GLS2, which encodes a glutaminase, are inversely associated with EMT. GLS2 down-regulation was correlated with reduced mitochondrial activity and glutamine independence even in low-glucose conditions. Restoration of GLS2 expression in GLS2-negative breast cancer cells rescued mitochondrial activity, enhanced glutamine utilization, and inhibited stem-cell properties. Additionally, inhibition of expression of the transcription factor FOXC2, a critical regulator of EMT in GLS2-negative cells, restored GLS2 expression and glutamine utilization. Furthermore, in breast cancer patients, high GLS2 expression is associated with improved survival. These findings suggest that epithelial cancer cells rely on glutamine and that cells induced to undergo EMT become glutamine independent. Moreover, the inhibition of EMT leads to a GLS2-directed metabolic shift in mesenchymal cancer cells, which may make these cells susceptible to chemotherapies. Identifying bioenergetics that facilitate the epithelial to mesenchymal transition (EMT) in breast cancer cells may uncover targets to treat incurable metastatic disease. Metastasis is the number one cause of cancer-related deaths; therefore, it is urgent to identify new treatment strategies to prevent the initiation of metastasis. To characterize the bioenergetics of EMT, we compared metabolic activities and gene expression in cells induced to differentiate into the mesenchymal state with their epithelial counterparts. We found that levels of  Highly proliferative tumors consume glucose and amino acids, including glutamine, to sustain the metabolic demands that support biomass production . GlutamiGLS2 gene [In mammals, including humans, two enzymes, GLS and GLS2, catalyze the conversion of glutamine to glutamate. GLS and GLS2 have distinct patterns of expression and regulation in different organs and tumor types . GLS is LS2 gene ,10.Many tumors depend on glutamine for growth, and glutamine addiction is associated with GLS ,11. PharGLS2 expression and the promotion of glutamine independence even in low-glucose conditions and in the presence of GLS. In addition, we observed that GLS2 re-expression enhanced glutamine consumption and reduced sphere formation. The transcription factor FOXC2 is critical to maintaining mesenchymal and stem-cell properties [GLS2 expression in breast cancer patients and found that, in line with our data, high GLS2 expression is inversely correlated with the EMT gene signature. Further, we found that GLS2 copy number deletions were over-represented in the basal breast cancer subtype; a subtype with poor clinical outcomes and high metastatic potential [GLS2 expression correlates with improved overall survival in breast cancer patients.Considerable evidence suggests that the epithelial to mesenchymal transition (EMT) program contributes to the development of therapy resistance and metastasis ,22,23,24operties ,28 and hoperties ,36,37,38otential . In suppGLS was induced and GLS2 was suppressed compared to control epithelial cells, even though both have the capacity to convert glutamine to glutamate . In this analysis we observed that the 143 patients who had lost one copy of GLS2 exhibited a corresponding reduction in GLS2 RNA expression . In line with results obtained from analysis of cell lines, expression of GLS2 is inversely correlated with GLS in 52% of the cancer types analyzed, including breast cancer  induced to undergo EMT by stable expression of the transcription factor Snail relative to the vector-expressing control cells A. The SnTo further characterize nutrient utilization, we measured glucose utilization and the glycolytic index in cells induced to undergo EMT compared to the control cells C,D. To a13C into GSH in HMLER cells that expressed FOXC2 than control cells after 6 h of growth in the presence of the 13C-labeled glutamine  displayed reduced mitochondrial membrane potential , impaireGLS2 suppression metabolically supports the EMT, we overexpressed GLS2 in the SUM159 breast cancer cell line, a line that has mesenchymal characteristics; experiments were also performed with the same cells expressing control vector  correlates with reduced survival compared to patients whose tumors express GLS2 (red line) based on a hazard ratio of 0.625 (p = 0.019) (GLS expression based on a hazard ratio of 1.499 (p = 0.045)  expressed GLS2 and GLS in an inverse relationship similar to that observed in breast cancer (GLS2 expression (blue circle) relative to GLS expression is a predictor of improved survival (risk coefficient < 0) in breast cancer and in 69% (22 out of 32) of the other cancers that we analyzed (GLS expression (red circle) relative to GLS2 expression correlates with poor survival in 66% (21 out of 32) of the cancers we analyzed  A. Notabl= 0.045) B. We thet cancer C. Based analyzed C. Higheranalyzed C.GLS expression and negatively correlates with GLS2 expression. The highly metastatic basal subtype is enriched with GLS amplifications and GLS2 deletions, but in less aggressive subtypes such as luminal A and luminal B, there are more frequent GLS2 amplifications and GLS deletions. Previous studies have described the opposing roles that GLS and GLS2 have in cancer [GLS and GLS2 has been described in the glioblastoma cell line T98G [GLS transcription is directly induced by oncogenes KRAS and MYC, and up-regulation of GLS contributes to glutamine addiction in primary tumors [We previously identified changes in metabolite abundances, particularly in glutamine and glutamate, that occur with the onset of EMT . Here wen cancer ,45,46,47ine T98G . GLS tray tumors ,45,46,48We sought to understand how the distinct expression patterns of these two glutaminases might metabolically benefit the EMT. We metabolically characterized cells that were induced to undergo EMT and assessed their abilities to utilize glucose and glutamine compared to their epithelial counterparts. We observed that cells that have undergone EMT were viable in low glucose conditions, even in the absence of glutamine, unlike epithelial control cells. We reason that this metabolic flexibility enables cancer cells to survive during the metastatic cascade where they encounter conditions of fluctuating nutrient availability. It has been previously demonstrated that, depending on the substrate used for energy synthesis, some cancer cells acquire metabolic flexibility upon transformation . HoweverWe hypothesized that the loss of GLS2 expression upon the induction of EMT is responsible for the glutamine-independent phenotype and reduced mitochondrial activity. To test this, we over-expressed GLS2 in the GLS2-negative and EMT-enriched breast cancer cell line SUM159. We found that GLS2 over-expression led to an enhancement of mitochondrial activity, glutamine consumption, and GSH and ATP production even in the presence of GLS. Furthermore, this enhanced mitochondrial activity mediated by GLS2 led to a decrease in secreted lactate and reduced the frequency of mammosphere formation. Notably, these effects occurred without altering the expression of EMT markers implying that the metabolic program alone can significantly impact EMT-induced stem-cell properties.FOXC2, ZEB1, or Snail. In future studies it would be valuable to test if inhibiting GLS while over-expressing GLS2 confers a more robust attenuation of EMT properties or tumorigenic potential.We have previously demonstrated strong evidence that the EMT program imparts stem-cell properties to cancer cells ,26,28. EGLS2 are associated with improved survival over 5 years (p = 0.019); however, GLS levels were not correlated with survival (p = 0.045). These findings indicate that the inverse relationship between GLS2 and FOXC2 may have a stronger predictive value for outcomes in breast cancer than the correlation between GLS2 and GLS and warrants further validation.We, and others, have established the functional link between EMT and FOXC2 ,27,28,30Considerable evidence links the EMT program with the development of resistance to chemotherapy and tumor relapse . For exa2. All cell lines derived from HMLE cells were cultured in MEGM (Lonza CC-3051)/DMEM F12  (1:1) supplemented with penicillin and streptomycin (Gibco/Life Technologies), insulin , hEGF (Sigma 9644), hydrocortisone (Sigma H0888), and bovine pituitary extract. MCF7 and MDA-MB-231 cells were cultured in DMEM/F12  supplemented with 10% fetal bovine serum (FBS) (Sigma), and penicillin and streptomycin. SUM159 cells were cultured in Ham’s F12 (Corning 10-090-cv) medium containing 10% FBS and penicillin and streptomycin. HEK293T cell lines were cultured in DMEM  supplemented with 10% FBS and penicillin and streptomycin. MCF10A cells were cultured in DMEM/F12 (Corning 10-090-CV) supplemented with 5% horse serum (Sigma H1138), 20 ng/mL human epidermal growth factor (Sigma E9644), 0.5 µg/ml hydrocortisone, 100 ng/mL cholera toxin (Sigma C-8052), 5 µg/mL insulin, and penicillin and streptomycin.All cell lines were cultured at 37 °C in 5% CO4 cells per well into 96-well PM-M2 plates . Cells were cultured in 50 µL of Biolog M1 medium (Biolog 72301) supplemented with penicillin and streptomycin, 200 mM glutamine, and 5% FBS. After 24 h, 10 µL of Biolog MA redox dye (Biolog 74351) was added to each well, and the plate was placed in the Biolog OmniLog2.3 plate reader to obtain kinetic data at 15 min intervals for 24 h. Data were analyzed with Biolog software packages PMM_Kinetic and PMM_Parametric to obtain the average of the initial rate values at 8 h. Each cell line was tested in triplicate.Cells were plated at 1 × 1050 was calculated using GraphPad Prism software.Cells were plated at 3000 cells per well in 96-well plates in growth medium. After 24 h, medium was removed by aspiration, and cells were washed with PBS. Medium was replenished with 100 µL of glucose and glutamine-free DMEM containing 12 mM, 3 mM, 1.5 mM, 0.8 mM, 0.4 mM, or 0 mM of glucose (Sigma). Each concentration was evaluated in four replicates. After 24 h, 20 µL of CellTiter 96 Aqueous One Solution  was added to each well. After incubation at 37 °C for 60 min, absorbance at 490 nm was determined. Raw data were converted to relative percent viabilities, transformed to log, and the IC2. After 12 h, the medium was changed to Seahorse base medium  supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate and adjusted to pH 7.4. The probe cartridge was prepared to have 0.5 µM oligomycin in port “A”, 0.25 µM FCCP in port “B”, and 0.25 µM Rotenone with 0.25 µM Antimycin in port “C”. For the glycolysis stress test, the probe cartridge was prepared to have 20 µL of 50 mM glucose in port “A”, 11 mM oligomycin in port “B”, and 650 mM 2-deoxyglucose in port “C”. ECAR was normalized to mpH/min. Oxygen consumption rate and extracellular acidification rate were measured by the Seahorse XFe96 Analyzer (Seahorse Biosciences).For the MitoStress test, cells were seeded at 15,000 cells per well in an XF96 cell culture plate with the appropriate medium. The XF96 probes were calibrated with 200 µL of calibrant solution and incubated at 37 °C in the absence of CO4 per well in a 96-well plate. After 24 h, an ATP standard curve was prepared