Escherichiacoli, confers resistance to multiple antibiotics in Pseudomonas fluorescens by directly regulating the MDR efflux pumps EmhABC and MexCD-OprJ. Moreover, we show that phosphorylation of the conserved Asp52 residue in RstA greatly enhances RstA-DNA interaction, and regulation of the multidrug resistance by RstA/RstB is dependent on the phosphorylation of the RstA Asp52 residue by RstB. Proteome analysis reveals RstA/RstB also positively regulates the efflux pump MexEF-OprN and enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. Our results suggest that, by coupling the expression of multiple efflux pumps and anaerobic nitrate respiration, RstA/RstB could play a role in defense against nitrosative stress caused by anaerobic nitrate respiration.Multidrug resistance (MDR) efflux pumps are involved in bacterial intrinsic resistance to multiple antimicrobials. Expression of MDR efflux pumps can be either constitutive or transiently induced by various environmental signals, which are typically perceived by bacterial two-component systems (TCSs) and relayed to the bacterial nucleoid, where gene expression is modulated for niche adaptation. Here, we demonstrate that RstA/RstB, a TCS previously shown to control acid-induced and biofilm-related genes in IMPORTANCE Microenvironmental hypoxia typically increases bacterial multidrug resistance by elevating expression of multidrug efflux pumps, but the precise mechanism is currently not well understood. Here, we showed that the two-component system RstA/RstB not only positively regulated expression of several efflux pumps involved in multidrug resistance, but also promoted expression of enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. These results suggested that, by upregulating expression of efflux pumps and pyoverdine biosynthesis-related enzymes, RstA/RstB could play a role in promoting bacterial tolerance to hypoxia by providing protection against nitrosative stress. A TCS ctrans , 6. The trans .Escherichia coli  superfamily efflux pump EmhABC is characterized to be a key determinant to the resistance toward multiple antibiotics including ampicillin, chloramphenicol, and tetracycline . Interestingly, two mutant strains with deletion of either Pf6N2E2_463 or Pf6N2E2_464 showed high cofitness values with above-mentioned three mutant strains  from E. coli. We next analyzed the cofitness data of ΔPf6N2E2_463 and found that three more mutant strains devoid of genes encoding other efflux pumps  have high cofitness values with the ΔPf6N2E2_463 strain  are likely to be involved in antibiotic resistance by regulating multiple efflux pumps including the EmhABC , MexCD , and Bcr-like (Pf6N2E2_3484) pumps in P. fluorescens.The similarity of fitness under different growth conditions between any two different deletion strains has been defined as the cofitness , and twosistance , we ther strains , suggest3 strain . The genP. fluorescens 2P24  with E. coli RstA/RstB proteins among the ORFs of P. fluorescens 2P24. Deletion of either of these two genes leads to increased susceptibility to multiple antibiotics including ampicillin, gentamicin, chloramphenicol, kanamycin, lomefloxacin, and tetracycline (mexC (PFLP_03468) deletion mutant, along with the emhABC deletion mutant, also showed decreased resistance to multiple drugs, yet with a slightly different antibiotic spectrum, suggesting different substrate specificities for these two efflux systems. The mutant devoid of the MFS transporter PFLP_00760, a homologous protein of Bcr, did not show significantly altered susceptibility to all the tested antibiotics  compared with that of the wild-type strain  that this DNA sequence weakly interact with RstA fused with an N-terminal maltose binding protein tag (MBP-RstA). As the DNA-binding ability of responsive regulators is generally dependent on phosphorylation of a conserved Asp residue, we performed an in vitro phosphorylation assay in which the MBP-RstA protein was incubated with acetyl phosphate, a high-energy phosphoryl-donor known to phosphorylate the conserved Asp residues in responsive regulators (P) interacted with the upstream sequence of emhA in a concentration dependent manner as shown by the EMSA  in total reaction mixtures of 20 μl each. Download FIG S1, TIF file, 1.0 MB.EMSAs of MBP-tag or phosphorylated MBP-tag protein with Copyright © 2021 Li et al.2021Li et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli  and tested its DNA-binding capacity. It was revealed that, compared with its wild-type counterpart, MBP-RstAD52A had significantly compromised DNA-binding affinities toward the upstream sequences of emhABC and mexCD-oprJ regardless of the acetyl phosphate treatment . This is most likely due to the labile nature of phosphorylated Asp, which has an extremely short half-life and was notoriously difficult to capture. We next performed a multiple sequence alignment and identified that the Asp52 residue of RstA was strictly conserved among responsive regulators from various bacterial species . The corE. coli , AlgR , gentamicin (4 folds), kanamycin (2 folds), and lomefloxacin (>2 folds) (rstB-kinase domain (ΔrstB-KD) also led to increased antibiotic susceptibility. Consistently, the expression levels of emhA, emhC, and mexC were significantly decreased in the rstAD52A, rstAD52E, and ΔrstA mutant strains compared with the wild-type strain  . Moreovee strain . It is irstB (ΔrstB-SD) had little effect on the antibiotic resistance , and hierarchical clustering using z-scored intensities of the 275 proteins quantified with high confidence  showed that four replicates of the samples of P. fluorescens and its derivatives ΔrstA were correctly clustered together  with fold change greater than 1.5 and P value less than 0.05 was used as thresholds, resulting in 33 upregulated proteins and 153 downregulated proteins  are involved in tight control of the expression levels of these efflux pumps. Disruption of the regulatory network that leads to upregulation of the efflux pumps have been commonly found in clinical isolates exhibiting the MDR phenotype. Being a close relative of P. aeruginosa, P. fluorescens produces a variety of antimicrobials and at the same time intrinsically resistant to multiple antibiotics, yet questions remain how the antibiotic resistance in P. fluorescens is regulated. Our work demonstrated that the TCS RstA/RstB contributes to antibiotic resistance of P. fluorescens by activating expression of the EmhABC and MexCD-OprJ efflux pumps, highlighting the important roles of certain TCSs in modulating intrinsic antibiotic resistance. In E.coli and Salmonella, RstA was reported to regulate genes related to biofilm formation and iron acquisition (6TACA) was not found in the promoter regions of major antibiotic efflux pumps in these bacterial species . The sequence alignment was performed using the Basic Local Alignment Search Tool of the NCBI website and the Multalin page .The homozygous cofitness data of Pf6N2E2_2823, Pf6N2E2_2824, Pf6N2E2_2825, Pf6N2E2_463, Pf6N2E2_464, Pf6N2E2_1660, Pf6N2E2_1661, and Pf6N2E2_3484 deletion strains were acquired from the online Cofitness Browser Database (E. coli strains were grown at 37°C in Luria-Bertani (LB) broth. P. fluorescens 2P24 was grown at 28°C in King’s B (KB) medium  medium . Ampicil10.1128/mSystems.00911-21.1TABLE S1Table S1, DOCX file, 0.02 MB.Strains and plasmids used in this study. Download Copyright © 2021 Li et al.2021Li et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. fluorescens 2P24 by conjugating with the E. coli strain WM3064 to replace the target gene locus. In brief, after transformed with the suicide plasmid, the WM3064 and the wild-type P. fluorescens 2P24 strains were mixed in the same dilution rates and plated on a KB agar plate for 6 h to 8 h. Following scraping little bacterial plaque in the double-stress  KB medium, proper dilutions were plated on a double-stress KB agar plate and single crossover strains were validated and cultured in the KB medium for 8 h. Under a 10% sucrose (wt/vol%) stress, mutant strains were confirmed by PCR amplification after the second crossover.The in-frame deletion mutants were constructed via double-crossover homologous recombination with the suicide plasmid pK18Km as reported previously . For eac5/ml with KB broth and inoculated to each well. After incubated at 28°C for 20 h, the MIC readings of the wells were detected by a microplate reader at a wavelength of 600 nm   of each antibiotic. Strains of log phase (OD600 = 0.8) were diluted to a final concentration of 10 BioTek) .lacZ fusions as previously reported  and a recipient strain (P. fluorescens 2P24) as mentioned above. As reported by Miller before  coloration, evaluated by absorbances of OD550, OD420, and OD650, was recorded to measure the activity of β-galactosidase . All samples were tested in three duplicates and assays were performed at least three times.We used a kanamycin resistance plasmid pRG970Km to construct reported . Purifier before , the Lac600 of 0.25, then 4 ml cultures were harvested by centrifugation at 4°C. After total RNA extraction  and cDNA preparation , qRT-PCR were performed using the SYBR Premix Ex Taq  and CFX96 real-time PCR detection system , all according to the manufacturer’s instruction. PCRs were set up using equivalent amounts of cDNA derived from the same amount of input RNA. The concentrations of all nucleic acid samples were measured using a NanoDrop 2000 spectrophotometer  and the 260/280 ratio of samples is limited between 1.8 and 2.0. At least three biological replicates were performed. As described previously, 2-ΔCt method was used to analyze relative expression levels of target genes and the expression level of 16S in each individual sample was measured as the internal control. The equation used in our study isOvernight cultured bacterial cells were diluted (1:50) in fresh KB medium and grown to the ODE. coli BL21(DE3) cells, which were developed in LB medium at 37°C supplemented with 50 μg/ml ampicillin. The protein expression was induced by 0.2 mM IPTG when OD600 reached 0.6 and cultured at 16°C for another 20 h. After harvested by centrifugation at 5,000 g for 10 min, the cells were resuspended in a column binding buffer containing 20 mM Tris–HCl at pH 8.