and relative water content (LRWC); increased chlorophyll a (Chl a) and b (Chl b) contents; improved fluorescence parameters; enhanced antioxidant enzyme activity and content; and reduced malondialdehyde (MDA) content. Expressions of all stress-related and enzyme-related genes were up-regulated. The F3′H gene (flavonoid synthesis pathway) exhibited similar up-regulation under 10 mmol·L−1 H2O2 treatment. Correlation and principal component analyses showed that 5 mmol·L−1 H2O2 could significantly alleviate the toxic effect of salt stress on Tartary buckwheat. Our results show that exogenous 5 mmol·L−1 H2O2 can alleviate the inhibitory or toxic effects of 150 mmol·L−1 NaCl stress on Tartary buckwheat by promoting growth, enhancing photosynthesis, improving enzymatic reactions, reducing membrane lipid peroxidation, and inducing the expression of related genes.We aimed to elucidate the physiological and biochemical mechanism by which exogenous hydrogen peroxide (H Fagopyrum esculentum Moench, 2n = 8) is an annual or perennial grain crop used as food and forage [F. tataricum), sweet buckwheat (F. esculentum), and golden buckwheat (F. cymosum), in addition to wild species [Buckwheat  and potassium (K+) ions is unbalanced [In natural biological populations, interactions between genotype and environment affect the phenotype of plants. In plants with the same genotype, different environments can lead to various phenotypic differences ,10. Withbalanced . Therefo2O2), superoxide anion (O2−), and hydroxyl radical (OH−) is involved in signaling cascades, and this determines the growth and development process and stress response [2O2 is an important regulatory component of plant signal transduction. It is not only a free radical produced by oxidative stress products, but also helps to maintain plant cell homeostasis [2O2 can regulate the expression of various genes in physiological metabolism, including the genes encoding antioxidant enzymes, regulating biotic and abiotic stress response proteins [2O2 has a concentration-dependent effect on physiological and biochemical processes. That is, high concentrations of H2O2 produce an oxidative stress reaction in plants, leading to cell damage [2O2 acts as a signal molecule participating in tolerance to various abiotic [2O2 is involved in the regulation of physiological activities such as seed germination [Reactive oxygen species (ROS) are biochemical substances that play a vital role in seed dormancy and germination. In particular, the intracellular homeostasis of hydrogen peroxide  could significantly increase the root growth of Tartary buckwheat at the germination stage [−1, H2O2 can significantly promote the germination of Tartary buckwheat under 50 mmol·L−1 NaCl treatment [2O2 were applied to the leaves of Tartary buckwheat to explore the growth, photosynthesis, antioxidant enzyme activity, membrane lipid peroxidation, and expression of related genes of Tartary buckwheat under NaCl stress. The physiological metabolic mechanism by which H2O2 regulates the salt tolerance of Tartary buckwheat was evaluated through correlation and principal component analyses to provide a scientific theoretical basis for exogenous H2O2 treatment to improve the salt tolerance of Tartary buckwheat.As a multifunctional signaling molecule, Hon stage , and at 2O + H2O) and 5H + N (5 mmol·L−1 H2O2 + 150 mmol·L−1 NaCl) treatments was significantly better than that under NaCl (150 mmol·L−1 NaCl + H2O) and 10H + N (10 mmol·L−1 H2O2 + 150 mmol·L−1 NaCl) treatments, and the root growth with 5H + N was better than that with the other three treatments.Under NaCl stress, plant height (PH), stem fresh weight (SFW), stem dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW) of Tartary buckwheat were significantly decreased by 28.22%, 51.90%, 44.63%, 59.60%, and 48.19%, respectively, compared with what they were under the CK treatment A–E. UndeCompared with the CK, the leaf area (LA) and leaf length (LL) of Tartary buckwheat increased significantly by 24.75% and 18.60%, respectively, under 5H + N treatment A,B, and Compared with CK, Chl a, Chl b, and Car were significantly reduced by 40.63–69.33% and 31.69–55.11%, respectively, under the NaCl stress and 10H + N treatments A. Under The total chlorophyll content included Chl a and Chl b. As shown in The change trends of PSII maximum photochemical efficiency (Fv/Fm), effective quantum yield (Fv’/Fm’), non-photochemical quenching coefficient (NPQ), and effective electron transfer rate (ETR) of Tartary buckwheat were similar A,B,E,F. PSII and qP of Tartary buckwheat were similar  were observed under 10H + N treatment  under NaCl stress, and its minimum value (17.194) under 5H + N treatment A. ComparThe PEPC activity of