and 100 µL of CellTiter-Glo reagent was added to each test sample and standard. After incubating at room temperature for 10 min, the luminescent signal was recorded with a luminometer.Cells were plated in triplicate at 1 × 105 cells were plated in triplicate in 6-well plates. After 24 h, growth medium was removed by aspiration, and cells were washed with PBS. Cells were lysed with 250 µL of RIPA buffer. A glutamate standard curve was prepared in a 96-well plate. Test wells contained 25 µL of sample, 175 µL of reaction buffer, and 2 µL of L-GLDH enzyme. The absorbance at 340 nm was measured at time 0 and in 40 min increments until the signal plateaued.Samples were analyzed with the Glutamine/Glutamate Determination Kit (Sigma GLN-1) following the manufacturer’s recommendations. Briefly, 5 × 10Cells were plated at 10,000 cells per well in 6 replicates in a 96-well plate. After 24 h, reduced glutathione was assayed with GSH-Glo Glutathione Assay (Promega V6911). Briefly, the growth medium was removed from each well, and cells were washed with PBS. A GSH standard curve was prepared, and cells were incubated with GSH-Glo reagent. After 30 min, Luciferin detection reagent was added to each well and incubated for 15 min. The luminescence was measured using a luminometer.Cells were plated on glass coverslips at 50% confluency. After 24 h cells were stained with 20 nM MitoTracker Red CMXRos (Life Technologies) under the same culture conditions. After 30 min, cells were fixed with 4% paraformaldehyde. After washing with PBS, coverslips were treated with 5% glycine in PBS for 15 min. Actin was stained with 488 Phalloidin , and the nuclei were stained with DAPI. Fluorescent images were obtained with a Zeiss fluorescent microscope. The fluorescence intensities of 100 cells were analyzed and quantified by ImageJ software to obtain the relative fluorescence intensity (RFI).13C-Glutamine (17 mg) was added to 50 mL of HMLE medium and filtered. HMLER cells expressing FOXC2 or HMLER control cells were seeded into wells of 10 cm plates at 70% confluency; each cell type was analyzed in four replicates. After 24 h, medium was aspirated, and 5 mL of labeled glutamine solution was added to the cells. A sample was collected immediately after addition. After 6 h, medium was removed by aspiration. Cells were washed with ice-cold PBS and 1 mL of methanol/water (1:1) was added to the cells. Cells were scraped into 15 mL Falcon tubes and immediately flash frozen by submerging tubes into liquid nitrogen. Samples were stored at −80 °C until metabolite extraction. Cell pellets were thawed at 4 °C and subjected to freeze–thaw cycles in liquid nitrogen and ice three times to rupture the cell membrane. Following this, 750 µL of ice-cold methanol/water (4:1) containing 20 µL of internal standards were added to each cell extract. Next, ice-cold chloroform/water (3:1) was added. Organic and aqueous layers were separated. The aqueous portion was deproteinized using a 3-KDa filter , and the filtrate containing metabolites was dried under vacuum . Prior to mass spectrometry, the dried extracts were re-suspended in 100 µL methanol/water (1:1) containing 0.1% formic acid and analyzed by liquid chromatography-mass spectrometry (LC/MS). U-Liquid chromatography was performed using an Agilent 1290 Series HPLC system equipped with a degasser, binary pump, thermostatted auto sampler, and column oven . All samples were kept at 4 °C. A 5 µL aliquot of sample was delivered to a 4.6 mm i.d. × 10 cm Amide XBridge HILIC column (Waters) at 300 µL/min. Chromatography was performed using 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Gradients were run starting from 85% solvent B to 30% B from 0–3 min, 30% B to 2% B from 3–12 min, 2% B was held from 12–15 min, 2% B to 85% B from 15–23 min, and 85% B was held for 7 min to re-equilibrate the column.13C incorporation into glutamine by measuring the expected precursor/product ion pairs: M + 0: 147.08/130.1, M + 1: 148.08/131.1, M + 2: 149.08/132.1, M + 3: 150.08/133.1, M + 4: 151.08/134.1, and M + 5 152.08/135.1. SRM was used to determine 13C incorporation into GSH by measuring the expected precursor/product ion pairs: M + 0: 308.0911/76, M + 1: 309.0911/76, M + 2: 310.0911/76, M + 3: 311.0911/76, M + 4: 312.0911/76, and M + 5: 313.0911/76. Mass isotopomer distribution was calculated and corrected for natural abundance.Samples were analyzed by a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies). Metabolites were measured using positive ionization mode with an electrospray ionization (ESI) voltage of 3000 eV. Approximately 9–12 data points were acquired per detected metabolite. Selected reaction monitoring (SRM) was used to determine 6 cells were evaluated in triplicate. Cells were collected using trypsin. All cell pellets were stored at −80 °C until analysis. Cell pellets were thawed at 4 °C and subjected to freeze–thaw cycles in liquid nitrogen and ice three times to rupture the cell membrane. Following this, 750 µL of ice-cold methanol/water (4:1) containing 20 µL of internal standards were added to each cell extract. Next, ice-cold chloroform/water (3:1) was added. Organic and aqueous layers were separated. The aqueous portion was deproteinized using a 3-kDa filter , and the filtrate containing metabolites was dried under vacuum (Genevac EZ-2plus). Prior to mass spectrometry, the dried extracts were re-suspended in 100 µL methanol/water (1:1) containing 0.1% formic acid and analyzed by LC/MS.For profiling of metabolites associated with glycolysis and TCA pathways, 5 × 102) 4 µm, 100 Å, 2.1 × 150 mm column  at 200 µL/min. Chromatography was performed using 5 mM ammonium acetate in water at pH 9.9 (solvent A) and 100% acetonitrile (solvent B). Gradients were run starting from 80% solvent B to 2% B over a 20 min period followed by 2% B to 80% B for a 5 min period, further followed by 80% B for a 13 min period. The flow rate was gradually increased during the separation from 200 µL/min (0–20 min) to 300 µL/min (20.1–25 min) to 350 µL/min (25–30 min) to 400 µL/min (30–37.99 min) and was then returned to 200 µL/min (5 min). Samples were analyzed using a 6495 QQQ triple quadrupole mass spectrometer using SRM. Metabolites associated with glycolysis and TCA pathways were measured using negative ionization mode with ESI voltage of −3500 eV. Approximately 9–12 data points were acquired per detected metabolite.Liquid chromatography was performed using an Agilent 1290 Series HPLC system equipped with a degasser, binary pump, thermostatted auto sampler, and column oven . All samples were kept at 4 °C. An aliquot of 5 µL of sample was analyzed. Samples were delivered into the spectrometer via normal phase chromatographic separation using a Luna Amino  according to the manufacturer’s instructions. RNA was quantified with a Thermo Scientific Nanodrop 2000c instrument. RNA sample purity was verified based on the ratio of absorbance at 260 nm to that at 280 nm. RNA samples were normalized to 1 µg. qRT-PCR experiments were run in triplicate, and the mean was used for the determination of mRNA levels. Relative quantification of the mRNA levels was performed using the comparative Ct method with human Cells were plated at 50% confluency on glass coverslips in 6-well plates. After 24 h, the medium was removed aspiration, and cells were washed with PBS. Cells were fixed in 4% paraformaldehyde at room temperature for 20 min and then washed with PBS. Cells were quenched with 4% glycine for 15 min at room temperature and washed with PBS. Cells were blocked with 5% BSA, 0.1% Triton-X100 in PBS for 1 h at room temperature. After three washes with PBS, cells were incubated with (1:200) primary GLS2 (LGA) antibody in 1% BSA for 2 h at room temperature. Cells were washed three times with PBS and then incubated with a secondary antibody at 1:1000 in 1% BSA for 1 h at room temperature in the dark. Cells were washed with PBS and nuclear stain DAPI was added to the cells at 1:1000. After 5 min in the dark. Cells were washed three times with PBS, and coverslips were mounted on glass slides with DAKO mounting solution and incubated at 4 °C overnight to dry. Fluorescent images of the slides were taken with the same exposure time for each slide. The anti-GLS2 was generously provided by Dr. José M. Matés and Dr. Javier Márquez, University of Málaga, and was generated as previously described .FOXC2 mutants were generated as described previously [HMLER-derived cell lines that express Snail and FOXC2, were generated as described previously . The S36eviously . The HMLGLS and GLS2 in the EMT-74 panel samples and compared the expression in the epithelial or mesenchymal state using a Student’s t-test. As controls, we evaluated expression of an epithelial marker (CDH1), mesenchymal markers , and a housekeeping gene (GAPDH).We had previously assembled the EMT-74 data set, a panel of 74 samples across six data sets representing the gene expression profiles of cells before and after an EMT . We examhttp://gdac.broadinstitute.org/, January 2016 version). For copy number and RNA expression association analysis, the LinkedOmics portal (http.linkedomics.org) was used. A threshold for copy number data was set by the GISTIC2 algorithm. Primary solid tumors were used for analysis. Statistical analysis was performed using the computing environment R (version 3.4). The one-way analysis of variance (ANOVA) was performed using aov function and p-value was obtained. Loss of one or two copies of a gene was considered as a deletion whereas gain in one or two copies of gene was considered as amplification.The copy numbers and RNA-seq data for 1075 patients in TCGA was downloaded from the Firehose of the Broad Institute .In a 96 well plate, 1 × 104 cells per well in 6 well-plates. Aliquots stained with Trypan Blue were counted manually every 24 h for 72 h. Cells were plated in triplicate at 1 × 10Mammosphere culture medium was made with 1% methylcellulose and MEGM without bovine pituitary extract. In a 96-well low attachment plate, 500 cells per well in six replicates were cultured for 14 days. Every 3 days, 50 µL of fresh media was added. Mammospheres were photographed, and spheres with a diameter greater than 80 µm were counted.GLS2 and FOXC2 and EMT score was performed across pan-cancer using the Spearman correlation. The normalized RNA expression data for TCGA samples was downloaded from Broad Firehose release 28 January 2016 run  ([https://doi.org/10.7908/C11G0KM9). Primary tumor samples but not normal tissue or metastases samples were selected for analysis. The correlation of the RSEM-normalized RNA expression values between genes in 33 TCGA cancer types was determined using the correlation function cor.test in R . The EMT score was calculated by single sample gene set enrichment analysis (ssGSEA) using normalized RNA expression of each cancer type and Hallmark gene set (MSigDB database version 6.2) by GSVA package (version 1.3).The correlation between expression of #11383)  package in R was used for the survival analysis and Cox coefficient was calculated for each cancer type. Visualization was performed using ggplot2 (v3.2.1) and survminer (v0.4.6) packages