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, and lysed by sonication. The debris was removed via centrifugation at 12,000 g for 30 min and subsequently the supernatant was loaded onto an amylose resin affinity column  for purification of the MBP protein or the MBP-tagged proteins, which were eluted by the binding buffer supplemented with 10 mM maltose. The purified proteins, MBP-RstA, MBP-D52A, and MBP, were concentrated and stored at –80°C for further usage.The RstA gene was amplified and cloned into pMAL-c2X expression vector containing an additional maltose-binding protein (MBP) tag. The RstA bearing D52A mutation expression plasmid was obtained via overlap PCR. All these recombinant plasmids and the untreated pMAL-c2X were transformed to in vitro as a standard reaction reported before  with a final concentration of 100 μg/ml and incubated for a further 1 h at 30°C. Then the buffer was replaced with 20 mM Tris-HCl (pH 7.4) by a centrifugal filter . As for control groups, the protein was processed with the same method except acetyl phosphate was removed from the dilution buffer.The proteins were phosphorylated d before . In brieemhA and emhC, designated as FAM-emhA and FAM-mexC, were amplified and purified for EMSA and DNase I footprinting assays. For EMSAs, 50 ng of purified promoters were incubated with different amounts of proteins for 30 min at room temperature. In a total volume of 20 μl, the reaction mixture contained 50 mM Tris-HCl at pH 7.5, 10 mM MgCl2, 10% (vol/vol) glycerol, 0.5 mM EDTA, 50 mM KCl, and 3 μM bovine serum albumin (BSA) to prevent unspecific binding  were collected and resuspended in 6 M Urea, 100 mM DTT, and 100 mM Tris-HCl (pH 7.0), and proteins were digested with trypsin overnight. The total peptide was desalted with C18-column and dissolved into 0.1% formic acid (FA) at a final concentration of 100 ng/μl. For MS analysis, 1 μg peptide of each sample was subjected to nanoflow liquid chromatography-tandem mass spectrometry analysis on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) coupled online to an EASY-nLC 1200 system in the data-dependent mode. Using a 150 mm × 75 μm C18 column with 2 μm particles, peptides were separated on a 90-min nonlinear gradient: 5%–35% buffer B for 60 min, 35%–80% buffer B for 20 min, and 100% buffer B for 10 min  at 300 nL/min constant flow rate. Source voltage and current were set to 2.5 KV and 100 A, respectively. All MS measurements were performed in the positive ion mode and acquired across the mass range of 300–1,800 m/z. Raw mass spectrometry files were analyzed by ProteinDiscovery (version 2.3)  was set to 0.01 for both peptide and protein identifications. Further bioinformatics and statistical analyses were performed using the software Perseus   and MS/M Perseus .P. fluorescens 2P24 have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD018793 (The MS raw files and proteome sequences of XD018793 .10.1128/mSystems.00911-21.4TABLE S4rstA deletion strain. Download Table S4, DOCX file, 0.03 MB.Up- and down-regulated proteins in the Copyright © 2021 Li et al.2021Li et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


THADA gene, neglecting the rare variants and other genes in 2p21; thus, this study was designed to investigate additional variants and novel susceptibility genes in 2p21. A total of 159 NSCL/P patients and 542 controls were recruited in the discovery phase, whereas 1830 NSCL/P patients and 2,436 controls were recruited in the replication phase. After targeted region sequencing, we performed association and burden analyses for the common and rare variants, respectively. Furthermore, RNA-seq, proliferation assay and cell cycle analysis were performed to clarify the possible function of the candidate gene ZFP36L2. Association analysis showed that four SNPs were specifically associated with non-syndromic cleft lip only (NSCLO) and two SNPs were associated with both NSCLO and NSCL/P. Burden analysis indicated that ZFP36L2 was associated with NSCLO . Moreover, SNPs in the ZFP36L2 targeted gene JUP were also associated with NSCLO. ZFP36L2 also inhibited cell proliferation and induced G2 phase arrest in the GMSM-K cell line. Therefore, we proposed that ZFP36L2 is a novel susceptibility gene of NSCLO in the 2p21 locus, which could lead to NSCLO by modulating cell proliferation and cycle.rs7590268 present on the 2p21 locus was identified to be associated with non-syndromic cleft lip with or without cleft palate (NSCL/P) in several populations, including the Chinese Han population, indicating that 2p21 was a susceptibility locus for NSCL/P. However, previous studies have only identified common single-nucleotide polymorphism (SNP) within the  Non-syndromic orofacial clefts (NSOFCs), which usually occur without any other physiological abnormalities, are one of the most common birth defects. NSOFCs are commonly divided into non-syndromic cleft lip with or without cleft palate (NSCL/P) and non-syndromic cleft palate only (NSCPO), which have been historically regarded as etiologically distinct phenotypes, because they differ in epidemiology and family patterns, as well as in the developmental origin of the lip and palate . NSCL/P,Individuals with NSCL/P are usually exposed to a series of problems early in life, such as difficulties in feeding and ear infections, which impose a heavy burden on both the affected families and society. Additionally, NSCL/P impact the patients’ quality of life throughout their life span, although surgical repair, speech therapy, dental care, and psychological support are available . TherefoIn the past few decades, several environmental factors have been identified to be associated with NSCL/P, including smoking, drinking, drug consumption (such as antiepileptic agents), and lack of dietary folic acid during early pregnancy . AlthougBased on the first meta-analyses of NSCL/P, which combined the data from two large GWAS, including 666 European trios and 795 Asian trios, 2p21 was proposed to be susceptibility region for NSCL/P for the first time . SubsequTHADA gene. Although THADA did not involve in craniofacial development according to the currently published literature, variations within it have been associated with many diseases, which may be attributable to its large size (370 kb), the effect of regulatory elements, or the other adjacent genes  < 0.01, which have a larger effect than common variants and could partially compensate for the missing heritability . Additiont genes . Notablyrst time ; howeverZfp36l2 phosphorylation influences the production of inflammatory mediators by regulating Mitogen-activated protein kinase (Mkp)-1 mRNA expression -type zinc finger tandem protein . It is won K. T. . Additio silence . Furtheropoiesis .via association analysis, burden analysis, and a series of functional analyses  in the 2p21 locus by targeted sequencing, followed by interpretation http://www.novogene.com/).In this study, we performed a two-phase case-control analysis, including an initial discovery phase and a subsequent replication phase. In the discovery phase, 159 unrelated patients with NSCL/P (80 NSCLP and 79 NSCLO patients) were selected from the Cleft Surgery Department of the West China College of Stomatology, Sichuan University. All of them self-identified as Han Chinese, and they did not have any other congenital anomalies. The whole-genome sequencing data of 542 Han Chinese normal controls (sequenced using Illumina Hiseq platform with an average coverage of 39.89) included in this phase were obtained from the Novogene internal database  and 2,255 normal controls from two GWAS , as wellHuman subject study protocols were approved by the Hospital Ethics Committee of West China Hospital of Stomatology, Sichuan University; these protocols conformed to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines. Written informed consent was obtained from recruited individuals of consenting age and from parents on behalf of their participating children (WCHSIRB-D-2016-012R1).® spectrophotometer , with good quality output of ratio of optical density at 260 nm to the optical density at 280 nm (OD260/OD280) values ranging from 1.8 to 2.0.Peripheral blood samples were collected from all the participants and their parents, from which DNA was extracted using the salting out method and then stored in Tris-EDTA buffer. The quality of the isolated genomic DNA was verified by electrophoresis on 1% agarose gel to exclude the possibility of DNA degradation or RNA/protein contamination. Furthermore, the DNA purity and concentration were detected using a NanoPhotometerAccording to the linkage disequilibrium (LD) structure shown by the CHB/JPT HapMap, the targeted region for deep sequencing was around rs7590268, based on ranges from chr2:43,417,119 to 43,838,705 (GRCh37/hg19), including exons, introns, and the intergenic region . Sequenchttp://genetics.bwh.harvard.edu/pph2/) (http://www.mutationtaster.org/)  software . Then, Su/pph2/)  and Mutaer.org/) .p < .000001) were removed from subsequent association analysis, which was conducted by PLINK (version 1.9)  MAF <0.01 in CHB and CHS  from the 1,000 Genome database and Novogene internal database (http://www.novogene.com/); 2) MAF <0.001 in the Genome Aggregation Database (GnomAD); and 3) at least two prediction tools indicate the rare variation to be damaging , the thresholds for damaging in each tool were set as follows: SIFT scores <0.05, Polyphen2_HDIV scores ≥0.957, Mutation Taster indicates “Disease causing”, CADD scores >10. Fisher’s exact test or Pearson chi-square test was performed between the cases and controls.Variants with call rates >95% were divided into two groups: common variants (MAF ≥0.01) and rare variants (MAF <0.01). Furthermore, we performed the Hardy-Weinberg equilibrium (HWE) test at each SNP. SNPs which deviated from HWE (ion 1.9) . The p vConsidering the important role of oral epithelium in facial morphogenesis, as well as its known association with NSCL/P, a human oral epithelial cell line  was selected for functional analysis in our study H. . GMSM-K ZFP36L2 (NM_006887.4) (siRNA-ZFP36L2: 5′-GCCUUCUACGAUGUCGACUTT-3′) and negative control siRNA (siRNA-negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′) were both designed and synthesized by GenePharma . Moreover, ZFP36L2 was cloned into the pcDNA3.1 overexpression plasmid   targeting , China) , and theZFP36L2 for 48 h. Then, the cells were collected and RNA-seq was performed using the BGISEQ-500 platform . Two biological replicates were included within each group. Differential gene expression analysis was performed using DEseq2 method , and GO enrichment analysis was performed on DEGs using DAVID 6.8  on a LightCycler 480 System . All experiments were performed in triplicate at least, each with three technical replicates. The results were calculated using equation 2−ΔΔCt. The primers used are shown in RNA was extracted using a Tissue/Cell RNA extraction Kit  48 h after transfection, and was then reverse-transcribed to cDNA using PrimeScript™ RT reagent Kit . RT-qPCR was performed using TB Green4 cells/100 μl. After 24, 48, and 72 h, 10 μl of Cell Counting Kit-8 (CCK-8)  was added to each well. Then, 3 h later, OD of each well was measured at a 450 nm wavelength. Measurements at each time point were replicated three times having five replicates each. Results are shown as mean ± SEM. Statistical analysis was performed using the unpaired two-tailed t-test in GraphPad Prism eight software.Upon transfection, GMSM-K cells were seeded into 96-well plates at a density of 2 × 105 transfected GMSM-K cells were plated in 6-well plates. After 48 h, cells were collected for further cell cycle analysis, in which cells were fixed in 70% ethanol at 4°C overnight, washed twice with cold phosphate buffer saline, and re-suspended in 0.5 ml PI staining reagent (25 μl propidium iodide (20X), 10 μl RNase A (50X), and 0.5 ml sodium citrate buffer) . The cells were then incubated in the dark at 37°C for 0.5 h. Samples were detected using an Attune NxT flow cytometer . FCS files were downloaded and analyzed using FlowJo software (version 10.4). For each group, results are shown as the mean ± SEM of three replicates. Statistical analysis was performed using the unpaired two-tailed t-test in GraphPad Prism eight software.Approximately 2 × 10In the discovery phase, from the 159 NSCL/P cases, we identified a total of 2,352 