Tartary buckwheat in the CK treatment was significantly higher than under NaCl stress and 10H + N treatment B. PEPC dFtNHX1, FtSOS1, FtNAC6, FtNAC9, FtWRKY46, and FtbZIP83 were highest in the 5H + N treatment. Relative expression levels of all these genes were greater than 10, with the expression of FtNHX1 being the highest, followed by that of FtbZIP83 and FtNAC9. The lowest expression of all genes was observed in FtNAC6 (0.145) under NaCl stress , followed by MnSOD (2.962) and GR (1.503), and the relative expression levels of genes under 5H + N treatment were significantly higher than they were under NaCl stress and 10H + N treatment  than they were under 10H + N treatment  than they were under NaCl stress (PEPC did not differ significantly between NaCl stress and 5H + N treatment (All genes reached their highest expression level under 5H + N treatment B. PEPC sreatment B. The rereatment B. For CAl stress B. Howevereatment B.F3′H was lowest under NaCl stress (0.059), and highest under the 10H + N treatment (1.047); this was significantly higher than the relative expression levels in the NaCl and 5H + N treatments, by 18.47 times and 4.70 times, respectively. The relative expression levels of F3H and CHS showed a decreasing trend, and the relative expression levels were highest under NaCl stress  also contained abscisic-acid-responsive element (abscisic acid responsiveness), methyl-jasmonate-responsive element (MeJA responsiveness), low-temperature-responsive element (low-temperature responsiveness), and other hormones and stress elements, in addition to a large number of light responsiveness elements (light responsiveness). The F3′H promoter sequence contained two salt stress response elements  at 986 base pairs (bp) and 1908 bp, respectively , followed by the correlations between Car and Chl b and between Fs and Fv’ (r = 0.97). (p < 0.05). Fm was significantly positively correlated with PH, RF/DW, SDW, and other growth parameters (p < 0.05), and PEPC was significantly positively correlated (p < 0.05) with leaf indices such as LL and LA (p > 0.05); SOD, GR, and fluorescence parameters such as Fo, Fv’, Fm’ were significantly lower, and POD showed a low correlation with most enzyme activity/content. In addition, = 0.97). A. Plant L and LA A. FigureIn this experiment, principal component analysis was carried out on 28 indices of morphology, physiology, and biochemistry. The two principal components extracted by PCA explained 73.3% and 12.3% of the variation B. The CK+ and K+ concentrations in the soil, which result in hyperosmotic conditions that prevent plants from absorbing water, causing adverse effects such as stunted growth [2O2 is a relatively stable, freely diffusing, and long-lived reactive oxygen molecule [2O2 has different regulatory effects in different varieties of plants and different environments. In the present study, the growth of Tartary buckwheat under 5H + N treatment was significantly better than that under the CK, NaCl stress, and 10H + N treatments. Compared with NaCl stress, the PH, SFW/SDW, and RFW/RDW of Tartary buckwheat were significantly increased under 5H + N treatment (p < 0.05), particularly the RFW, which increased nearly 1.5-fold , a significant positive correlation between PEPC activity and leaf morphology , and a significant negative correlation between MDA content and enzyme activity and leaf index . PC1 and PC2 identified through principal component analysis explained 73.3% and 12.3% of the variation, respectively  of Xichang Institute of Agricultural Sciences, Liangshan Prefecture, Sichuan Province, China.−1 NaCl [−1 H2O2 was used for foliar spraying in this experiment. The transplanted Tartary buckwheat was cultured in a culture room under specific conditions . At 40 days after transplanting (DAT), growth indices and photosynthesis parameters were measured, and sampling was used to measure different physiological and biochemical indices. The schematic diagram of the experimental design is shown in Tartary buckwheat variety “Chuanqiao-2” seeds with uniform size and no pests and diseases were selected. Seeds were rinsed with distilled water, sterilized with 1% NaClO for 10 min, then rinsed with double-distilled water (DDW) several times. The sterilized and rinsed seeds were sown on the surface of 2 layers of qualitative filter paper in a sterile Petri dish (90 mm diameter), and placed in a constant-temperature incubator  for 7 days. At 7 d after sowing (DAS), the Tartary buckwheat seedlings with uniform growth were transplanted into plant pots  containing mixed nutrient soil (soil:substrate = 1:1) that had been sterilized by high temperature. At 20 d after transplanting (DAT), each pot was irrigated with 2 liters (L) of Hoagland