in R.Survival analysis was carried out using vital status and overall survival (days from diagnosis to last follow-up) for the TCGA pancancer patients. Only patients with 5 years of follow-up were selected and used for the analysis. Cox proportional-hazards model implemented in p-values were adjusted for multiple testing using the Benjamini Hochberg method, significance was set at an adjusted p-value < 0.001. Metabolic enzymes and transporters were identified using a previously published comprehensive gene set [Gene expression analysis was performed on a previously published dataset as previously described in . Analysigene set .t-test.Unless otherwise stated, all samples were assayed in triplicate. All in vitro experiments were repeated at least three independent times, and the animal study included 10 mice in each group. Unless otherwise indicated, data are presented as means ± standard deviation (SD), and significance was calculated using the Student’s unpaired two-tailed Cell pellets were obtained by trypsinization. Cell pellets were lyzed with RIPA buffer (Sigma R0278) containing phosphatase inhibitor  and protease inhibitor (Roche 11697498001) and vortexed vigorously then incubated on ice for 30 minutes, spun down, and cell debris removed. After quantification by Bradford assay, 50 µg of protein was denatured in β-mercaptoethanol at 100 °C for 10 minutes. Samples were loaded onto a 10% SDS-agarose gel and run at 110 V. For blots, antibodies were used at 1:1000 in 5% milk in TBST. Antibodies used were anti-Snail , anti-FOXC2 , anti-ZEB1 , anti-actin , and anti-tubulin . To quantify the protein band intensity, densitometry analyses were performed by evaluating band intensity of mean gray value using ImageJ software . In brieGLS2 expression relative to GLS is a predictor for improved survival and higher GLS2 expression negatively correlates with an EMT signature. Our data provide insights into how metabolic alterations occur in parallel to the onset of the transcriptional program that supports EMT and its aggressive features. Since we found that inhibition of FOXC2 changes the metabolism of cancer cells by promoting mitochondrial activity, future work is warranted to test whether combination treatment strategies can be employed to take advantage of altered metabolic states in cells that have features of EMT.In conclusion, we demonstrate here that a critical regulator of the EMT, FOXC2, also induces metabolic flexibility in breast cancer. Furthermore, we showed that GLS2 expression and mitochondrial metabolism are altered upon induction of EMT. The expression of GLS2 and mitochondrial metabolic program were restored when FOXC2 expression or activity were inhibited. We also demonstrated that in multiple cancer types, including breast cancer, higher


Previous studies reported that URB597 (URB) had therapeutic potential for treating chronic cerebral hypoperfusion (CCH)-induced neuroinflammation and autophagy dysfunction. However, the interaction mechanisms underlying the CCH-induced abnormal excessive autophagy and neuroinflammation remain unknown. In this study, we investigated the roles of impaired autophagy in nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 inflammasome activation in the rat hippocampus and the underlying mechanisms under the condition of induced CCH as well as the effect of URB treatment.N-acetylcysteine (NAC), (7) BCCAo+URB, (8) BCCAo+URB+3-MA, (9) BCCAo+URB+CQ, (10) BCCAo+URB+minocycline, (11) BCCAo+URB+NAC. The cell localizations of LC3, p62, LAMP1, TOM20 and NLRP3 were assessed by immunofluorescence staining. The levels of autophagy-related proteins , NLRP3 inflammasome-related proteins , microglial marker (OX-42) and proinflammatory cytokines (iNOS and COX-2) were evaluated by western blotting, and proinflammatory cytokines (IL-1β and TNF-a) were determined by ELISA. Reactive oxygen species (ROS) were assessed by dihydroethidium staining. The mitochondrial ultrastructural changes were examined by electron microscopy.The CCH rat model was established by bilateral common carotid artery occlusion (BCCAo), and rats were randomly divided into 11 groups as follows: (1) sham-operated, (2) BCCAo; (3) BCCAo+autophagy inhibitor 3-methyladenine (3-MA), (4) BCCAo+lysosome inhibitor chloroquine (CQ), (5) BCCAo+microglial activation inhibitor minocycline, (6) BCCAo+ROS scavenger CCH induced microglial overactivation and ROS accumulation, promoting the activation of the NLRP3 inflammasome and the release of IL-1β. Blocked autophagy and mitophagy flux enhanced the activation of the NLRP3-CASP1 inflammasome pathway. However, URB alleviated impaired autophagy and mitophagy by decreasing mitochondrial ROS and microglial overactivation as well as restoring lysosomal function, which would further inhibit the activation of the NLRP3-CASP1 inflammasome pathway.These findings extended previous studies indicating the function of URB in the mitigation of chronic ischemic injury of the brain. A substantial amount of evidence indicates that chronic cerebral hypoperfusion (CCH), a state of chronic cerebral blood flow reduction, is associated with several cerebrovascular and neurodegenerative disorders including Alzheimer’s disease (AD), carotid stenosis/occlusion, cerebral arteriovenous malformation, dural arteriovenous fistula, moyamoya disease and cerebral small vessel disease –6. In ouUnder conditions of cerebral ischemic insult, neuroinflammatory changes, such as the activation of microglia and astrocytes and the release of proinflammatory cytokines, initially serve as protective mechanisms. However, the unabated release of these proinflammatory cytokines subsequently interrupts the balance between proinflammatory and anti-inflammatory cytokines, contributing to neuronal damage. Chronic neuroinflammation is tightly associated with neuronal loss, neuronal cell death and accumulation of reactive oxygen species (ROS) , 12–14. Autophagy, an evolutionarily conserved cellular process, facilitates the continual delivery of damaged proteins and organelles to the lysosome for degradation, which is essential for cell survival and the maintenance of cellular homeostasis . AutophaTo clarify these issues, in the present study, we attempted to investigate the roles of impaired autophagy involved in NLRP3 inflammasome activation and the underlying mechanisms under conditions of CCH as well as treatment with URB.N-acetylcysteine , (7) BCCAo+URB , (8) BCCAo+URB+3-MA, (9) BCCAo+URB+CQ, (10) BCCAo+URB+minocycline, (11) BCCAo+URB+NAC. After 12 weeks, all rats were euthanized by deeply sedating them with 100% O2/5% isoflurane, followed by decapitation. Their brains were immediately removed for experiments or stored at − 70 °C for later use.Male Sprague-Dawley rats, weighing 230–250 g, were obtained from the experimental animal center of Shanghai SIPPR-BK Laboratory Animal Co. Ltd. . Prior to the experiments, the rats were housed under standard conditions of temperature (23 ± 1 °C), humidity (60%) and light , with free access to food and water. After 1 week of acclimatization, the rats were randomly divided into 11 groups (five rats per group) as follows: (1) sham-operated, (2) bilateral common carotid artery occlusion (BCCAo), (3) BCCAo+autophagy inhibitor 3-methyladenine , (4) BCCAo+lysosome inhibitor chloroquine (CQ Sigma), (5) BCCAo+microglial activation inhibitor minocycline (Sigma-Aldrich), (6) BCCAo+ROS scavenger Experimental protocols were performed according to Chinese legislation on the use and care of laboratory animals and were approved by the Animal Laboratory Center of Tongji University School of Medicine.The maximum chronic hypoperfusion phase in a rat model of CCH induced by BCCAo may persist for 12 weeks , which wURB , CQ , minocycline , NAC  and 3-MA  dosages were selected based on previous studies , 23–25. Guide catheters were implanted into both cerebral ventricles  . Daily iBriefly, according to our previous study , coronalg for 10 min at 4 °C. The pellet was resuspended and disrupted in 1.5 mL ice-cold disruption buffer. After centrifugation at 1000×g for 10 min at 4 °C, the supernatant was collected and centrifuged at 6000×g for 10 min. The pellet (containing mitochondria) was resuspended in 750 μL mitochondrial purification buffer and added to the top of a mitochondrial purification buffer layer . After centrifugation at 14,000×g for 15 min, a pellet or band containing mitochondria formed in the lower part of the tube and was transferred to a new tube. The suspension was washed three times with 1.5 mL mitochondrial storage buffer by centrifuging at 8000×g for 10 min. The highly purified mitochondria were resuspended in mitochondrial storage buffer. Fresh mitochondria were used for membrane potential detection. Mitochondrial and cytosolic fractions were stored at − 70 °C until further use.The mitochondrial fraction was isolated using a Qproteome Mitochondria Isolation Kit . Briefly, 20 mg of tissues were cut into pieces and homogenized in 2 mL lysis buffer with protease inhibitor solution. The supernatant (containing cytosolic proteins) was collected after centrifugation at 1000×Samples (20 μg of protein) were separated by 8%, 10%, or 12% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and 0.1% Tween-20 in Tris-buffered saline for 1 h and then incubated overnight at 4 °C with primary antibodies against the following proteins: microtubule-associated protein 1 light chain 3 , p62 , lysosomal-associated membrane protein 1 , parkin , BCL-2/adenovirus E1B (19 K)-interacting protein , NLRP3 , CASP1 , IL-1β , OX-42 , cyclooxygenase-2 , inducible nitric oxide synthase , voltage-dependent anion-selective channel 1  and β-actin . The membrane was then incubated with the appropriate horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies for 1 h at room temperature. Protein bands were detected by applying SuperSignal-enhanced chemiluminescent substrate solution . The proteins were quantified by OD ratio using β-actin as the control. In the case of mitochondria, VDAC1 was employed as the loading control.The hippocampus was dissected from the brain and then was serially dehydrated, embedded in paraffin and cut into 5-μm thick coronal sections for immunofluorescence. Briefly, paraffin-embedded sections were deparaffinized and washed three times with PBS for 5 min. Then, the sections were immersed in EDTA-Tris solution (pH 9.0) for 30 min at 98 °C for antigen retrieval and rinsed three times with PBS for 5 min. Subsequently, the slides were incubated with 10% non-immune goat serum for 30 min at room temperature to block non-specific staining. After that, the slides were incubated in humidified chambers at 4 °C overnight with primary antibodies as follows: LC3 , p62 , LAMP1 , TOM20  and NLRP3 . The next day, after washing these sections in PBS, tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-mouse/rabbit secondary antibodies  were applied for 1 h at 37 °C. Finally, all stained specimens were observed under a confocal laser scanning microscope . For the quantitative analysis, the average score of five randomly selected areas was calculated using National Institutes of Health (NIH) Image Pro Plus 6.0 software.The amounts of tumor necrosis factor (TNF)-α and IL-1β in the brain tissues were quantified using an enzyme-linked immunosorbent assay (ELISA) kit , according to the manufacturer’s instructions. Briefly, equal amounts of proteins were loaded into all wells, followed by measurements of optical density (OD) in a plate reader at a wavelength of 450 nm, with analyses for concentrations based on a standard curve. For convenience, all results were expressed as pg/mg protein.Reactive oxygen species (ROS) were determined by dihydroethidium (DHE) staining as described previously , 28. BriP < 0.05 were used as the criterion for statistical significance.The data in our study are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test. Values of In our previous studies , 11, we The inflammasome is a cytosolic multi-protein complex composed of NLRs, adaptor protein ASC and CASP1 . The NLRAutophagy mainly consists of three sequential steps including autophagosome formation, transportation to the lysosomes and degradation in the lysosomes, and each step may have diverse functions . LC3, p6Prior evidence has shown that microglial overactivation may lead to the activation of the NLRP3-CASP1 inflammasome pathway and the release of IL-1β –36. AddiTo observe the ultrastructural changes of the mitochondria, brain sections were examined by electron microscopy. In the sham group, we found mitochondria with prominent cristae and an intact membrane. After the induction of CCH, abnormal mitochondrial swelling and vague cristae were observed, which was mitigated by URB treatment. However, the autophagy inhibitor 3-MA and lysosome inhibitor CQ seemed to increase the numbers of these abnormal mitochondria Fig. , indicatAccording to the mitophagy pathways , damagedN-acetylcysteine (NAC) significantly decreased the increased protein levels of NLRP3, cleaved CASP1 and cleaved IL-1β induced by CCH (Fig. Mitochondria play a critical role in the activation of the NLRP3 inflammasome . ROS, maCCH Fig. , therebyCCH Fig.  and 10b.CCH Fig. . MoreoveCCH Fig. . The aboOwing to the complex mechanisms involved in autophagy and neuroinflammatory responses, the interactions between autophagy and neuroinflammation are controversial. The autophagy proteins function as inducers and suppressors of inflammatory responses, while inflammatory signals could promote or inhibit the process of autophagy . FocusinWe previously demonstrated that CCH impaired cognitive function and neuronal plasticity . IL-1β iP62, the described adaptor protein for autophagic transportation, is involved in the autophagy-mediated inhibition of NLRP3 inflammasome activation , 43. IntCCH induced mitochondrial dysfunction, which would promote ROS generation. ROS accumulation may activate the NLRP3-CASP1 inflammasome pathway. According to our electron microscopy results, the autophagy inhibitor 3-MA and lysosome inhibitor CQ further increased the number of CCH-induced abnormal mitochondria, indicating that defective mitophagy may exacerbate the damage to mitochondrial integrity. Subsequently, we confirmed the existence of impaired mitophagy flux after CCH. The mitophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, which may activate NLRP3-CASP1 inflammasome signaling . Under tWhen pathologically insulted, microglia can transform into an “activated” state. Appropriate activation of microglia can be beneficial to neuronal survival, but excessive activation of microglia may aggravate neuronal damage . EvidencThe tight interactions between ROS accumulation and microglial overactivation and autophagy are mainly reflected in two ways: the induction of autophagy by ROS accumulation or microglial overactivation –51 and tIn conclusion, our data revealed that CCH-induced microglial overactivation and ROS accumulation promoted the activation of the NLRP3 inflammasome and the release of IL-1β. In addition, blocked autophagy and mitophagy flux may contribute to the activation of the NLRP3-CASP1 inflammasome pathway. However, URB might alleviate impaired autophagy and mitophagy by decreasing mitochondrial ROS and microglial overactivation as well as restoring lysosomal function, which would further inhibit the activation of the NLRP3-CASP1 inflammasome pathway. These findings extend and underscore the potential therapeutic effects of URB in chronic cerebral ischemic insults.


Plasmodium falciparum has been reported in many malaria-endemic regions particularly in African countries. Genetic polymorphism of the P. falciparum Na+/H+ exchanger (pfnhe1) is considered to influence QN susceptibility. Here, ms4760 alleles of pfnhe1 were analysed from imported African P. falciparum parasites isolated from returning travellers in Wuhan, Central China.Quinine (QN) remains an effective drug for malaria treatment. However, quinine resistance (QNR) in pfnhe1 gene were determined using nested PCR with DNA sequencing.A total of 204 dried-blood spots were collected during 2011–2016. The polymorphisms of the pfnhe1 ms4760 haplotypes. In total, 28 distinct ms4760 alleles containing 0 to 5 DNNND and 1 to 3 NHNDNHNNDDD repeats were identified. For the alleles, ms4760–1 , ms4760–3 , and ms4760–7  were the most prevalent profiles. Furthermore, 5 undescribed ms4760 alleles were reported.Sequences were generated for 99.51% (203/204) of the PCR products and 68.63% (140/204) of the isolates were analysed successfully for the pfnhe1 ms4760 polymorphisms from imported P. falciparum isolates in Wuhan. Pfnhe1 may constitute a good genetic marker to evaluate the prevalence of QNR in malaria-endemic and non-endemic regions.The study offers an initial comprehensive analysis of  Quinine (QN), a natural quinoline derivative from Cinchona bark, has been widely used in malaria-endemic regions for several centuries to treat severe malaria cases or malaria in the first trimester of pregnancy . FollowiPlasmodium falciparum chloroquine resistance transporter (pfcrt) [pfmdr1) [pfmrp1) [pfdhfr) [pfdhps) [pfnhe1) [pfK13) [pfmdr1 [pfcrt [pfmrp [pfnhe1 [pfmdr1 [pfcrt [86Y184 wild-type allele in pfmdr1 and 49.43% (87/176) of the isolates carried the pfcrt K76 T mutation, suggesting moderate sensitivity to QN in Africa [pfnhe1 in QNR is still limited. A previous study demonstrated that pfnhe1 might be involved in QNR [pfnhe1 gene, SNPs at codons 790, 894 and 950 and microsatellite variations in three different repeat sequences  located on chromosome 13 of the P. falciparum genome have been identified [To date, some genes associated with drug resistance have been identified, including  (pfcrt) , multidr[pfmdr1) , multidr[pfmrp1) , dihydro[pfdhfr) , dihydro[pfdhps) , sodium/[pfnhe1) , and kel [pfK13) . However [pfK13) , 21. Alt [pfmdr1 , pfcrt [1 [pfcrt , 23, pfmt [pfmrp  and pfnh [pfnhe1 . QNR-ass [pfmdr1  at codonn Africa . Howeverd in QNR . For theentified , 27. Anoentified .pfnhe1 in patients who were infected with P. falciparum returning from different African countries and assessed whether pfnhe1 can be used as a molecular marker of QNR.In light of these reports, we investigated the prevalence of P. falciparum returning from Africa and confirmed by the Wuhan Centers for Disease Prevention and Control (CDC) during 2011–2016. These samples were examined using One Step Malaria HRP2/pLDH (P.f/Pan) tests  and Giemsa-stained thick and thin peripheral blood smear examinations. The identities of Plasmodium spp. were confirmed by real-time fluorescent quantitative PCR. Genomic DNA (gDNA) of uncomplicated P. falciparum isolates was extracted from DBSs by using a TIANamp Blood DNA Kit  according to the manufacturer’s instructions. To characterize the microsatellite repeats in pfnhe1, the ms4760 region (482 bp) of the pfnhe1 gene was amplified by nested PCR in 204 samples.In total, 204 dried-blood spots (DBSs) were collected from patients who were infected with 2+), 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), and sterile ultrapure water to a final volume of 20 μl. The gene target was amplified under the following conditions for the first run: initial denaturation at 95 °C for 3 min; followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 5 min. For the second round of PCR, 1.0 μl primary PCR products were amplified with a 50 μl reaction system, including 25 μl 2× NovoStar Green PCR Mix, 1.0 μl forward primer (10 μM), 1.0 μl reverse primer (10 μM), and 22 μl H2O. Secondary run conditions were as follows: initial denaturation at 95 °C for 3 min; followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min.DNA was amplified by nested PCR . The primary primers including NHE-A and NHE-B, and the secondary primers containing NHE-C and NHE-D were described in a previous study –31. No nhttps://plasmodb.org/plasmo/) under Gene ID.: PF3D7_1303500.Five microliters (5 μl) of nested PCR products were resolved by electrophoresis in 1% agarose gels, and examined for quality under a UV light. Samples with bright bands were selected for DNA sequencing . Sequences were then translated using the Edit Sequence tool and aligned using the MEGALIGN programme with DNAstar  software. The amino acid sequences were compared with the reference sequence from PlasmoDB  and GraphPad Prism (version 5.01). plicable . SpecifiP. falciparum malaria were collected and tested. All 204 samples were successfully amplified by nested PCR. Sequences were generated for 99.51% (203/204) of the PCR products and included 63 poor-quality sequencing products. Finally, 68.63%  of PCR products were analysed for pfnhe1 ms4760 haplotypes. In the 140 studied samples, the number of DNNND repeats (Block II) ranged from zero to five, with one, two and three repeats being more common and accounting for 27.86% (39/140), 42.14% (59/140) and 23.57% (33/140) of these samples, respectively . The ms4760 alleles in the parasite isolates from Nigeria, Congo, Angola, and Liberia accounted for 47.86% (67/140) of the samples. Furthermore, a combination of samples from Nigeria , Congo , Liberia , and Angola  was responsible for 67.86% (19/28) of the alleles. Although only 8 samples were included in the analysis for Mozambique, various ms4760 alleles  were detected. The same phenomenon can be observed for Guinea, Ghana, Zambia, Cameroon and Uganda . No significant associations were seen among years for the other alleles (P > 0.05). A significant association between ms4760–1 and ms4760–7 was also observed in 2015 . No significant associations were seen among the alleles for other years (P > 0.05).A total of 28 different ms4760 alleles were observed among the 140 imported African pfnhe1 polymorphisms from imported P. falciparum isolates in Wuhan during 2011–2016 was described. The number of DNNND repeats in Block II and NHNDNHNNDDD repeats in Block V ranged from 0 to 5 and 1 to 3, respectively. A total of 28 distinct ms4760 alleles were observed, including 23 referenced alleles [pfnhe1 microsatellite sequence polymorphisms was found (28 genotypes for 140 isolates). Previous studies observed more or less ms4760 microsatellite profiles of pfnhe1, including 8 ms4760 alleles in 71 P. falciparum isolates from Southeast Asia, Africa, and Latin America [In the present