Single-nucleotide variants (SNVs) and 552 insertion/deletions (indels), including variants located in exons, splice sites, introns, UTRs, and intergenic regions . For comp > .000001) were recruited for association analysis. When compared with the p value 1.60E-04, which was adjusted for multiple corrections (p = .05/312), only rs201795193  was significantly associated with NSCL/P.A total of 312 common SNPs (MAF ≥0.01) that passed the threshold of HWE  to test their associations with NSCL/P, NSCLP, and NSCLO (p = .05/7 = 7.14E-03). Among these, four SNPs, including rs201795193 , rs12990267 , rs199721109 , and rs74343467  were specifically associated with NSCLO; the other two SNPs, rs6544660 and rs12478601, were associated with both NSCLO  and NSCL/P , where the significant signal of NSCL/P might be driven by the NSCLO since both SNPs show lower p values in NSCLO than that in NSCL/P  with nd NSCLO . Six outn NSCL/P . All theTHADA and four rare variants of ZFP36L2 were enrolled in the burden analysis, details about these variants were shown in the ZFP36L2 at 2p21 were significantly associated with increased risk for NSCL/P , NSCLP , and NSCLO   . InteresZFP36L2 exons among 204 patients with NSCLO and 181 normal controls. The rare variants were filtered using the same criterion enrolled into the burden analysis, and the result indicated that ZFP36L2 was also associated with NSCLO . Based on these findings, we further verified that the 2p21 locus was a risk factor for NSCLO and that the ZFP36L2 gene in 2p21 is a susceptibility gene for NSCLO in the Han Chinese population.Replication of the burden analysis was conducted by sequencing ZFP36L2 in the etiology of NSCLO, we performed RNA sequencing on human oral epithelial cell line (GMSM-K) with or without ZFP36L2 knockdown. Two biological replicates were included for each group.To investigate the potential roles of ZFP36L2 was knocked down  in total, among which 270 genes were upregulated and 57 genes were downregulated when ked down . GO analked down    .NGFR, TES, JUP, PLAU, FAS, PTK2B, TNFRSF14, SAT1), “apoptotic signaling pathway”  and “positive regulation of cell migration”  that were influenced by ZFP36L2. A total of 4,682 SNPs were recruited; we obtained the most SNPs in the SEMA5A gene, followed by TIAM1, SEMA3C, PTK2B, and FGF1 genes , two SNPs within the JUP gene were significantly associated with NSCLO: rs4479305  and rs9913846  , and none were identified to be associated with NSCL/P or NSCLP  . Meanwhior NSCLP .Jup was expressed in the medial nasal processes in E10.5 mouse embryos  .Our evidence can be summarized into two parts, the first being bioinformatics-related. Association analysis based on a large cohort and stringent significance threshold showed that most SNPs in 2p21 locus were associated with NSCLO, from which we inferred that 2p21 was a susceptibility locus for NSCLO. We annotated the six statistically significant SNPs using HaploReg v4.1 (reg.php) . The T areg.php) . FurtherZFP36L2 was carried out to further determine its association with NSCLO. We found that Zfp36l2 gene was expressed in the facial processes related to mouse lip development  associated with the term “regulation of cell proliferation” were expressed in the medial nasal, lateral nasal or maxillary processes. However, only two genes (Mllt11 and Ngfr genes) associated with the term “apoptotic signaling pathway” and three genes  associated with the term “positive regulation of cell migration” were expressed in the three upper lip-related processes (JUP, which was influenced by ZFP36L2 (ZFP36L2 might lead to NSCLO by influencing cell proliferation, and our study proved that ZFP36L2 negatively regulates cell proliferation and induces cell arrest in the G2 phase of GMSM-K. Our results were consistent with those of previous studies, that is, ZFP36L2 inhibited cell proliferation through a cyclin D-dependent and p53-independent pathway (J. ZFP36L2 on the cell cycle, the results are slightly varied. Fat-Moon Suk et al. observed that ZFP36L2 led to G1 phase arrest and thus resulted in a decreased S phase (ZFP36L2 might be a key protein in S phase progression control in the case of genome instability (ZFP36L2 affects the cell cycle, but its effects may be slightly modified in different cell types.We attempted to address how the rocesses  (Brunskirocesses . Further ZFP36L2 . Therefothway J. . As for  S phase , whereastability . In our ZFP36L2 gene in the etiology of NSCLO and proposed that ZFP36L2 is a novel susceptibility gene for NSCLO among the Han Chinese population. Further research is required to address limitations of this study. All samples recruited in our study were from the Han Chinese population; due to the genetic heterogeneity among different populations, further verification should be conducted in other populations. Additionally, to clarify the role of ZFP36L2 in NSCLO intuitively, follow-up experiments in animal models are necessary.In summary, our study comprehensively illustrated the important role of the


Colorectal cancer stem cells (CSCs) have the potential for self-renewal, proliferation, and differentiation. And LGR5 is a stem cell marker gene of colorectal cancer. Curcumin can suppress oncogenicity of many cancer cells, yet the effect and mechanism of curcumin in LGR5(+) colorectal cancer stem cells (CSCs) have not been studied. In this study, we studied the effect of curcumin on LGR5(+) colorectal CSCs using the experiments of tumorsphere formation, cell viability and cell apoptosis. Then autophagy analysis, RNA-Seq, and real-time PCR were used to identify the mechanism responsible for the inhibition of LGR5(+) colorectal CSCs. Our results showed that curcumin inhibited tumorsphere formation, decreased cell viability in a dose-dependent manner, and also promoted apoptosis of LGR5(+) colorectal CSCs. Next, we found curcumin induced autophagy of LGR5(+) colorectal CSCs. When LGR5(+) colorectal CSCs were co-treated with curcumin and the autophagy inhibitor (hydroxychloroquine), curcumin-induced cell proliferation inhibition decreased. In addition, we also found that curcumin inhibited the extracellular matrix (ECM)-receptor interaction pathway via the downregulation of the following genes: GP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC. Further, these genes were transcriptionally regulated by TFAP2A, and the high expression of TFAP2A was associated with poor prognosis in colorectal cancer. In conclusion, curcumin suppressed LGR5(+) colorectal CSCs, potentially by inducing autophagy and repressing the oncogenic TFAP2A-mediated ECM pathway. Colorectal cancer (CRC) is the world’s fourth most deadly cancer, and the second and third most common cancer diagnosed in women and men, respectively . AdvanceCurcuma longa. It has been demonstrated that curcumin possesses extensive therapeutic activities against multiple ailments, such as inflammation, metabolic syndrome, liver disease, arthritis, and neurodegenerative disease [Curcumin is a polyphenol extracting from  disease . In the  disease –9. For i disease ; curcumi disease . Other s disease . HoweverIn addition, recent studies have revealed that LGR5 is one of the few cell surface markers used to identify and isolate actively cycling stem cells in the colonic crypt . Furthervitro studies including the tumorsphere formation, cell viability and cell apoptosis, to illuminate the effect of curcumin on LGR5(+) colorectal CSCs. Then, we used autophagy analysis, RNA-Seq, and real-time PCR to identify the mechanism of the inhibition in LGR5(+) colorectal CSCs.To investigate the effects and the mechanism of curcumin on LGR5(+) colorectal CSCs, we used SW620 cell line (ATCC CCL-227) was obtained from the American Type Culture Collection . Recombinant human epidermal growth factor  and recombinant human basic fibroblast growth factor  were purchased from PeproTech, Inc. . Dulbecco's modified Eagle's medium (DMEM)/F12 medium, B-27 (Lot No.17504-044), and N-2 (Lot No.17502-048) supplements and recombinant human leukemia inhibitory factor  were purchased from Gibco . Curcumin (Lot No.C1386), hydroxychloroquine  and rapamycin (Lot No. V900930) were purchased from Sigma .3/well) were seeded in flat-bottom ultra-low attachment culture plates  in DMEM/F12 medium supplemented with 20 ng/mL EGF, 20 ng/mL FGF, B-27 (1 ×) supplement, N-2 (1 ×) supplement, and 10 ng/mL LIF. Then, the cells were incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air [SW620 cells  and then LGR5(+) cells were enriched by magnetic activated cell sorting. Fluorescence-activated cell sorting was performed using BD FACSAria Cell Sorter  .The total protein of LGR5(+) SW620 CSCs, LGR5(-) SW620 CSCs, and SW620 CSCs was extracted, and 50 μg of the total protein was used for electrophoresis. We transferred the electrophoresis products onto polyvinylidene fluoride membranes, and the membranes were blocked with 5% skim milk for one hour. After washing with Tris-buffered saline with Tween-20 , the membrane was incubated with primary antibodies LGR5  and β-tubulin , and shaken at 4 °C overnight. Next, the polyvinylidene fluoride membranes were washed thrice with TBST, incubated with a secondary antibody at room temperature for 1 h and washed with TBST again. Finally, immunoreactive products were visualized using enhanced chemiluminescence  and quantified using ImageJ software .LGR5(+) colorectal CSCs were incubated with different concentrations of curcumin  for 48 h. Then, sphere formations of different groups were observed under inverted light microscope. The culture medium (DMEM/F12) was then discarded and 100 μL fresh culture medium was added, along with 10 μL CCK-8 reagent  into each well, and incubated at 37 °C for one hour. The optical density was measured at 450 nm using a microplate reader .In this study, the Annexin V-APC (PI) Apoptosis Analysis Kit  was used to analyze cell apoptosis. After t10 PFU/mL), and treated with different concentrations of curcumin (1 μM and 5 μM) or rapamycin (100 nM) for 48 h. In this study, autophagy inducer rapamycin served as a positive control group. Then, the expression of mCherry and GFP was visualized using confocal fluorescence microscopy  at 100 × magnification. Autophagy flux was evaluated by observing the number of yellow and red puncta.Because the green fluorescent protein (GFP) signals can be quenched and the monomeric red fluorescent protein (mRFP) signal is more stable in the acidic environment of the lysosome, autolysosomes, and autophagosomes were labeled with mRFP (red) or GFP (green), respectively. The LGRAfter treated with curcumin (1 μM and 5 μM) or rapamycin (100 nM) for 24 h, the LGR5(+) colorectal CSCs were washed twice with ice-cold PBS, fixed using 2.5% glutaraldehyde for 60 min at room temperature, and post-fixed in 1% osmium tetroxide for 30 min. As described previously , 20, allAccording to our previous studies, 20, HCQ2FC|≥ 1. These genes were analyzed by clustering expression patterns, and were classified according to the involved biological processes and molecular functions and associated cell components, using the Gene Ontology database. In addition, KOBAS (http://kobas.cbi.pku.edu.cn/home.do) was used to analyze the pathway enrichment analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG).Total RNA of the LGR5(+) colorectal CSCs treated with and without