nutrient solution containing 150 mmol·L−1 NaCl ,60. BaseAfter the plants were uprooted, the surface soil was washed, and the plant height (PH) was measured directly using a ruler, and expressed in cm. The stem fresh weight (SFW) and root fresh weight (RFW) were measured using a thousand-point electronic balance, and then samples were placed in an oven at 105 °C for 30 min, and dried at 65 °C to a constant weight. Stem dry weight (SDW) and root dry weight (RDW) were measured with a thousand-point electronic balance and expressed in mg.2 and cm, respectively. Relative water content (LRWC) was determined according to Su et al. [Leaf area (LA) and leaf length (LL) were determined with an LI-3000C portable leaf area analyzer , and are expressed in cmu et al. , using tv/v) ethanol, soaked in dark conditions for 72 h, and then the absorbance at 663 nm, 645 nm, and 470 nm was measured using a microplate reader . Chl a, Chl b, and Car were determined using the following formulae:The contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) were determined according to Hossain et al. . Briefly−2·s−1, and an atmospheric CO2 concentration of 380 ± 5 mmol·mol−1. The measurements were conducted between 10 a.m. and 2 p.m. on a sunny day. Parameters were calculated using the following formulae:The following parameters were measured in the third top-down leaf of the plant’s fully expanded leaves according to the method of Lu et al. , using tBiochemical indicators were estimated using the BOXBIO kit  following the manufacturer instructions.−1.Catalase activity (CAT), peroxidase activity (POD), superoxide dismutase activity (SOD), ascorbate peroxidase activity (APX), and glutathione reductase activity (GR) were estimated according to Kong et al. . The corg at 4 °C to collect the supernatant and then placed on ice for measurement. A microplate reader  was used at 265 nm and 412 nm, respectively, after adding related reagents. The absorbance values for AsA and GSSG are expressed in nmol·g−1 and ug·g−1, respectively.Ascorbic acid content (AsA) and oxidized glutathione content (GSSG) were measured according to Rama and Prasad . Homogen−1.Malondialdehyde content was measured according to Hodges et al.  We took −1.PEPC activity was measured according to Aoyagi and Bassham . The samA 0.1 g sample of Tartary buckwheat leaves was fully ground in liquid nitrogen, and then total RNA was extracted using the E.Z.N.A. Plant RNA Kit . The integrity of extracted RNA was checked by 1% agarose gel electrophoresis, and RNA purity and concentration were measured using a spectrophotometer . RNA was reverse-transcribed into the first-strand cDNA using the Hiscript II Q RT Supermix for qPCR Kit  in a 20 μL reaction system.FtH3 as the internal control gene, and the amplification primers are shown in 2O. qPCR was performed using CFX96 Real-Time System . The relative expression levels of target genes compared to the internal control gene were calculated using the 2−ΔΔCt method [Primer Premier 5.0  software was used to design specific primers for qPCR, with t method .http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the presence of cis-acting elements in promoter sequences (the upstream 2000 bp) of key genes of the flavonoid synthesis pathway.The PlantCare website (We accessed on 14 July 2021 to p < 0.05), multiple comparisons (Duncan), and correlation analysis (Pearson) were performed with IBM SPSS Statistics 26.0 software , and the results are expressed as the mean ± SD. Column charts were drawn using GraphPad Prism7.0 software . Principal component analysis plots were made using Origin 2019b software. Correlation heatmaps and cis-acting element visualizations were generated using TBtools v1.09876 [Analysis of variance (ANOVA, v1.09876 .2O2 could promote the growth of Tartary buckwheat under NaCl stress, increase the accumulation of chlorophyll content, enhance electron transfer and transformation during photosynthesis, effectively improve enzymatic reactions, reduce cell membrane lipid peroxidation, induce or activate the expression level of related genes, alleviate the toxic effect of NaCl stress on Tartary buckwheat, and promote normal physiological metabolism and biochemical reactions in Tartary buckwheat. A concentration of 5 mmol·L−1 H2O2 produced the optimal promoting effect on Tartary buckwheat under 150 mmol·L−1 NaCl stress. Appropriate concentrations of H2O2 can alleviate the inhibitory effect of salt stress, but the mechanisms and signaling pathways of H2O2-mediated salt tolerance still need to be further dissected in detail, as well as how H2O2 regulates related genes to activate defense systems to alleviate salt stress.In this study, compared with CK and NaCl treatments, spraying exogenous H