study, the African distribution of ms4760  alleles –35 and f America , and 27  America .pfnhe1 polymorphisms and QN susceptibility in vitro [pfnhe1 have been analyzed, especially in Block II and V [50 of QN for parasites with 3 DNNND repeats was significantly higher than those with 1 or 2 repeats [Although no valid molecular marker of QNR is currently available, several studies have studied the connection between in vitro , 37–40. in vitro . SequencII and V . StudiesII and V . The samII and V , 42. Fur repeats . Thus, ipfnhe1 polymorphisms in isolates from western Kenya [50 and NHNDNHNNDDD repeats [pfnhe1 in Thailand isolates [pfnhe1 ms4760 microsatellite in several studies [Several studies have demonstrated the different relationship between QN susceptibility in vitro and rn Kenya , 37. The repeats , 37. In isolates  and Indiisolates . Thus, Q studies , 42 but pfnhe1 ms4670 microsatellites in QNR may be dependent on the genetic background of the P. falciparum parasites [P. falciparum isolates from Africa have reduced susceptibility to QN. Thus, attention should be paid to this issue.These conflicting findings suggest that the role of arasites  and/or tarasites . Ms4760–arasites , 28 and arasites . Accordipfnhe1 in Africa. However, the sample sizes in this study were relatively small, particularly in 2011. Therefore, in vitro and in vivo studies with large-scale samples need to be considered for elucidating the role of pfnhe1 ms4760 as a molecular marker of QNR.Among the 140 studied sequences, West Africa displayed the highest number of different alleles, followed by South Africa, Central Africa, East Africa, and North Africa. While several alleles were shared between different countries, others appeared restricted to specific regions. Our genotyping data are consistent with previous findings, showing three profiles  as the predominant alleles. The newly observed profiles were from West Africa (4 isolates), Central Africa (2 isolates), East Africa (1 isolate) and South Africa (1 isolate), which suggests great abundance in the genetic diversity of pfnhe1 ms4760 polymorphisms from imported P. falciparum isolates in Wuhan. It demonstrated that parasite isolates from Africa are moderately diverse. In addition, continuous surveillance for molecular markers of QNR is highly recommended.This primary study offers a comprehensive analysis of


Ligustrum vulgare L. leaves and selected isolates from it. The antiradical activity of the water infusion was determined using DPPH, ABTS and FRAP tests. Oleuropein and echinacoside concentrations in the water infusion were determined by HPLC. Water infusion, echinacoside and oleuropein were used for an antilipoxygenase activity assay using lipoxygenase isolated from rat lung cytosol fraction. Activity of 8-LOX, 12-LOX and 15-LOX were monitored through formation of 8-HETE, 12-HETE and 15-HETE, respectively. The water infusion exhibited the highest activity against all lipoxygenases, followed by oleuropein. Echinacoside was ineffective against LOXs in lower concentrations, while higher concentration showed similar inhibition on 8-LOX and 12-LOX. 15-LOX was affected more and the presence of echinacoside remarkably decreased its activity.The aim of the study was a HPLC evaluation of the lipoxygenase activity inhibiting activity of a water infusion of  Ligustrum have been traditionally used worldwide in folk medicine. In the folk medicine of Azerbaijan, the use of common privet (Ligustrum vulgare L.) (LV) leaves is linked to the treatment of hypertension [in vitro assay of inhibitory activity on lipoxygenase was chosen.Many species of genus rtension , and is rtension . Common rtension . Chinesertension , antivirrtension  or anti-rtension . Most ofrtension . In connA variety of redox active compounds have been identified as inhibitors of lipoxygenases. Lipoxygenase (LOX) is the key enzyme in the biosynthesis of leukotrienes, which are postulated to play an important role in the pathophysiology of several inflammatory diseases since the products of lipoxygenase catalyzed oxygenation  seem to be involved in inflammatory reactions (rheumatoid arthritis and psoriasis). There is also good evidence that leukotrienes are mediators of asthmatic responses, glomerular nephritis, myocardial ischemia and cancer .Ligustrum vulgare L. leaves, and that of two isolates from the infusion, echinacoside and oleuropein . The concentrations of oleuropein and echinacoside in the water infusion were determined by HPLC and the antiradical activity of the samples was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 2-2′-azinobis-(3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical and ferric reducing ability of plasma (FRAP) tests.(2), 5-HETE and 12-HETE have been found to stimulate the proliferation of various cancer cells and to promote tumor development. In contrast, 13-hydroxyoctadecadienoic acid and 15-HETE, appear to inhibit the growth of colon and prostate cancer cells. Increased production of leukotriene LTB(4), 5-HETE, and 12-HETE has also been reported in the mucosa of colon cancer patients and the saliva of oral cancer patients. Because eicosanoids appear to have both negative and positive modulatory effects, they seem to be vital in the prevention of inflammation-related diseases, including cancer [Eicosanoids have potent and diverse biological activities in cell proliferation, tissue repairing, blood clotting, blood vessel permeability, and immune cell behavior. These compounds also play a very important role in the pathogenesis of inflammation and cancer. For example, prostaglandin PGEg cancer .Ligustrum vulgare L. leaves and its components echinacoside and oleuropein on lipoxygenase, where activity of LOX was monitored as an increase in the UV absorbance at 234 nm that reflected the formation of hydro-peroxylinoleic acid [50 values of 82.2 μmol L−1 for oleuropein, 296 μmol L−1 for echinacoside, 0.388 mg L−1 for water infusion of LV leaves and competitive character of inhibition of all tested inhibitors. In the present study we evaluate the influence of mentioned infusion and its constituents on some different lipoygenases by an HPLC method.We have previously reported the inhibitory activity of a water infusion of eic acid . In thatL. vulgare leaves. The analyzed sample contained 72.29 μg of echinacoside and 145.45 μg of oleuropein in 5 mg of lyophilizate. Standardized sample was further used for determination of the inhibitory activity on lipoxygenase isolated from rat lung cytosol fraction and antiradical activity using DPPH, ABTS and FRAP tests because many antioxidants are known inhibitors of this enzyme . 15-LOX was more sensitive, and the presence of echinacoside decreased its activity to 47%. The different inhibitory effectiveness of the isolated compounds and water infusion on lipoxygenase activity could be influenced by the presence of other constituents in the prepared infusion. It is known that plant polyphenols exhibit inhibitory effects on prooxidative enzymes such as xanthine oxidase, myeloperoxidase or lipoxygenases and this fact may additionally contribute to the potential inhibitory effect of the water infusion observed in our work.Activity of lipoxygenase was affected in a dose-dependent manner, based on used inhibitor, as shown in Ligustrum vulgare L. is an abundant source of iridoids and phenolics [L. vulgare  leaves which include secoiridoids and tyrosol derivatives, and both hydroxycinnamates and flavonoid glycosides. Oleuropein, ligustaloside A and ligustaloside B, and ligstroside constitute the relevant secoiridoids class.henolics . HPLC-DAL. vulgare leaves comprises quercetin 3-O-rutinoside, luteolin glucosides, namely luteolin 7-O-glucoside and luteolin 4'-O-glucoside and both apigenin 7-O-glucoside and apigenin 7-O-rutinoside. Hydroxycinnamates are represented by p-coumaric acid and echinacoside. Appreciable amounts of hydroxytyrosol and its glucoside were also detected in L. vulgare leaves. Antilipoxygenase activity can be potentiated with the activity of flavonoids (namely apigenin and luteolin derivatives) which exhibit remarkable activity against LOX [Ligustrum vulgare [. This is in good correlation with literature data. Oleuropein is one of the most active substances in virgin olive oil [(4) generation at the 5-lipoxygenase level with decreasing effectiveness hydroxytyrosol > oleuropein > caffeic acid > tyrosol.The flavonoid composition of inst LOX . Apigeni vulgare , togethe vulgare  such as live oil . In intaLigustrum vulgare leaves and its constituents are potent inhibitors of mammalian LOX.Interaction of plant phenolics with mammalian lipoxygenases merits particular attention because these enzymes are potential targets for the health-preserving effects. Our current results and previous work, as well as data from the literature suggest that water infusion of Ligustrum vulgare L. were collected in the Arboretum Mlyňany, Institute of Dendrobiology, Slovak Academy of Sciences in September 2006 and dried at room temperature. Samples were identified by Dr. Tomaško (Arboretum Mlyňany) and a voucher specimen is deposited there.Leaves of Ligustrum vulgare L. was prepared by hot distilled water maceration , and filtration after cooling. Cold infusion was lyophilized and from 100 g of an infusion 2.8582 g of lyophilizate sample were obtained. Analytical HPLC was performed with an Agilent Technologies HP 1050 Series with UV detector and autosampler . Compounds were separated on a 150 × 4.6 mm i.d., 5-μm particle, Zorbax Eclipse XDB C18 column (Agilent Technologies) using a gradient prepared from 0.1% aqueous H3PO4  and acetonitrile . The flow rate was 0.7 mL·min−1, the column temperature 28 °C and sample injection volume 20 μL. The gradient was: 0 min: 90% A + 10% B; 13 min: 78% A + 22% B; 23 min: 60% A + 40% B; 24 min: 90% A + 10% B. Echinacoside was purchased from PhytoLab  and oleuropein was isolated from the leaves of Ligustrum vulgare L. by the authors [Ligustrum vulgare L. leaves. The UV spectra of two marker compounds were given by an UV detector, whereby the maximum absorption was set at 330 nm for echinacoside and 240 nm for oleuropein.Water infusion from leaves of 2 = 0.9999) for echinacoside at concentrations of 1.42–100 μg·mL−1, Y = 22.559x − 9.9624 (r2 = 1) for oleuropein at concentrations of 1,27–100 μg·mL−1. The limits of detection (LOD) of standards were 1.09 μg·mL−1 for echinacoside and 1.08 μg·mL−1 for oleuropein. The limits of quantifications (LOQ) of standards were 1.42 μg·mL−1for echinacoside and 1.27 μg·mL−1for oleuropein. Good linear calibration curve was obtained for echinacoside at concentrations 1.69–72.29 μg·mL−1 and for oleuropein at concentrations 1.63–145.45 μg·mL−1 in the lyophilized infusion of L. vulgare. For the lyophilized infusion sample, LOD of echinacoside was 1.37 μg·mL−1 and for oleuropein 1.23 μg·mL−1, LOQ of echinacoside was 1.69 μg·mL−1 and for oleuropein 1.63 μg·mL−1. Thus, applied analytical procedure was suitable for the selected compounds quantification in Ligustrum vulgare.For two reference standards, good linear calibration curves were obtained as follows: Y = 21.183x − 10.086  as a source of