curcumin (5 μM), were extracted using TRIzol® Reagent  according to the manufacturer's instructions. To explore the mechanism of curcumin in LGR5(+) colorectal CSCs, RNA-Seq was performed. Firstly, Agilent 2100 Bioanalyzer  was used to confirm RNA quality, then RNA-Seq libraries were prepared from three biological replicates and sequenced on an Illumina HiSeq . Fastqc and RSEM were used to assess the quality of the reads and calculate the expression of the genes, respectively . The scrNext, Cytoscape plugin iRegulon was used to identify master regulators that targeted these significantly expressed genes . TranscrTFAP2A and GAPDH was quantified with SYBR mixture , using a real-time PCR machine . The primers used were the following: for TFPA2A, TFAP2A F: 5′-CGTGTCCCTGTCCAAGTCCAA-3′ and TFAP2A R: 5′-GACCCGGAACTGAACAGAAGA-3′ [A CRC cDNA microarray (MecDNA-HColA095Su01) containing 15 paired cancer and non-CRC tissues and 65 CRC tissues was purchased from Shanghai Outdo Biotech Company . The mRNA expression of GAAGA-3′ ; for humTFAP2A expression with overall survival in CRC, the expression of TFAP2A was divided into two categories: high expression (ΔCT < 11.1) and low expression (ΔCT ≥ 11.1), and the correlation was analyzed by the Kaplan–Meier method with a log-rank test or Wilcoxon test.To investigate the correlation between p < 0.05, **p < 0.01, ***p < 0.001.All data were expressed as the mean ± standard deviation of at least three individual experiments. The data were analyzed by one-way analysis of variance (ANOVA) using the SPSS Statistics 26.0 . The following values were considered statistically significant: *Human LGR5(+) colorectal CSC models provide an opportunity to study the effect and mechanism of antitumor drugs. SW620 cells were cultured in flat-bottom ultra-low attachment culture plates in DMEM/F12 medium supplemented with 20 ng/mL EGF, 20 ng/mL FGF, B-27 (1 ×) supplement, N-2 (1 ×) supplement, and 10 ng/mL LIF. After sphere formation was observed, cells were labeled with anti-LGR5 MicroBeads, then LGR5(+) cells were enriched by magnetic activated cell sorting. The results of western blotting revealed that LGR5 was highly expressed in LGR5(+) SW620 CSCs, but was hardly detected in LGR5(-) SW620 CSCs  colorectal CSCs, LGR5(+) SW620 CSCs were treated with curcumin . We found that curcumin reduced tumorsphere formation in a dose-dependent manner  was 39.36 ± 2.36%, whereas the apoptosis rate of control cells was 5.07 ± 0.62%  colorectal CSCs was unclear. Thus, we transfected the curcumin (1 μM and 5 μM) treated-LGR5(+) colorectal CSCs with Ad-mRFP-GFP-LC3 adenovirus, and then detected the two different fluorescent signals to monitor the autophagic flux status post-treatment with curcumin. In this study, autophagy inducer rapamycin served as a positive control group, then we found both yellow and red puncta were increased in cells treated with either curcumin or rapamycin, indicating that curcumin can induce autophagy in LGR5(+) colorectal CSCs Fig. , suggestAs shown in Fig. In order to identify mechanisms of curcumin in LGR5(+) colorectal CSCs, RNA-Seq was used, and the result revealed that there were 1098 significantly differentially expressed genes in curcumin-treated cells and control cells, with 363 upregulated and 735 downregulated genes. These significantly differentially expressed genes between curcumin-treated cells and control cells were shown in Fig. GP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC , we found that the ECM–receptor interaction pathway included these ten significantly negatively expressed genes and were associated with shorter survival of CRC patients ; logrank p = 0.049)  cells involved in ‘Pathways in cancer,’ ‘PI3K-Akt signaling pathway,’ ‘MAPK signaling pathway,’ ‘Wnt signaling pathway,’ ‘NF-kappa B signaling pathway,’ ‘ECM-receptor interaction,’ and ‘Lysosome’ (Data not shown). In addition, there were ten significantly negatively expressed genes involved in ECM-receptor interaction, including TFAP2A in LGR5(+) colorectal CSCs treated with curcumin was significantly downregulated .To investigate the role of ge Table . After tColorectal cancer (CRC) is the world’s fourth most deadly cancer, and evidence shows that rare CSCs have the potential for self-renewal, proliferation, and differentiation in CRC . LGR5 waIn this study, we observed that curcumin inhibited tumorsphere formation, decreased cell viability in a dose-dependent manner. Curcumin also promoted apoptosis of LGR5(+) colorectal CSCs. In addition, curcumin can induce autophagy in many tumor cells. For example, curcumin inhibited proliferation, induced the autophagy and apoptosis in gastric cancer cells . SimilarGP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC in the ECM–receptor interaction pathway, which is also associated with shorter survival of CRC patients. As previously reported, the ECM acts as a structural support and provides stem cells with signals to regulate its function via reciprocal interactions between cells and the components of the ECM [Next, the RNA-Seq data showed that curcumin decreased the expression of  the ECM . At pres the ECM ; curcumi the ECM . HoweverTFAP2A-mediated ECM-receptor interaction. TFAP2A is a member of the AP-2 family of transcription factors, and this family is composed of TFAP2A/AP-2α, TFAP2B/AP-2β, TFAP2C/AP-2γ, TFAP2D/AP-2δ, and TFAP2E/AP-2ε [TFAP2A has been reported in different cancers [TFAP2A is overexpressed in human nasopharyngeal carcinoma and promotes tumorigenesis by influencing the HIF-1α/VEGF/PEDF pathway [TFAP2A expression is associated with poor prognosis in gastric cancer [TFAP2A is linked with melanoma through the regulation of cell adhesion molecules [TFAP2A is increased in more “stem-like” cancers [TFAP2A expression was markedly elevated in CRC compared with that in noncancerous colorectal tissues, and was associated with poor prognosis in CRC. Previous studies have found that curcumin induced apoptosis via reducing the expression of AP-2γ in human malignant testicular germ cells, whereas the pretreatment with the proteasome inhibitor MG132 blocked both the curcumin-induced reduction of AP-2γ and antiproliferative effect. Nevertheless, the effect of TFAP2A in CRC has not yet been reported. In future, the role of TFAP2A and other genes in curcumin-induced apoptosis of colorectal CSCs will be studied.After identifying the master regulators that targeted the aforementioned genes, we found that curcumin inhibited LGR5(+) colorectal CSC proliferation by downregulating 2E/AP-2ε . The abe cancers . For exa pathway . Howeverc cancer , and theolecules . In addi cancers . Our resTFAP2A-mediated ECM pathway. Lastly, while our study entailed mostly in vitro experiments, further studies, especially in vivo, are needed to understand the mechanism of effect of curcumin in LGR5(+) colorectal CSCs and to evaluate this potential therapeutic approach to CRC. In addition, the correlation between TFAP2A-mediated ECM pathway and autophagy also deserves further study.In summary, we found that curcumin suppresses the proliferation of LGR5(+) colorectal CSCs by inducing autophagy and transcriptionally repressing the oncogenic


This study aims to explore metabolic remodeling in plasma and tissue samples in patients with glioblastoma through an integrated targeted and untargeted metabolomics-based strategy. We report phospholipids, sphingomyelins, acylcarnitines, and acylglycerols as key impaired metabolic classes in glioblastoma.(1) Background: Glioblastoma is the most common malignant brain tumor in adults. Its etiology remains unknown in most cases. Glioblastoma pathogenesis consists of a progressive infiltration of the white matter by tumoral cells leading to progressive neurological deficit, epilepsy, and/or intracranial hypertension. The mean survival is between 15 to 17 months. Given this aggressive prognosis, there is an urgent need for a better understanding of the underlying mechanisms of glioblastoma to unveil new diagnostic strategies and therapeutic targets through a deeper understanding of its biology. (2) Methods: To systematically address this issue, we performed targeted and untargeted metabolomics-based investigations on both tissue and plasma samples from patients with glioblastoma. (3) Results: This study revealed 176 differentially expressed lipids and metabolites, 148 in plasma and 28 in tissue samples. Main biochemical classes include phospholipids, acylcarnitines, sphingomyelins, and triacylglycerols. Functional analyses revealed deep metabolic remodeling in glioblastoma lipids and energy substrates, which unveils the major role of lipids in tumor progression by modulating its own environment. (4) Conclusions: Overall, our study demonstrates in situ and systemic metabolic rewiring in glioblastoma that could shed light on its underlying biological plasticity and progression to inform diagnosis and/or therapeutic strategies. Glioblastoma (GBM) is the most common malignant brain tumor in adults . Its incFrom January 2020 to November 2020, twenty-nine patients with histologically documented glioblastoma were selected for the study. All patients were referred to our neurosurgery department, and resection or biopsies of the tumor were performed in all cases. Plasma and tissue samples were collected at surgery before initiating any treatment. Medical charts were reviewed, and the following data were collected: gender, age at diagnosis, past medical history, revealing symptoms, radiological characteristics, type of surgery performed, and histological and biological characteristics of the tumor. All samples used in this study belong to a declared collection located in the Pathology Department (Prof. Annie Laquerrière) of Rouen University Hospital in accordance with the relevant guidelines and regulations and with the permission of the local authorities. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.®p180 analysis are included in the kit or provided by Biocrates Life Science AG .All reagents, internal and calibration standards, quality controls, test mix, and a patented 96-well filter plate required for the AbsoluteIDQSample preparation was carried out according to the manufacturer’s protocol ,45. Brie® p180 kit is a fully automated assay based on phenyl isothiocyanate (PITC) derivatization of the target analytes in bodily fluids using internal standards for quantitation. Amino acids and biogenic amines are determined in LC-MS mode, acylcarnitines, phospholipids, sphingomyelins, and the sum of hexoses are analyzed in flow injection analysis (FIA). The analyses were performed following the instructions of the kit manufacturer using the liquid chromatography instrument prominence Shimadzu UFLC System  coupled to the 4000 Qtrap mass spectrometer  with an electrospray ion source. A system suitability test was conducted before each batch of the samples  to warm up the LC-MS/MS system and check the inter-day performance of the system. Data acquisition and processing were performed using the Analyst 1.5 software . Twenty microliters of the sample extract were used in the flow injection analysis (FIA) in positive mode to capture acylcarnitines, glycerophospholipids, and sphingolipids, while hexoses were monitored in a subsequent negative mode run. All FIA injections were carried out using the Biocrates MS Running Solvent. All metabolites were identified and quantified using isotopically labeled internal standards and multiple reaction monitoring (MRM) as optimized and provided by Biocrates Life Sciences AG .The AbsoluteIDQv/v) water/acetonitrile at 10 mg/L and used for ion mobility cell calibration.Water, acetonitrile, isopropanol, and formic acid were optima LCMS grade and purchased from Fisher Chemicals. Ammonium formate was LCMS grade . Leucine Enkephalin  at a concentration of 2 mg/mL  was used as a reference for mass measurements. Poly-DL-alanine was prepared in 50:50 (2 evaporated. The resulting phase was re-suspended in 200 µL ACN/H2O/IPrOH (1:1:1). A pooled quality control (QC) was prepared by mixing 5 µL of each plasma sample. Four QC dilutions at 1:2, 1:4, 1:8, and 1:16 were prepared from the QC sample. Samples were stored at −20°C before use.Human plasmas were prepared using a liquid biphasic approach. Lipids were extracted from 100 µL of plasma by adding 280 µL chloroform, 140 µL methanol, and 84 µL water. The mixtures were vortex-mixed, cooled in ice for 15 minutes, and centrifuged 10 min at 12,000 rpm. The bottom phase was removed, transferred to glass UPLC vials, and N2O (40/60) containing 10 mM ammonium formate; mobile phase B consisted of IPrOH/ACN (90/10). Both solvents contained 0.1% formic acid. The flow rate was maintained at 100 µL min−1. A mobile phase gradient separation was performed over 20 min according to the following steps: 10% B at 0 min, 56% B at 2 min, 75% B at 10 min, 99% B at 12–15 min, 10% B at 16–20 min. Sample analysis order was randomized to avoid the potential for confounding critical variables with analytical run order effects.The liquid chromatography separation was performed with a UHPLC system . Separation was carried out at 50 °C using a 1.0 × 100 mm Acquity UPLC HSS T3 column (Waters), with a particle size of 1.8 µm, equipped with a 0.2 µm prefilter. Mobile phase A consisted of ACN/Hm/z 50−1200 was used. The source and IMS parameters are summarized in + at m/z 556.2771 and [M-H]− at m/z 554.26202. Sodium formate solution was used for external instrument calibration. The Synapt G2 HDMS was equipped with a traveling wave “Triwave™” geometry, including an ion mobility cell (IMS T-wave). The TOF analyzer was operated in the V resolution mode with an average mass resolution of m/Δm 20,000 . Data acquisition of an ion mobility experiment consisted of 200 bins. The cross-collision section (CCS) values, obtained in nitrogen, were experimentally determined using singly charged Poly-DL-alanine oligomers as the TWIM calibrant species for ESI+. The CCS values were derived according to previously reported procedures ) was used for external calibration before each analysis.MALDI matrices, including 2.5-dihydroxybenzoic acid (DHB) and 9-aminoacridine hydrochloride monohydrate (9-AA), were purchased from Sigma-Aldrich . Sodium trifluoroacetate  at 0.1 mg mL−1 in MeOH/H2O 50:50 [v/v]) was deposited with the following parameters: nozzle temperature 80 °C, nozzle velocity 1200 mm min−1, N2 pressure 10 psi, N2 flow rate 2 L min−1, number of passes 12, flow rate 100 µL min−1, and track spacing 3 mm. In negative-ion mode, 9-AA matrix  was sprayed using the following parameters: nozzle temperature 90 °C, nozzle velocity 1200 mm min−1, N2 pressure 10 psi, N2 flow rate 3 L min−1, number of passes 2, flow rate 120 µl min−1, track spacing 3 mm, and drying time 30 s between passes. Each slide was vacuum-dried before analysis.Fresh frozen 10-µm sections of human brain tissue were mounted on conductive ITO-coated glass slides 75 × 25 mm  and stored at −80 °C until analysis. Matrix solutions were applied with an automatic micro sprayer HTX TM-Sprayer  as previously described . In the m/z 150–1000 via MALDI source. Thus, the instrument was auto calibrated during image acquisition. In positive-ionization mode, calibration was performed by assigning C7H6O4 , C7H15NO3 , C7H6O4 , C5H14NO4P , C9H17NO4 , C14H8O6 , C21H12O9 , C40H80NO8P , and C42H82NO8P . In the negative-ion mode, the assigned peaks were C13H10N2 , C10H15N5O10P2 , C10H16N5O13P3 , C41H80NO8P , and C47H83O13P . Data size was set at 2 million points for a transient length of 0.87 s, and spectra were acquired with a 97% data file reduction. A single MSI measurement was performed for each specimen. Images were captured using FTMS control and FlexImaging  software. Images were processed with SCiLS Lab Pro software . The total ion current method was used for normalization, and m/z intervals were automatically set at ±1 ppm. Images were viewed using both FlexImaging and SCiLS Lab software .Data were acquired on a FTICR instrument  equipped with a 12-Tesla superconducting magnet and a dynamically harmonized ICR cell. This instrument was also equipped with both a laser desorption ionization source  and an electrospray (ESI) source. Each MALDI spectrum for each position was the result of 1 scan and 500 consecutive laser shots. Spectra were acquired over a 100 µm raster. Before imaging analyses, the instrument was externally calibrated in the required mode by NaTFA infusion via an ESI source, then internally calibrated by assigning known metabolites from Preliminary assignments based on accurate mass measurements were performed using the mass spectrometry databases, METLIN  and HMDB®  according to the manufacturer’s instructions. The sample was eluted in a final volume of 30 µL and stored at −20 °C. Double-stranded DNA quantification was performed by a fluorometric method using a Quantit™ PicoGreen® dsDNA Assay kit  and a Twinkle LB970 microplate fluorimeter . For each sample, cfDNA quantification was performed in duplicate from 2 µL of eluate, and normalization was performed using a standard calibration curve of known concentrations of standard dsDNA (from 0 to 10 ng). Overall, cfDNA concentration reflects both cell-free genomic DNA as well as circulating tumor DNA (ctDNA).Circulating cell-free DNA was extracted from blood samples right before surgery and as previously described . Brieflyt-tests or Mann–Whitney U depending on the normal distribution of the data. The Limma package [p < 0.05 was considered statistically significant. For network analysis, the first step was to compute several glasso-based networks (GLN) [Ion intensities or absolute concentrations were log-transformed and Pareto-scaled . Missing package  was used package , and p <ks (GLN) . Three kks (GLN) . Using tks (GLN)  and the ks (GLN) . The modks (GLN) . All datThe mean glioblastoma showed a male predominance of 82%. The mean age at diagnosis was 66 ± 11.4 years for females and 64 ± 11.1 years for males. Regarding the performed surgery, it consisted of a biopsy in 56.7% and resection in the other cases. The mean controls age was 39 ± 12.5 years for females and 37 ± 10.9 years for males. A cohort overview is presented in To analyze the data, an exploratory approach was first used. This unsupervised analysis aimed to track samples’ clustering trends based on their underlying metabolic profiles. We unveiled these grouping trends using Spearman correlations and hierarchical clustering of the samples. The results showed a clear separation of two distinct groups related mainly to control and GBM samples p-values, the top metabolites are presented in To go further in the analysis, we performed a differential analysis between the two groups GBM versus Control, using each of the three datasets. The analysis identified 176 annotated metabolites that were differentially expressed between GBM and control samples. The plasma-based comparison yielded 148 metabolites, including 75 phospholipids , 20 sphingomyelins, 15 acylcarnitines, 14 amino acids, 12 triacylglycerids, 10 biogenic amines, 1 cholestan steroid, and 1 diacylglycerol. The tissue-based comparison unveiled 28 metabolites, including 17 phospholipids , 5 acylcarnitines, 1 diacylglycerol, 2 sphingomyelins, 2 triacylglycerols, and 1 steroid ester. The summary results are presented in p-value < 0.01) are presented in Given the opportunity to have in-tissue metabolomics with matched-plasma samples, we compared the plasma-based and tissue-based metabolic profiles and found ten lipids that overlap between the two lists. These include, phosphatidylcholines LPC(18:0), PC(36:5), PC(40:6), PC(32:1), sphingomyelins SM(33:1), SM(36:2) and triacylglycerides TG(56:7), TG(52:2), TG(52:4), TG(52:3). The boxplots of the top five lipids are presented in p-value < 0.05 and abs(Spearman rho) > 0.25. The positively correlated metabolites included Ornithine (Spearman rho = 0.52), Lysine (Spearman rho = 0.49), Phenylalanine (Spearman rho = 0.46), Propionylcarnitine (Spearman rho = 0.45), Carnitine (Spearman rho = 0.45), Hexadecenoylcarnitine (Spearman rho = 0.42), Tetradecadienylcarnitine (Spearman rho = 0.4), Hexenoylcarnitine (Spearman rho = 0.35), Spermine (Spearman rho = 0.3), SM C18:1 (Spearman rho = 0.28), Butenylcarnitine (Spearman rho = 0.29), SM C16:1 (Spearman rho = 0.29), and Asymmetric dimethylarginine (Spearman rho = 0.27). The negatively correlated metabolites included two biogenic amines Serotonin (Spearman rho = −0.68) and Methionine sulfoxide (Spearman rho = −0.29). A network visualization is presented in Given the rising interest in using circulating free DNA (cfDNA) in oncology , we alsoUsing the targeted plasma metabolomic dataset, different correlation networks were built using control, disease, or both samples. Using these correlation networks, 29 patient-specific metabolic signatures were extracted . These pDespite a sustained research effort in the fight against glioblastoma, the prognosis of this condition remains devastating, and its metabolic signaling pathways are barely understood. To tackle this challenge, omics-based technologies could shed light on this aggressive disease. Metabolic phenotyping through metabolomics is one of the promising tools to interrogate functional metabolic readouts.In this study, we identified four metabolites’ classes as proxies of deep metabolic remodeling in plasma and tissue samples from glioblastoma patients, which included phospholipids, acylcarnitines, sphingomyelins, and Triacylglycerols. Some of the highlighted metabolites in our study have been previously reported. This is the case for a number of amino acids, such as asparagine, glutamine, lysine, citrulline, or valine, for example  38,39,4,439,41. In summary, our study demonstrated the potential of systems-based metabolomics strategies to holistically interrogate biological plasticity in glioblastoma and parse the role of lipids in tumor progression by the modulation of its own environment. The next step of our work would be the study of therapeutic modulations of these metabolic signatures in order to probe treatment effectiveness. Applying these plasma metabolic patterns could also inform clinical practice to adapt treatment before MRI or clinical modification. A better understanding of metabolic impairments underlying glioblastoma spread would also drive the development of targeted therapies to prevent glioblastoma from promoting its own extension to the surrounding tissues. Such systems-based strategies highlight the importance of multi-omics and multimodal investigations to understand glial lesions to pave the way to more personalized therapies and, ultimately, achieve the promise of precision medicine.