LOX was isolated according to procedure of Kulkarni et al. . Brieflyet al. .S)-hydroxyeicosatetraenoic acid (8-HETE), 12(S)-HETE and 15(S)-HETE were purchased from Cayman Pharma s.r.o .Arachidonic acid  was used as a substrate prepared in a solubilized state as described . StandarPurified enzyme was analyzed for its ability to metabolize AA according to the literature . Incubat−1, the column temperature 28 °C and sample injection volume 50 μL. The gradient was: 0 min: 85% A + 15% B; 12 min: 100% A; 25 min: 85% A + 15% B; 27 min. UV detection at 210, 234 and 280 nm was used.Analytical HPLC was performed with an Agilent Technologies HP 1,050 Series with UV detector and autosampler (Agilent Technologies). Compounds were separated on a 250 × 4.0 mm i.d., 5-μm particle, Nucleosil 120-5 C18 100A column  guarded with a Zorbax XDB C18, 12.5 × 4.6 mm pre-column (Agilent Technologies) using a gradient prepared from acidified methanol  and deionized water (component B). The flow rate was 0.2 mL·minn-hexane the sample was analyzed by HPLC using the apparatus described above. Compounds were separated on a Zorbax Rx-SIL 150 × 2.1 mm i.d., 5-μm particle column (Agilent Technologies) using an isocratic elution with mobile phase prepared from n-hexane and acidified 2-propanol . The collected fraction was evaporated under a nitrogen stream. After reconstitution in eak area . For HPL2 = 0.9925) for 8-HETE at concentrations of 0.139–2 μg·mL−1, Y = 604x − 177.91 (r2 = 0.998) for 12-HETE at concentrations of 0.144–4 μg·mL−1 and Y = 487.85x − 178.57 (r2 = 0.9979) for 15-HETE at concentrations 0.233–4.02 μg·mL−1. The LOD of standards were 0.134 μg·mL−1 for 8-HETE, 0.135 μg·mL−1 for 12-HETE and 0.195 μg·mL−1 for 15-HETE. The LOQ of standards were 0.139 μg·mL−1for 8-HETE, 0.144 μg·mL−1for 12-HETE and 0.233 μg·mL−1 for 15-HETE.For three reference standards, good linear calibration curves were obtained as follows: Y = 893.78x − 279.47 (rt-test (pt), The values represent the means ± standard deviation (SD).The statistical significances of all calculated values were determined by paired Student′s Ligustrum vulgare leaves and two its constituents. These results indicate that the tested samples may contribute to the activity of L. vulgare when used as an anti-inflammatory agent in traditional medicine. However, the prospective therapeutic value and the real clinical efficacy of this plant should be proved by further in vitro and in vivo experiments.In summary, this study and previous work reports the efficient inhibition of rat lung cytosolic fraction lipoxygenase by a water infusion of


N-methyl-D-aspartate receptor (NMDAR) antagonists prevented the enhancing effect of MA on γ oscillations, whereas none of them affected baseline γ strength. Protein kinase A, phosphoinositide 3-kinase and extracellular signal-related kinases inhibitors had no effect on MA. We propose that the DR1/DR2-Akt-NMDAR pathway plays a critical role for the MA enhancement of γ oscillations. Our study provides an new insight into the mechanisms of acute MA on MA-induced psychosis.Gamma frequency oscillations  have been suggested to underlie various cognitive and motor functions. The psychotomimetic drug methamphetamine (MA) enhances brain γ oscillations associated with changes in psychomotor state. Little is known about the cellular mechanisms of MA modulation on γ oscillations. We explored the effects of multiple intracellular kinases on MA modulation of γ induced by kainate in area CA3 of rat ventral hippocampal slices. We found that dopamine receptor type 1 and 2 (DR1 and DR2) antagonists, the serine/threonine kinase PKB/Akt inhibitor and  Neuronal synchronization at γ frequency band  emerges from activated neuronal networks, consisting of mutually connected principal cells and inhibitory cells . γ oscilAberrant γ oscillations have been considered as a biomarker of a variety of neurological and psychiatric disorders, including Alzheimer’s disease , ADHD B, schizopin vitro by the glutamate receptor agonist kainate (KA)  . Althougate (KA) , at low ate (KA) , which s. When all pedal reflexes were abolished, the animals were perfused intracardially with chilled (5°C), oxygenated artificial cerebrospinal fluid (ACSF) in which the sodium chloride had been replaced by iso-osmotic sucrose. This sucrose-ACSF contained (in mM): 225 sucrose, 3 KCl, 1.25 NaH2PO4, 24 NaHCO3, 6 MgSO4, 0.5 CaCl2, and 10 glucose (pH: 7.4). Horizontal slices (400 μm) of rat brain were cut at 5°C in the sucrose-ACSF, using a Leica VT1000S vibratome  and stored at room temperature at the interface between recording ACSF and humidified carbogen (95% O2–5% CO2) until transferred to the recording chamber. The recording ACSF contained (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 24 NaHCO3, 2 MgSO4, 2 CaCl2, and 10 Glucose (pH: 7.4).All animal use procedures were approved by the Ethics Committees at the Xinxiang Medical University for the Care and Use of Laboratory Animals, and all efforts were made to minimize animal suffering and the number of animals used. Ventral hippocampal slices were prepared from Sprague Dawley rats . The animals were anesthetized by intraperitoneal injection of Sagatal . Field potentials were amplified by Neurolog NL106 AC/DC amplifiers  and band-pass filtered between 0.5 Hz and 2 kHz using Neurolog NL125 filters (Digitimer). Electromagnetic interference from the mains supply was eliminated from the recordings with the use of Humbug 50 Hz noise eliminators (Digitimer Ltd.). The recordings were digitized at a sample rate of 5–10 kHz using a CED 1401 plus ADC board .Data were analyzed off-line using Spike-2 software (Cambridge Electronic Design). Power spectra were generated to provide a quantitative measure of the frequency components. Power spectra were constructed for 60 s epochs using a fast Fourier transform algorithm. The area under the curve between 20 and 60 Hz was used to quantify the γ power.t-test. Comparisons between the effect of a drug and changes in time-matched controls were made with unpaired Student’s t-test. The effect of MA concentrations was tested by one-way ANAOVA.All statistical tests were performed using SigmaStat software . Since data sets were not different from normal distributions, results are expressed as mean ± standard error of mean. Comparisons within slices were made with paired Student’s P < 0.05.Effects were considered statistically significant, if Methamphetamine was purchased from the National Institute for Food and Drug Control . The selective DR1 antagonist SCH23390, DR2 antagonist raclopride, the mitogen-activated protein kinase inhibitor U0126 and the PI3K inhibitor wortmannin were purchased from Tocris Cookson Ltd. . The Akt/PKB inhibitor triciribine PKA inhibitor H89, NMDAR antagonist D-AP5 and the kainate receptor agonist kainate (KA) were obtained from Sigma-Aldrich (United Kingdom).Stock solutions, at thousand times the final concentration, were made up in water or in DMSO, and stored in individual aliquots at −20°C. Final solutions were prepared freshly on the day of the experiment.in vitro. The CA3 network was activated by perfusing hippocampal slices with 200 nM KA  and a peak frequency of 26.6 ± 1.0 Hz  = −2.94, P = 0.026] for 2 μM, by 152 ± 32%  for 100 μM MA. The MA-induced increase in γ power was concentration-dependent . Further application MA (20 μM) increased γ power by 120 ± 44% of H89 . These results indicate that PKA was not involved in the MA-induced effect on γ oscillations.Because DR1 and DR2 mediate PKA activity in opposite ways, we tested the PKA inhibitor H89 on the MA-mediated enhancement of γ oscillations. Application of H89 (10 μM) alone had no effect on γ power  = −0.474, P = 0.660, t(5) = −2.778, P = 0.039, t(19) = −0.161, P = 0.874]. The results show that the ERK was not involved in MA-mediated enhancement of γ oscillations.Because MA induces ERK phosphorylation in the hippocampus  and ERK t(7) = −1.359, P = 0.223]. Further application of MA (20 μM) increased γ power by 137 ± 40% from wortmannin . These results indicate that PI3K was not involved in MA-mediated enhancement of γ.Because PI3K is implicated in the effects of the MA analog phentermine , we testBecause MA activates Akt , we testt(7) = 0.104, P = 0.920, t(7) = −0.612, P = 0.560, Application of triciribine (5 μM) did not affect γ power [103 ± 4% of baseline, t(6) = −1.438, P = 0.200, t(6) = −1.065, P = 0.328, The NMDA receptor plays a critical role in excitatory synaptic neurotransmission and plasticity, and NMDAR antagonists can cause hallucinations and increases in γ oscillations in hippocampus, nucleus accumbens and prefrontal cortex . We testin vitro, is dependent on both DR1 and DR2 activation, Akt activity and NMDAR activity, but not on ERK, PI3K or PKA activity.Our results demonstrate that MA increases hippocampal γ oscillation strength Methamphetamine increases dopamine concentrations in the tissue . Both DRThe sensitivity to Akt inhibition reported here, suggests that MA enhancement of γ oscillations involves Akt signaling. This is in line with the observation that acute MA increases the Akt phosphorylation in cultured hippocampal neurons  and thatInstead of PI3K, ERK may be an alternative upstream molecule of Akt . HoweverTaken together, our observations indicate that the MA-mediated enhancement of γ oscillations acts through DR1/DR2-mediated Akt activation . Our resin vitro (in vivo (In addition to dopamine, MA also increases tissue levels of norepinephrine and serotonin. However, if anything, norepinephrine suppresses kainate-induced oscillations in vitro  and nore(in vivo . This ma(in vivo . Seroton(in vivo . Thus, aThe dependence on NMDAR activity of the MA-mediated enhancement of γ oscillations in our experiments, suggest a crucial role for NMDAR-linked signaling. This is in agreement with the MA-induced enhancement of NMDA currents in mesencephalic dopamine neurons  and withInterestingly, Akt activity is dynamically regulated by synaptic activity that is coupled to NMDA receptor activation . Akt-medA receptor density and GABAergic synaptic transmission (CA3 γ oscillations emerge from the interplay between fast-firing parvalbumin-expressing interneurons and pyramidal cells . Activatsmission . Alternasmission .in vitro kainate model, it is likely that similar MA-mediated enhancement γ oscillations will take place in vivo, where modulation by midbrain and brainstem inputs adds a level of complexity.Although our observations are based on the isolated γ oscillation-generating CA3 network in the The MA-induced enhancement of γ oscillations is in line with the observation that MA treatment increases γ oscillations in the NAc of freely moving rats  and thatIn addition to dopamine, MA increases norepinephrine and serotonin levels , which mIt is worth to mention that the enhancement of γ oscillations by acute MA is mechanistically different from the effect of other psychostimulants such as ketamine, which also increases cortical γ power and causes cognitive deficits and psychosis . In contYL, XX, HX, YW, and YJ conducted the experiments. YL, XX, XY, JW, and YZ analyzed the data. CL and RZ designed and directed the