The biological activities of curcumin in humans, including its antioxidative and anti-inflammatory functions, are limited by its naturally low bioavailability. Different formulation strategies have been developed, but the uptake of curcumin from these galenic formulations into and efflux from intestinal cells, which may be critical processes limiting bioavailability, have not been directly compared. Furthermore, little is known about their effect on P-glycoprotein activity, an important determinant of the pharmacokinetics of potentially co-administered drugs. P-glycoprotein activity was determined in LS180 cells, incubated with 30 or 60 µmol/L of curcumin in the form of seven different formulations or native curcuma extract for 1 h. All formulations inhibited P-glycoprotein activity at both concentrations. Curcumin uptake, after 1 h incubation of LS180 cells with the formulations (60 µmol/L), showed significant variability but no consistent effects. After 1 h pre-treatment with the formulations and further 8 h with curcumin-free medium, curcumin in cell culture supernatants, reflecting the efflux, differed between individual formulations, again without a clear effect. In conclusion, curcumin inhibits P-glycoprotein activity independently of its formulation. Its uptake by and efflux from intestinal cells was not significantly different between formulations, indicating that these processes are not important regulatory points for its bioavailability. Curcuma longa Linn. and has been used in Ayurvedic medicine due to its many reported health beneficial activities, including anticarcinogenic, anti-inflammatory, and anti-diabetic effects [The lipophilic phytochemical curcumin is the main curcuminoid from  effects . The dir effects ,3,4).More recently, curcumin has been investigated as a nutraceutical in the prevention and complementary therapy of diseases, such as cancer, chronic inflammatory disorders, Alzheimer’s disease, and many more . mdr1 gene, member of the family of ATP-binding cassette transporters, and is expressed in the liver, intestine, and kidney. Its overexpression in cancer cells is the main cause of multidrug resistance [The efflux transporter P-glycoprotein (P-gp) is a 170-kDa transmembrane protein, encoded by the sistance . Intestisistance ,22. Prevsistance ,25,26,27sistance ,31,32,33sistance ,30,34. Csistance ,18. The In a preceding human trial, we investigated the pharmacokinetics of curcumin, from seven different galenic formulations relative to that of native curcumin, and observed that only those with PS80 and γ-cyclodextrin significantly improved its bioavailability . Here, w2Odd; Longvida®; Verdure Sciences, Noblesville, IN, USA), a curcuma extract with turmeric oils , a curcuma extract with adjuvants , submicron-particle curcumin , phytosomal curcumin  a curcumin-γ-cyclodextrin complex  and curcumin incorporated into PS80 micelles consisting of 93% PS80 and 7% curcuminoid powder . For experiments, rhodamine 123 , verapamil hydrochloride (stock 50 mmol/L in H2Odd), elacridar (stock 2 mg/mL in DMSO) and PS80 were purchased from Sigma-Aldrich . Empty PS80 micelles were from AQUANOVA AG . β-Glucuronidase type H-1 from Helix pomatia (EC 3.2.1.31) was purchased from Sigma-Aldrich . Methanol, ethylacetate, ethanol and acetonitrile were HPLC grade.The following curcumin formulations were used for experiments: A native curcuma extract , liposomal curcumin , between passages 46 and 51. This cell line is recommended for studies on the regulation of P-gp and other efflux proteins . Cells w5 cells/well were seeded in 48-well plates and grown for 24 h at 37 °C and 5% CO2. Cells were incubated with all formulations normalized to concentrations up to 75 µmol/L, a positive control (0.1% Triton X-100), solvent controls , and a negative control (MEM Complete) for 1 h at 37 °C and 5% CO2. Then, substances were removed and cells were incubated with neutral red dye  diluted to 140 µmol/L in MEM Complete for further 2 h at 37 °C and 5% CO2. The dye was discarded, and neutral red destain solution  was added and incubated for 15 min at room temperature while shaking. Absorbance was quantified with a Synergy HT  at 580 nm. Medium control served as a reference for 100% viability.To ensure detectable amounts of curcumin in cell lysates and supernatants, in all experiments, formulations were normalized to the maximum curcumin concentration that did not show cytotoxic effects. To determine the appropriate concentration, neutral red uptake assays were conducted as described in Repetto et al., (2008) with minor modifications . Then, 35 cells/well were seeded in 48-well plates and grown for 24 h. Half of the cells were incubated with the formulations normalized to 30 µmol/L or 60 µmol/L curcumin, solvent controls , negative control (MEM Complete), or positive control  for 10 min at room temperature while shaking, followed by a 30 min incubation at 37 °C. Fluorescence intensity was read by a Synergy HT) at emission/excitation 485/529 and was normalized to protein content . Relative intracellular R123 was calculated by referring the quotient of fluorescence intensity with elacridar by fluorescence intensity without elacridar to the one from medium control cells.The P-gp activity assay was conducted as described before with modifications . Brieflyil as in ) and 1 µ6 cells/well were seeded in 6-well plates and grown for 24 h. Cells were treated with the formulations, normalized to 60 µmol/L curcumin, and medium control for 1 h at 37 °C and 5% CO2. For stability control, all solutions were incubated in parallel without cells. Aliquots were frozen at −80 °C before and after 1 h incubation. For the cellular uptake experiments, supernatants were frozen at −80 °C after 1 h incubation. Cells were washed with PBS, scratched off the plate, washed again with PBS and lysed by an incubation on ice with 20 µL lysis buffer -aminomethan hydrochloride) pH 8; 1% Nonidet P-40; 4% protease inhibitor). Cell lysates were sonicated for 1 min, centrifuged at 11,900× g for 5 min at 4 °C. Aliquots were used to quantify the protein amount by Bradford protein assay [Additionally, 2 × 10in assay . All sam6 cells/well were seeded in 6-well plates and grown for 24 h. Cells were incubated with substances as described for the cellular uptake experiments (see 2.5). After 1 h incubation at 37 °C and 5% CO2, supernatants were removed, cells were washed with PBS and incubated with curcumin-free MEM Complete for further 8 h at 37 °C and 5% CO2. After 8 h, supernatants were stored at −80 °C. Equal to the cellular uptake experiments, cells were washed with PBS, scratched off the plate, washed again with PBS and lysed with 20 µL lysis buffer. Cell lysates were sonicated for 1 min, centrifuged at 11,900× g for 5 min at 4 °C and aliquots for protein quantification were stored with all other samples at −80 °C until further analysis. To observe curcumin efflux over time, representatively, efflux experiments were conducted with native and micellar curcumin as described above with a pre-incubation for 1 h and further incubations with curcumin-free MEM Complete for 6, 8, and 24 h.For cellular efflux experiments, 2 × 10Helix pomatia (10 mg/mL in 0.1 M sodium acetate) for 45 min at 37 °C. For curcumin extraction from all samples on ice, 3 mL of extraction solvent  were added and mixed for 30 s. After a centrifugation at 1690× g for 5 min at 4 °C, 2 mL of the upper layer were transferred to a clean tube. The extraction was performed three times. After the second and third extraction, 3 mL of the upper layer were collected. Combined supernatants were evaporated and resuspended in 75 µL methanol for HPLC analysis. Next, 20 µL of each sample were injected into a Shimadzu-HPLC system  equipped with a Reprosil-Pur C18 AQ column  kept at 40 °C. Curcuminoids were quantified with a fluorescence detector . Mobile phase (55% H2Odd adjusted to pH 3 with perchloric acid and 45% acetonitrile) was delivered at a flow rate of 1.4 mL/min. External standards for curcumin , demethoxycurcumin  and bis-demethoxycurcumin  were used. All peaks were integrated with LabSolutions software .For quantification of curcumin in cell lysates, supernatants, and stability controls, curcumin was extracted as described before . Becausen = 3), each consisting of technical duplicates. A one-way ANOVA and Dunnett’s post-hoc test with the medium control group serving as the reference, were used to compute differences between group means of P-gp activity. Differences between the formulations, regarding intra- and extracellular curcumin concentrations, in uptake and efflux experiments were assessed by one-way ANOVA, followed by Tukey’s post-hoc tests. The same tests were applied to assess differences between different time points, within the formulations, in time-dependent efflux experiments with native and micellar curcumin. Curcumin stability was confirmed by testing for differences between concentrations before and after 1 h incubation by t-tests. For all statistical tests, differences were considered significant at p < 0.05.All statistical analyses were conducted with GraphPad Prism 9 . Data are presented as arithmetic mean ± SD or SEM, as indicated. All experiments were conducted in biological triplicates  inhibited P-gp activity, but not when formulated as empty PS80 micelles (p = 0.7851). In concentrations of 30 µmol/L curcumin (p = 0.9956), significantly increased R123 accumulation.After the co-incubation of LS180 cells with the P-gp substrate R123 and 60 µmol/L curcumin from the different formulations, intracellular R123 accumulation was significantly increased compared to medium control A. Conseqcurcumin B, all fop = 0.0116) and empty PS80 micelles (p = 0.0022) increased intracellular R123. The solvent control DMSO had no effect on P-gp activity at any of the concentrations tested , between curcumin with turmeric oils and submicron-particle curcumin (p = 0.0210), and between curcumin with turmeric oils and micellar curcumin  and from 8 to 24 h (p < 0.0001 for micellar, p = 0.0002 for native curcumin; Significant differences were observed between curcumin with turmeric oils and adjuvants (To the best of our knowledge, the present study is the first attempt to compare the effects of different galenic formulations of curcumin on the transport activity of P-gp. All curcumin formulations and native curcumin increased the accumulation of the P-gp-substrate R123 in LS180 cells when they were co-incubated for 1 h. P-gp inhibitory actions of unformulated curcumin were previously described for the intestinal cell lines LS180 and Caco-2 ,26,27. AConsequently, the reported effects of native curcumin on the pharmacokinetics of drugs that are P-gp substrates ,30,34, sWe quantified the uptake of curcumin from the different formulations, into LS180 cells within 1 h, to better understand the similar P-gp-inhibitory activities of the formulations. Curcumin with turmeric oils showed significantly increased intracellular curcumin concentrations compared to all other formulations and native curcuma extract A, in agrA theoretically possible explanation for differences in cellular uptake would be different rates of degradation of unformulated, or formulated, curcumin in cell culture medium. Curcumin is instable at neutral to alkaline pH and its degradation at pH 7.0 to 7.4, similar to the conditions in our experiments, was reported to occur within 1 h ,47,48. TEfflux of curcumin and its conjugated metabolites from intestinal cells back to the intestinal lumen, facilitated by efflux transporters, has been proposed as a critical factor limiting its bioavailability ,46. The Curcumin concentrations in supernatants decreased significantly from 6 to 8 h and from 8 to 24 h . The oppIntracellular metabolism is another factor that might contribute to decreasing curcumin concentrations with time. As observed in human and rat intestinal and hepatic microsomes, curcumin is mainly conjugated to curcumin glucuronide and curcumin sulfate, but also reduced to dihydro-, tetrahydro-, hexahydro-, or octahydrocurcumin . In humaIn conclusion, native and formulated curcumin inhibited P-glycoprotein activity in cultured intestinal cells, without significant influences of the galenic formulation. Furthermore, cellular uptake by, and efflux from, intestinal cells were not significantly affected by its formulation, even though significant differences in the bioavailability of different curcumin formulations, front and foremost micellar curcumin, have been reported. Consequently, the presented data suggest that intestinal uptake and efflux may not be important regulatory points, limiting the bioavailability of the direct and indirect antioxidant curcumin. Nevertheless, the inhibition of P-glycoprotein activity by curcumin warrants further investigation, as it may pose the risk of undesired interactions with prescription drugs.


Seizure score and seizure latency were observed. Mitochondrial redox status as well as ATP and uncoupling protein 2 was recorded. Moreover, glutamate homeostasis was unveiled. The present findings demonstrate the TMZ-attenuated Li/PIL seizure score and latency. It improved mitochondrial redox status, preserved energy production mechanisms, and decreased reactive astrocytes evidenced as decreased glial fibrillary acidic protein immune-stained areas in hippocampal tissue. In addition, it modulated phosphorylated extracellular signal-regulated kinases (p-ERK1/2) and p-AMP–activated protein kinase (p-AMPK) signaling pathways to reflect a verified anti-apoptotic effect. Consequently, it upregulated mRNA expression of astroglial glutamate transporters and reduced the elevated glutamate level. The current study demonstrates that TMZ exhibits robust anti-seizure and neuroprotective potentials. These effects are associated with its ability to modulate mitochondrial redox status, boost p-ERK1/2 and p-AMPK signaling pathways, and restore glutamate homeostasis in hippocampus.Mitochondrial oxidative status exerts an important role in modulating glia–neuron interplay during epileptogenesis. Trimetazidine (TMZ), a well-known anti-ischemic drug, has shown promising potential against a wide range of neurodegenerative disorders including epilepsy. Nevertheless, the exact mechanistic rationale behind its anti-seizure potential has not been fully elucidated yet. Herein, the impact of TMZ against mitochondrial oxidative damage as well as glutamate homeostasis disruption in the hippocampus has been investigated in rats with lithium/pilocarpine (Li/PIL) seizures. Animals received 3 mEq/kg i.p. LiCl It optimizes oxygen demands xidation . Potentixidation . TMZ wasxidation . It imprxidation , conservxidation . It alsoxidation  and hind disease . TMZ pro disease  and prev disease . The exaThe current study was, therefore, conducted to investigate whether TMZ could modulate mitochondrial redox status and glutamate homeostasis in the hippocampi of rats following lithium/pilocarpine (Li/PIL) injection. It also explored the impact of such effects on the incidence and intensity of induced seizures.Adult male Wistar rats each weighing 180 ± 200 g were obtained from the National Research Centre in Cairo and housed at constant temperature and humidity at the animal facility of the Faculty of Pharmacy, Cairo University. Seizure induction was done from 9 am to 12 pm to diminish circadian effects on seizure vulnerability. The investigation fulfilled the Guide for the Care and Use of Laboratory Animals of the Ethical Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University (Permit number: PT2715).3) and pilocarpine (PIL) were purchased from Sigma-Aldrich, MO, United States, while trimetazidine dihydrochloride (TMZ) was purchased from Rameda Pharmaceutical Company, Cairo, Egypt.Lithium chloride  20 h later, for the induction of seizures  received TMZ  for five consecutive days. Group III (Li/PIL control) received LiClseizures . Group IImmediately after PIL injection, rats were placed individually in Plexiglas cages and were observed for 30 min. Each rat was assigned a convulsive score from 0 to 5 based on the Racine scale , where bn = 12/group) were isolated and divided into two subsets. The hippocampi in the first subset (n = 6/group) were homogenized in cold phosphate buffer saline . In the second subset (n = 6/group), the hippocampi were frozen at −80°C to be used in Western blot and quantitative real-time PCR (qRT-PCR) analyses.Four hours after PIL injection, rats were euthanized by decapitation under light anesthesia, and their brains were carefully excised. Brains of three rats from each group were preserved in 10% formalin to be used for histological examinations and immunohistochemical staining. The hippocampi of the remaining rats -1-piperazineethanesulfonic acid) HEPES buffer was added to the supernatant and centrifuged at 10,000 × g for 30 min. HEPES buffer was added to the mitochondria pellets after discarding the supernatant and recentrifuged for 10 min at 10,000 × g, then the supernatant was discarded and PBS was added to the final mitochondria-rich fraction pellets , an end product of lipid peroxidation, using the specific colorimetric kit  and expressed as nmol/mg protein. Reduced glutathione (GSH) was measured according to the method of The ATP content was estimated in the total hippocampal homogenate using the BioVision's ATP colorimetric assay kit . The assay utilizes the phosphorylation of glycerol to generate a product that is easily quantified colorimetrically at 570 nm and expressed as pg/mg protein.The total tissue homogenate was used to determine glutamate and cytochrome c (Cyt c) levels using ELISA assay kits . Their results were expressed as µg/mg protein and pg/mg protein, respectively. In addition, UCP2 was measured in the mitochondrial rich fraction by a specific ELISA kit  and expressed as pg/mg protein.p-nitroaniline (pNA) from caspase-3 sequence-specific peptides. pNA has an absorption peak which is easily measured colorimetrically at 405 nm and used to express caspase-3 activity as nmol pNA/h/mg protein.Caspase-3 activity was estimated in the total hippocampal homogenate using the caspase-3 colorimetric assay kit . The assay depends on the dissociation of yellow group p-AMPK) (Thy 172), or Beta actin (β-actin) primary antibodies . Next, the membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies . Finally, the bands were established with the Chemiluminescence Reagent Kit , and their intensity was analyzed by Chemi Doc™ Imaging System with Image Lab™ software version 5.1 . All values were normalized to that of β-actin and presented as fold-change.Following total hippocampal protein quantification by using the Bradford kit , 20 μg protein of each sample was separated by SDS/polyacrylamide gel electrophoresis, then transferred onto polyvinylidene difluoride membranes . A blocking solution comprising 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, and 3% bovine serum albumin (BSA) was then added to the membranes at room temperature for 2 h to avoid nonspecific binding of the antibodies before incubating them overnight at 4°C with one of the following primary antibodies: p-extracellular signal-regulated kinases (p-44/42 ERK1/2) (Thr202/Tyr204), p-AMP-activated protein kinase  was used to extract total RNA from the total hippocampal tissue, which was then measured spectrophotometrically at 260 nm. Equal amounts of the extracted RNA were then reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit . To assess the gene expression of glial glutamate transporters, GLAST and GLT-1, qRT-PCR was performed using ABI PRISM 7500 Fast Sequence Detection System . The sequences of the PCR primer pairs used were the following: For GLAST, F:5’- CCAGTGCTGGAACTTTGCCT -3′, R: 5′- TAAAGGG CTGTACCATCCAT -3′, for GLT-1 F:5’-ACAAAAAGCAACGG AGAAGAGCC -3′, R: 5′- TACGGTCGGAGGGCAAATCC -3′, and β-actin . In brief, 1 μg of the total RNA was mixed with 50 μM oligo (dT) 20, 50 ng/μL random primers, and 10 mM dNTP mix in a total volume of 10 μL in an optical 96-well plate using universal cycling conditions (5 min at 95°C followed by 45 cycles of 5 s at 95°C and 10 s at 60°C). The relative expression of the target genes was obtained using the 2−ΔΔCT formula . All valBrain samples fixed in 10% neutral buffered formalin were trimmed and processed in serial grades of ethanol, cleared in xylene, infiltrated with synthetic wax, and embedded out into Paraplast tissue-embedding media. 3- to 5-μ-thick sagittal sections were cut by a rotatory microtome. The sections were stained with Harris Hematoxylin and Eosin (H&E) as a general tissue examination staining method. Hippocampal neurons were outlined, then the pathological changes in the different hippocampal regions were examined at high power (×400 magnification) in each group. The Lesion scoring system for pathological changes in different hippocampal regions was performed . Each anDeparaffinized 5-μ-thick tissue sections were treated with 3% hydrogen peroxide for 20 min, washed by PBS, then incubated with anti–glial fibrillary acidic protein (GFAP) monoclonal antibody from Thermo Fischer Scientific, MA, United States (1:100) overnight. After that, tissue sections were washed by PBS and incubated with a secondary antibody HRP EnVision kit (DAKO) for 20 min and then with diaminobenzidine for 10 min. Finally, they were counter stained with hematoxylin, dehydrated, and cleared in xylene for microscopic analysis.Six random nonoverlapping fields from different hippocampal regions per tissue sample were analyzed for determination of average area % of expression of GFAP in immunostained sections, as well as mean intact neurons count in Cornu Ammonis regions  and Dentate Gyrus (DG) hilar cells. All micrographs and data were obtained by using a full HD microscope camera operated by the Leica application module for tissue sections analysis ”.p < 0.05. GraphPad Prism® software package, version 5 , was used to carry out all statistical tests.All data obtained, except for total histology scores, Racine scores, and seizure incidence, were expressed as mean ± standard deviation (SD). Results were analyzed using the two-way analysis of variance test (ANOVA) followed by the Tukey–Kramer multiple comparison’s test as a post-hoc test. Total histology scores and Racine scores were presented as median and analyzed using the Kruskal–Wallis test followed by Dunn's test as a post-hoc test. Incidence of Stage 3–5 seizures was compared using Fisher's exact probability test for all statistical tests, the level of significance was set at p < 0.0001) and noticeably reduced seizure incidence (p < 0.0001) and severity (p < 0.01) by 86.6 and 78.6%, respectively.As shown in p < 0.0001). Pretreatment with TZM, however, markedly increased their mRNA expression to reach 4-fold and 2.8-fold to that in the Li/PIL control group, respectively (p < 0.0001)  .p < 0.0001 On the contrary, rats pretreated with TMZ exhibited a normal level of glutamate in their hippocampi, p = 0.21, recording a decrease of 43.2%, as compared to the Li/PIL control group.As shown in Li/PIL induced astrocytes activation as demonstrated by the obvious increase in the immunostaining density of hippocampal GFAP , an effect that was largely prevented by TMZ recording a decrease of 69.5%, as compared to the Li/PIL control group .p-AMPK (0.55 ± 0.07 vs 1.015 ± 0.01) as compared to that in the normal control (p < 0.0001). TMZ induced a further increase in hippocampal p-ERK1/2 by 62.5% and prevented the drop in p-AMPK reaching 1.6-fold to that in the Li/PIL control group   along with a profound decline in ctively) .p < 0.0001). Pretreatment with TMZ effectively enhanced the mitochondrial TAC as well as GSH content to reach 1.5-fold and 1.44-fold, respectively, to that in the Li/PIL group (p < 0.001 and p < 0.0001). Consequently, TMZ reduced mitochondrial MDA production by 60% as compared to Li/PIL rats (p < 0.0001), showing a similar level to that in the normal control group p = 0.065  and GSH (35.9 ± 1.2 vs 57.1 ± 3.6) content along with the marked increase in mitochondrial MDA . However, TMZ was effective in impeding UCP2 expression in mitochondria-rich fraction by 65% as compared with the Li/PIL control group (p < 0.0001) and restoring hippocampal ATP, reaching a comparable content to the normal control group, p = 0.59, to reach 1.9-fold to that in the Li/PIL control group (Li/PIL increased UCP2 in mitochondria-rich fraction (6.9 ± 1.6 vs 1.02 ± 0.02) with a pronounced reduction in the hippocampal ATP (11.2 ± 2.3 vs 20.5 ± 1.7) content (ol group .p < 0.0001). Pretreatment with TMZ largely prevented these mitochondrial derangements recording a decrease by 50.8 and 60.4%, respectively, as compared to that in the Li/PIL control group  and, sequentially, in caspase-3 activity (153.6 ± 9.8 vs 47.5 ± 7.1), as a marker of apoptotic cell death as compared to the normal control group (ol group .Microscopical examination of the hippocampi from normal control rats showed the normal architecture of the CA regions  and DG rThe current investigation emphasizes the efficacy of TMZ in reducing the incidence and severity of seizures in rats subjected to Li/PIL. This anti-seizure potential was associated with a marked reduction in the hippocampal glutamate content along with a noticeable upregulation of the gene expression of astroglial transporters: GLT-1 and GLAST. These effects were correlated with the ability to preserve the integrity of neurons in the different hippocampal regions.