study. CL, YL, and MV wrote the manuscript. MV and CL revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C. rodentium is a widely used model for colonic infection and has been a major tool in fundamental discoveries in the fields of bacterial pathogenesis and mucosal immunology. Despite extensive studies probing acute C. rodentium infection, our understanding of the early stages preceding the infection climax remains relatively undetailed. To this end, we apply a multiomics approach to resolve temporal changes to the host and microbiome during early infection. Unexpectedly, we found immediate and dramatic responses occurring on the day of colonic infection, both in the host intestinal epithelial cells and in the microbiome. Our study suggests changes in cholesterol and carbon metabolism in epithelial cells are instantly induced upon pathogen detection in the colon, corresponding with a shift to primarily facultative anaerobes constituting the microbiome. This study contributes to our knowledge of disease pathogenesis and mechanisms of barrier regulation, which is required for development of novel therapeutics targeting the intestinal epithelium.The mouse pathogen  Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis , import , and efflux . We also detected interleukin 22 (IL-22) responses in IECs  on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosa-associated microbiome, which parallel innate immune responses.We used the mouse attaching and effacing (A/E) pathogen  Colonization levels plateau at 108 to 109 CFU/g of feces between 8 to 12 DPI, before bacterial shedding decreases as the infection starts to clear between 12 to 16 DPI, which we define as the steady-state and clearance phases, respectively , which plays an essential role during  the gut . IL-22 a the gut –14. IL-2trophils  and Th17trophils , 17, whiype mice , suggestlaudin-2 , 20.C. rodentium subverts IEC metabolism in order to evade innate immune responses and establish a favorable niche in the colon . This revealed a dramatic reprogramming of the crypt cellular composition, metabolism, and DNA replication and repair immediately as the pathogen starts to colonize the colon, which coincided with the expansion of mucosa-associated Enterobacteriaceae.Importantly, while studies of pathogen-host interactions are usually conducted at the acute phase of the infection, little is currently known about the temporal host responses culminating before the pathogen burden peaks. The aim of this study was to temporally resolve IEC and microbiome responses at the expansion phase of C. rodentium at the steady-state phase of the infection (8 DPI), including reprogramming of bioenergetics and metabolism , and 16S rRNA gene sequencing.We recently reported IEC responses to tabolism . Here, wC. rodentium shedding and tissue association during the expansion phase of infection. This revealed that shedding reached an average of 6.5 × 107 CFU/g by 4 DPI and 6.9 × 108 CFU/g by 6 DPI  but is able to adapt once initial colon colonization has occurred, thus reaching homogenous levels of colonization from 5 DPI onwards. Comparing tissue distributions of C. rodentium at 4 DPI  and 6 DPI  by immunohistochemistry revealed distinct differences in bacterial abundance and distribution  and DNA (blue) in colon sections from mice (n = 10) mock infected or infected with C. rodentium at 3, 4, or 6 DPI, as indicated. *, the same sample as appears in FIG S1, TIF file, 1.4 MB.Representative images of immunostaining of Copyright © 2019 Hopkins et al.2019Hopkins et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. rodentium infection on IECs during the expansion phase, we performed proteomic analysis of colonic IECs isolated from C. rodentium-infected mice at 4 and 6 DPI using mock-infected mice as controls. We included 5 mice per time point and monitored colonization levels up until and including the day of extraction; mice that did not reach the minimum number of CFU per gram of stool thresholds (1 × 107 CFU/g stool by 4 DPI or 1 × 108 CFU/g stool by 6 DPI) were excluded from further processing to reduce heterogeneity within each condition . Download FIG S2, TIF file, 1.2 MB.CFU-per-gram measurements were taken for mice used for the proteomics experiment. Mice that did not reach colonization levels of 1 × 10Copyright © 2019 Hopkins et al.2019Hopkins et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00062-19.3FIG S3n = 5) were inoculated with ∼1 × 109C. rodentium organisms, and one group of mice (n = 5) was simultaneously mock inoculated with PBS. (A) Intestinal epithelial cells were enriched from the mice at either 4 DPI or 6 DPI, and individual mice were pooled within the group at a 1:1 protein ratio. This was repeated twice more as independent biological repeats. Each separate condition/biological repeat was then labeled with a different TMT reagent and subjected to LC-MS/MS analysis. (B) Volcano plot of log2(FC) versus the –log10 showing the differential regulation of proteins in IECs during infection (combined 4- and 6-DPI data) compared to that of mock-infected samples. Download FIG S3, TIF file, 1.3 MB.Two groups of mice . Statistical analysis considering both the 4 and the 6 DPI data as infected samples identified 587 upregulated and 446 downregulated proteins compared to protein expression in the mock-infected samples , correlating with a previous in vitro study that showed that C. rodentium infection led to inactivation of FOXO3 in intestinal epithelia ..Mus musc10.1128/mBio.00062-19.4FIG S4FIG S4, TIF file, 0.8 MB.Bar plot showing targets of the named transcription factor in red, if it increased in abundance at 4 DPI, and in blue if it decreased in abundance at 4 DPI. Enrichment scores are shown in gray on the right axis. Targets are presented as percentages of the total number of targets of the transcription factor in question. Download Copyright © 2019 Hopkins et al.2019Hopkins et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. rodentium colonization is low, sporadic, and restricted to the upper surface of the crypt. While both expansion phase time points show downregulation of the TCA cycle, the pentose phosphate pathway, and pyruvate metabolism, only 6 DPI additionally shows upregulation of steroid hormone biosynthesis, suggesting that changes to IEC metabolism are initiated as early as the day of colonic colonization (4 DPI), but further aspects of metabolism become significantly affected as pathogen levels in the colon increase. Furthermore, downregulation of the TCA cycle and OXPHOS suggests a progressive shift of cellular bioenergetics during infection to aerobic glycolysis, which coincides with an increase in cell proliferation.To further resolve temporal changes, one-dimensional (1D) enrichment analysis was applied to 4 and 6 DPI samples separately, revealing early onset of a number of DNA repair pathways, with base excision repair specific to 4 DPI only . SignifiOn a protein-specific level, among the proteins ranked in the top 100 for most changed in abundance, we found a number of innate immunity and nutritional immunity proteins, including matrix metallopeptidase 9 (MMP9), Reg3β, Reg3γ, inducible nitric oxide synthase (iNOS), LCN-2, DMBT1, S100A8, and S100A9 . Further2 fold changes [log2FC], 1.53 and 1.11, respectively), with a more exaggerated increase at 6 DPI . Uniquely, in parallel with the increased abundance of proteins involved in cholesterol biogenesis and import, we found an increase in the cholesterol efflux transporters Abca1 and Abcg5, with a significant increase in Abca1 protein levels at both 4 DPI and 6 DPI   .10.1128/mBio.00062-19.5FIG S5n = 3). (B) Abca1 expression levels remain unchanged at 3 DPI compared to those in mock-infected samples. Data were determined by qRT-PCR analysis (n = 5). Download FIG S5, TIF file, 0.9 MB.(A) Fold changes in protein levels from levels in mock-infected samples from the same biological experiment involved in the cholesterol biosynthesis pathway. Data were determined by proteomic analysis  receptor (Idol) is regulated by liver X receptor (LXR), transcription factors which are considered to be mutually antagonistic . Unexpectedly, unlike with the proteomics data, this revealed that expression of Hmgcr was significantly downregulated at 6 DPI  and bioenergetic  activities.As we found dramatic changes in the metabolic landscape of the epithelium, we next analyzed whether this affected the composition of the mucosa-associated colonic microbiome. This revealed a significant decrease in alpha diversity at both 4 and 6 DPI compared to that in the control mice . WeighteDPI mice , there wDPI mice C and D. DPI mice . No signDPI mice , suggest10.1128/mBio.00062-19.6FIG S6C. rodentium infection. (B) Alpha diversity (observed OTUs) showing no significant decrease overtime, as analyzed with the Spearman correlation. (C and D) Jaccard (C) and Bray-Curtis (D) PCoA analysis showing no significant difference in community composition, analyzed by PERMANOVA. (E) Taxonomic composition of individual mice. (F and G) Relative abundances of species of the Citrobacter/Trabulsiella genus (F) and Enterobacteriaceae family (not further classified) (G). **, P < 0.05 by the Kruskal-Wallis test with Dunn’s multiple-comparison test. In all panels, a dot or bar represents an individual mouse. Download FIG S6, TIF file, 2.8 MB.(A) CFU-per-gram stool counts. 16S rRNA gene analysis of the stool microbiotas of uninfected mice and at 4 and 8 days after Copyright © 2019 Hopkins et al.2019Hopkins et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. rodentium dissemination from the gut. Protection of the mucosal surface is achieved via IL-22 signaling to the IECs, which respond by phosphorylation of STAT3, cell proliferation, expression of AMPs, and induction of nutritional immunity   . This ren (PCNA)  and C. INA . Thisn (PCNA) A and B. n (PCNA) , suggest10.1128/mBio.00062-19.7FIG S7C. rodentium (white), and DNA (blue) in colon sections from mice (n = 5) infected with C. rodentium . (B) Percentage of Ki67 of total crypt length. Each dot represents the mean percentage per mouse (n = 5). Significance was determined by Tukey’s multiple-comparison test. ns, not significant. Download FIG S7, TIF file, 1.5 MB.