+ around astrocytes accompanied with glutamate uptake reverses the working way of transporters and abruptly alters the harmonized glia–neuron interaction (Disrupting the complex neuroglial circuitry has been shown to robustly contribute to neurological disorders including epilepsy . Any chaeraction .In the current study, GFAP was used as a tool to assess astrogliosis during seizures. Rats injected with Li/PIL showed a massive expression of GFAP in the CA1, CA3, and hilar regions of the hippocampi, which were shown to be the same sites of injury by histopathological examination. Our findings harmonize with previous studies that revealed a rapid amplified GFAP expression in various animal models of epilepsy, including PTZ , electriIn the current study, enhanced GFAP expression was also accompanied with an increase in the hippocampal glutamate level as well as a marked reduction in mRNA expression of both GLT-1 and GLAST. In agreement with these results, astrocytes activation was coupled with decreased GLT-1 and extracellular glutamate flooding in models of peripheral nerve injury . Furtherp-ERK1/2 were found to be co-expressed in the hippocampi of mice subjected to PIL-induced status epilepticus after 6 h. However, GFAP, but not p-ERK1/2, was still expressed after 3 days (p-ERK1/2 increase in hippocampal neurons and astrocytes following kainic acid–induced seizures. These results go in line with the present study and suggest the possible contribution of p-ERK1/2 in the protective effect of astrogliosis in the early stages of epilepsy. In the same context, p-ERK1/2 was previously claimed to mediate GLT1 expression in astrocytes (p-ERK1/2 observed after TMZ may be a partial mechanism through which it could increase mRNA expression of astroglial transporters and, consequently, glutamate uptake.Interestingly, GFAP and r 3 days . Similartrocytes ; howevertrocytes . Coherenvia ERK1/2 activation (2+-activated K+ channels in response to redox status change may pose a plausible mechanism toward increasing hippocampal neuronal excitability after exposure to extracellular glutamate (Simultaneously, mitochondrial dysfunction associated with the obvious alterations in mitochondrial oxidative status exerts an important role in modulating glia–neuron interplay . Li/PIL tivation , the redtivation . Accorditivation  paralleltivation . Additiolutamate .in vitro study using rat retinal Müller cell line. It revered glial transporter inhibition and protected the retina from ischemia-induced excitotoxicity (p-AMPK, which largely participates in restoring neuronal energy balance (TMZ was effective in restoring mitochondrial oxidative homeostasis as evident by the enhanced TAC and restored GSH. Ultimately, it cured lipid peroxidation, an effect that reflected on mitochondrial efficacy in ATP production and, consequentially, function and expression of astroglial glutamate transporters. The activity of TMZ on glial uptake of glutamate was previously documented in an toxicity . In agretoxicity . This ef balance . AMPK ac balance  and nucl balance . In the  balance . In addi balance .2+ accumulation following excitotoxic insults was reported not only to participate in further mitochondrial ROS production and ATP synthesis inhibition but also inevitability to the activation of membrane transition pores and Cyt c release, leading to apoptotic neuronal death (via boosting ERK expression (Intracellular Caal death , as demoal death  is postual death . Furtherpression  to act ipression . In addipression .via endorsing high mitochondrial membrane potential to preserve Ca+2 homeostasis (via activation of both AMPK and ERK. Intriguingly, purified rat brain mitochondria have been shown to express binding sites for TMZ (in vitro studies advocated that TMZ could inhibit mitochondrial permeability transition pores' opening (In the present study, the success of TMZ in limiting seizure incidence was associated with a marked reduction in apoptotic neuronal death rate as manifested by the obvious decrease in hippocampal Cyt c and caspase-3 activity. The anti-seizure as well as the antiapoptotic effects of TMZ could be linked to the potentiating effect of ERK phosphorylation in both neurons and glial cells. In addition, the effect of TMZ on AMPK could participate in its antiapoptotic activity eostasis . In agre for TMZ . Former  opening  and thusp-ERK1/2/p-AMPK signaling. Also, it succeeded in reducing ATP-dependent energy disruptions as well as astrocytes activation. This was all reflected as a reduction in neuronal apoptosis and preserved cellular integrity.In conclusion, TMZ showed a promising anti-seizure potential that was evident on seizure score as well as on various biochemical indicators. The beneficial actions of TMZ could be attributed to its ability to decrease mitochondrial oxidative damage as well as glutamate accumulation, parallel to a positive modulation of


Pro) could contribute to reducing or suppressing SARS-CoV-2 replication. Using a targeted approach, we identified 17 plant products that are included in current and traditional cuisines as promising inhibitors of SARS-CoV-2 3CLPro activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CLPro activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (Curcuma longa) rhizomes, mustard (Brassica nigra) seeds, and wall rocket (Diplotaxis erucoides subsp. erucoides) at 500 µg mL−1 displayed significant inhibition of the 3CLPro activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC50 value of 15.74 µg mL−1 was calculated for turmeric extract. Commercial curcumin inhibited the 3CLPro activity, but did not fully account for the inhibitory effect of turmeric rhizomes extracts, suggesting that other components of the turmeric extract must also play a main role in inhibiting the 3CLPro activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CLPro inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC50 value of 41.43 µg mL−1. The current study identifies plant extracts and molecules that can be of interest in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials.Antiviral treatments inhibiting Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication may represent a strategy complementary to vaccination to fight the ongoing Coronavirus disease 19 (COVID-19) pandemic. Molecules or extracts inhibiting the SARS-CoV-2 chymotripsin-like protease (3CL Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a predominantly airborne transmitted coronavirus first identified in December 2019, which is the causal agent of Coronavirus disease 19 (COVID-19) . Since iMany vaccine projects have been developed and are underway to fight COVID-19 . SeveralPro), also known as main protease (MPro), is required for the proteolytic processing of the viral polyproteins during the maturation step, thus becoming an essential tool for the successful replication of the virus  = 2.0) to 500 µg mL−1 (log [turmeric] = 2.7), resulted in a very high inhibition of the SARS-CoV-2 3CLPro activity (−1 (log [curcumin] = 0.4–1.9). Higher concentrations of the compound could not be tested because of its insolubility in the reaction buffer. As expected, the co-incubation of 3CLPro with curcumin also showed a positive effect on the protease inhibition. Thus, concentrations over 30 µg mL−1 (log [curcumin] = 1.5) produced a residual protease activity below 50%, which decreased to 28.1% when the compound was tested at 75 µg mL−1 (log [curcumin] = 1.9)  .−1 (log (mustard) = 2.3) and 500 µg mL−1 (log (mustard) = 2.7) of the extract reduced the activity of SARS-CoV-2 3CLPro to 42.1% and 9.4%, respectively (−1 (log (mustard) = 2.0) of mustard seeds extract only inhibited such activity by 19.9% . The IC50 for the mustard seeds extract was 128.1 µg mL−1   = 0.7) and 500 µg mL−1  = 2.7) of extract, respectively. The co-incubation with 200 µg mL−1 of extract produced an inhibitory response of 56.8% compared with the absence of extract. For this material, the IC50 estimated from the dose–response curve was 257.4 µg mL−1   . In the e 0.937) .Pro inhibition activity. Allyl isothiocyanate is naturally released in plants from its precursor owing to the activity of the endogenous plant myrosinases [Pro with the purified sinigrin did not result in a significant reduction of the protease activity even at the highest concentration tested, 500 µg mL−1. By contrast, the co-incubation with allyl isothiocyanate at 500 µg mL−1  = 2.7) resulted in a complete inhibition of the 3CLPro activity. Therefore, the dose–response curve was obtained for allyl isothiocyanate. The response fitted to a sigmoidal curve  with an estimated IC50 of 41.43 µg mL−1 allyl isothiocyanate standard  that are rich in sinigrin ,57,58,61icrobial ,83 and aicrobial ,85,86. Bicrobial . These lS donors . In factS donors  previousPro. Extracts of turmeric have been identified as a candidate plant extract for reducing SARS-CoV-2 3CLPro activity, which could eventually affect the viral replication. In addition, other plant extracts, such as those of cruciferous plants containing sinigrin, which, after consumption, is degraded to allyl isothiocyanate, might be considered as well for their inhibition of SARS-CoV-2 3CLPro activity. The current study provides information that can help in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials. Thus, further studies following the results of this work should be addressed: (1) to chemically characterize the plant extracts with high potential inhibitory activity and to re-evaluate this capacity after the fractionation of the extracts, thus allowing the identification of the biomolecules responsible of such activity and possible synergistic effects; and (2) to evaluate this potential inhibition in cell-based studies where the virus, host cell, and plant extract interact, also determining the toxicity limit prior to conducting other pre-clinical and clinical trials.This work is a first targeted approach for the evaluation of plant extracts and containing natural compounds for the development of prophylaxis, adjuvant therapies, and drug treatments aimed at inhibiting the activity of SARS-CoV-2 3CL