(A) Representative images of immunostaining at 3 DPI of Ki67 (green), PCNA (red), Copyright © 2019 Hopkins et al.2019Hopkins et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. rodentium-infected mice by ELISA. LCN-2 and S100A8 levels increased significantly from baseline levels by 4 DPI and continue to increase up to 8 DPI , CXCL1 (B), and Reg3 expression levels remained unchanged at 3 DPI compared to levels in mock-infected samples. Determined by qRT-PCR analysis. Download Copyright © 2019 Hopkins et al.2019Hopkins et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the iNOS expression during C. rodentium infection which was statistically significant at 6 DPI and 8 DPI , it temporally reshapes the architecture and cellular composition all the way down to the stem cell niche, starting with depletion of goblet cells and then DCS cells and, finally, terminally differentiated enterocytes.Taken together, these results suggest that while in vivo bacterial infection at the protein level is relatively unexplored, with most temporal proteomics studies focusing on viral infection of cell culture, for example, hepatitis C virus (HCV) infection , when pathogen adhesion is sporadic; thus, polarization may be independent of direct bacterial contact and might represent a global epithelial response to diffusible signals, such as cytokines, or direct cell-to-cell communication between IECs.Surprisingly, despite low levels of shedding and sporadic adhesion at 4 DPI, we observed significant perturbations in the proteome, including the establishment of an inverse correlation between repression of central metabolic pathways and upregulation of processes involved in DNA damage repair and replication. Notably, previous proteomics work revealed similar patterns of upregulation of DNA replication, mismatch repair, and cell cycle, concomitantly with downregulation of steroid hormone biosynthesis, metabolic pathways, the PPAR signaling pathway, the TCA cycle, and OXPHOS within a group of cell lines categorized as the colorectal proteomic subtype 1 . This gr40–C. rodentium infection  was not induced until 8 DPI, although the corresponding proteins were found in higher abundance in infected IECs than in controls. These results suggest that LXR and SREBP2 regulation of their target genes in IECs is somewhat different from their well-studied activities in the liver, the major site where the bodily cholesterol homeostasis is controlled  but instead as soon as the colon is seeded with C. rodentium at 4 DPI. On the other hand, both proteomic and transcriptional profiling of Ido1 and iNOS show no statistically significant increase in levels at 4 DPI compared to levels in mock-infected controls, with iNOS being significantly induced by 6 DPI in proteomics  and qRT-PCR data  and nutritional immunity . However, while Tsai and colleagues have shown upregulation of Claudin-2 at 2 DPI , which wPCR data  and Ido1PCR data . These dListeria monocytogenes mouse infection , at the day of colonic colonization (4 DPI), no significant CCH was detected at this time point. Taken together, these results suggest that sensing the presence of a pathogen triggers robust colonic responses, which might potentially be a preparatory mechanism in case of pathogenic expansion. Indeed, early proliferation and STAT3 activation have recently been seen in the small intestine following nfection .C. rodentium infection, with significant reduction of mucin-containing goblet cells being observed at the peak of infection  (∼5 × 109 CFU/ml).Overnight cultures of Mice were inoculated by oral gavage with 200 μl of the concentrated inoculum, and the number of viable bacteria used as inoculum was determined by retrospective plating onto LB agar containing NAL. Mock-treated mice were orally gavaged with 200 μl PBS. Stool samples were collected daily after inoculation, and the number of viable bacteria per gram of stool was determined by serial dilution and plating onto LB agar containing NAL.g for 10 min), followed by two PBS washes. Enterocyte pellets were stored at −80°C for either proteomics or qRT-PCR experiments.At 4 or 6 DPI, a 4-cm segment of terminal colon was cut longitudinally, placed in 4 ml enterocyte dissociation buffer , and incubated at 37°C with shaking for 45 min. The remaining tissue was removed and stored at −80°C, and lifted enterocytes were collected by centrifugation  for 2.5 h and then submersed in 70% ethanol. Fixed tissues were processed, paraffin embedded, and sectioned at 5 μm by Lorraine Lawrence . Formalin-fixed, paraffin-embedded (FFPE) sections were then stained with either hematoxylin and eosin (H&E) or Alcian blue and periodic acid-Schiff (AB/PAS) stain, using standard techniques, or processed for immunofluorescence.H&E-stained sections were analyzed using a 20× lens objective on the Zeiss Axio Observer Z1 microscope. At least 20 well-oriented crypts from each section from each individual mouse were measured, and an average crypt length for each mouse was calculated, using ZEN 2.3 Lite software (Zeiss). Similarly, Ki67 staining was also measured by this method and divided by the total length of the crypt to calculate the percentage of Ki67 staining. The same protocol was used to analyze PCNA staining in the crypt. The density of goblet cells was calculated by counting the number of goblet cells per crypt and measuring the total crypt length.2O). Once cooled, slides were first blocked in PBS-TS supplemented with 10% normal donkey serum (NDS) for 20 min in a humid chamber, before being incubated with primary antibody diluted in PBS-TS with 10% NDS for 1 h. Ki67 (Abcam) was diluted 1:50, PCNA (Abcam) was diluted 1:500, and intimin 280B against porcine EPEC  twice for 10 min, 95% EtOH twice for 3 min, 80% EtOH once for 3 min, and PBS-0.1% Tween 20-0.1% saponin (PBS-TS) twice for 3 min. Subsequently, sections were then heated for 20 min in demasking solution (0.3% trisodium citrate-0.05% Tween 20 in distilled HStool samples were homogenized in PBS with 0.1% Tween 20 (PBST) using a vortex machine for 15 min. Samples were centrifuged at 16,000 rpm for 10 min, and the supernatant was extracted and stored at −80°C. LCN-2 concentration was determined using a DuoSet mouse lipocalin-2 ELISA or DuoSet mouse S100A8 ELISA (R&D Systems), according to the manufacturer’s instructions.RNA from enriched IECs was isolated with an RNeasy minikit (Qiagen) by following the manufacturer’s instructions. RNA was treated with RQ1 RNase-free DNase (Promega), and subsequently, cDNA was synthesized using a Moloney murine leukemia virus reverse transcription kit (Promega). Quantitative RT-PCR was performed using Fast SYBR green master mix (Thermo Fischer Scientific) and different primer pairs , 0.1% SDS, 10% isopropanol on ice, assisted with pulsed probe sonication. Samples were subsequently boiled at 90°C for 5 min on a shaking device at 300 rpm. Protein concentration was measured with the Quick Start Bradford protein assay (Bio-Rad) according to the manufacturer’s instructions. Aliquots containing 100 μg of total protein, with an equal contribution from each individual mouse within the group, were prepared for trypsin digestion. Samples were reduced with 5 mM tris-2-carboxyethyl phosphine (TCEP) and alkylated with 10 mM iodoacetamide (IAA). Proteins were then digested by adding 75 ng/μl trypsin to each sample and incubating the samples overnight. The resultant peptides were diluted up to 100 μl with 0.1 M TEAB buffer and labeled with a tandem mass tag (TMT) multiplex reagent vial (Thermo Scientific) according to the manufacturer’s instructions. Hydroxylamine was used to quench the reaction, and then all 11 samples were combined in equal amounts to a single tube. The sample was then dried with a centrifugal vacuum concentrator.18 column  on a Dionex Ultimate 3000 high-performance liquid chromatograph (HPLC) system. Mobile phase A was 0.1% ammonium hydroxide, and mobile phase B was 100% acetonitrile, 0.1% ammonium hydroxide. The TMT-labeled peptide mixture was reconstituted in 100 ml mobile phase A and was fractionated using a multistep gradient elution method at 0.2 ml/min as follows: isocratic for 5 min at 5% phase B, gradient for 35 min to 35% phase B, gradient to 80% phase B in 5 min, isocratic for 5 min, and reequilibrated to 5% phase B. Fractions were collected every 30 s and vacuum dried. LC-MS/MS analysis was performed on the Dionex Ultimate 3000 system coupled with the Orbitrap Fusion mass spectrometer (Thermo Scientific). Each peptide fraction was reconstituted in 40 μl 0.1% formic acid, and 7 μl was loaded to the Acclaim PepMap 100, 100-μm by 2-cm, 5-μm, 100-Å C18 trapping column at a 10-μl/min flow rate. The sample was then subjected to a gradient elution on the Acclaim PepMap rapid-separation LC  C18 capillary column at 45°C. Mobile phase A was 100% H2O, 0.1% formic acid, and mobile phase B was composed of 80% acetonitrile, 0.1% formic acid. The gradient separation method at a flow rate of 300 nl/min was as follows: gradient for 90 min from 10% to 38% phase B and for 10 min up to 95% phase B, isocratic for 5 min at 95% B, reequilibrated to 5% phase B in 5 min, and isocratic for 10 min at 10% phase B. Precursors between 375 and 1,500 m/z were selected, with mass resolution of 120,000, automatic gain control (AGC) of 4 × 105, and IT (injection time) of 50 ms, with the top speed mode in 3 s, and the precursors were isolated for collision-induced dissociation (CID) fragmentation with a quadrupole isolation width of 0.7 Th (Thomson unit). Collision energy was set at 35%, with AGC at 1 × 104 and IT at 50 ms. MS3 quantification was obtained with higher-energy collisional dissociation (HCD) fragmentation of the top 7 most abundant CID fragments isolated with synchronous precursor selection (SPS). Quadrupole isolation width was set at 0.7 Th, collision energy was applied at 65%, and the AGC setting was at 1 × 105 with IT at 105 ms. The HCD MS3 spectra were acquired for the mass range 100 to 500 with a resolution of 50,000. Targeted precursors were dynamically excluded for further isolation and activation for 45 s with 7-ppm mass tolerance.Offline high-pH reverse-phase (RP) peptide fractionation was performed using the Waters XBridge Cq value and a decoy database search. All spectra were searched against UniProt reference proteomes of Mus musculus and C. rodentium protein entries. The reporter ion quantifier node included a TMT 11-plex quantification method with an integration window tolerance of 15 ppm and integration method based on the most confident centroid peak at the MS3 level. Only unique peptides were used for quantification, with protein groups considered for peptide uniqueness. Peptides with an average reported signal-to-noise ratio of >3 were used for protein quantification. The enterocytes obtained from mock-infected mice were used as controls for log2 ratio calculations.The SEQUEST-HT search engine was used to analyze the acquired mass spectra in Proteome Discoverer 2.1 (Thermo Scientific) for protein identification and quantification. The precursor mass tolerance was set at 20 ppm, and the fragment ion mass tolerance was set at 0.5 Da. Spectra were searched for fully tryptic peptides with maximum 2 mis-cleavages. TMT6plex at the N terminus/K and carbamidomethyl at the C terminus were defined as static modifications. Dynamic modifications included oxidation of M and deamidation of N/Q. Peptide confidence was estimated with the Percolator node. The peptide FDR was set at 0.01, and validation was based on the P values were calculated using a single-sample t test in the Perseus proteomics tool  analysis pipeline . The 16S V4 region was amplified using 515F/806R primers and sequenced using 2× 250-bp paired-end sequencing (Illumina MiSeq). Sequences were analyzed using the Qiime2  was used to plot line graphs, bar charts, and dot plots. Volcano plots and distribution plots were drawn in RStudio with the ggplot2 and ggrepel packages.Data were analyzed by a paired two-tailed Student The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository  with the