Caenorhabditiselegans is a key model organism in these fields, owing to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single-sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for example, glycolysis and the tricarboxylic acid cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand; for instance, in research related to nutrition and aging. The nematode This article has an associated First Person interview with the joint first authors of the paper. Summary: We describe an integrated metabolomics and lipidomics method for analyzing metabolites in C. elegans using a single-sample preparation, which provides a robust way of obtaining comprehensive, high-quality metabolic data from biological samples. Considerable advances in high-performance liquid chromatography (HPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) make it possible to reliably detect tens of thousands of compounds . AdditioCaenorhabditis elegans nematodes have been used intensively to investigate genetics, development, as well as aging. C. elegans is a versatile model system as genetic influences can be tested with relative ease, owing to the availability of large repositories of mutants as well as RNA interference (RNAi) libraries. Moreover, genetic reference populations have been generated for C. elegans in which natural genetic variation is present at a level similar to that in the human population -MS -MS  and NMR  (GC)-MS . Drawbac (GC)-MS . Recent  (GC)-MS .Table 1.C. elegans, combining our previously reported metabolomics and lipidomics methods into a unified extraction, a variation of the popular Folch or Bligh and Dyer liquid-liquid extraction (LLE) method  method , and rel) method .C. elegans. This quality is not a given for extractions, as endogenous metabolites span a wide range of physicochemical properties, making it difficult to extract a large range of the metabolome with a single solvent. Additionally, polar solvents typically lack the ability to precipitate interfering proteins in biological samples, making a simple water extraction of polar metabolites impractical. An elegant way to remedy these issues is to use an LLE like the Bligh and Dyer method. In this case, a highly apolar solvent, e.g. chloroform, is used to precipitate protein and facilitate the breakdown of biological organization, while a polar solvent is added to extract polar metabolites in a separate layer. This type of extraction also doubles as a separation step, removing apolar compounds from the polar layer (and vice versa), thereby reducing ion suppression effects during MS analysis. Interestingly, this type of two-phase extraction was first applied for extracting lipids in the (predominantly) chloroform phase . Four example metabolites – pyruvate, cytidine monophosphate (CMP), adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+) – showed strong linearity across the range of worms before applying normalization for internal standard (Table S1). We then established which internal standards to use for each metabolite (Table S2). Selection of the internal standard was based on the combination of the lowest coefficient of variation for biological replicates after correction with internal standard , and highest Pearson correlation coefficient after correction across the number of worms (+ (Table S2).In order to enable validation, we used  the LLE A. Polar of worms F-I. ApplTable S1). We observed that 99% of polar metabolites had an RSD <30 between different extractions, while 97% of polar metabolites had an RSD <30 when repeatedly injecting a pool of those samples, indicating that our method is highly robust.Altogether, we established optimal internal standard conditions for each of the polar metabolites and found that each of these can be reliably measured in a sample of ∼2000 worms. Subsequently, we determined the analytical repeatability of our method using a repeated injection of a pooled sample throughout the analytical run. For each metabolite measured in these samples, a relative standard deviation (RSD) was calculated (Table S3). Lipids were separated using both a normal-phase (NP) and reversed-phase (RP) chromatography method and measured on a Q Exactive Plus mass spectrometer. Additionally, a pooled sample was injected ten times.Instead of a dedicated single-phase extraction we reported previously , we usedN-acyl group), resulting in good linearity  show the same trend, although to a lesser extent  consist of a glycerol backbone and three fatty acid tails. Owing to these uniformly apolar qualities, TGs of any chain length partition almost exclusively to the chloroform phase during the two-phase extraction. This results in a linear relationship between the number of worms and the measured abundance of TGs B. A simiinearity H. Finallof 0.667 I. This ir extent J.1) and a polar head group. Similar to PCs, LPC abundance is high and its detection reaches a plateau at higher worm numbers  when including ≤2000 worms. Other lysophospholipids are poorly detected in the chloroform phase of the current two-phase method, resulting in loss of linearity . Interestingly, despite containing two fatty acid side chains, phosphatidic acid (PA), and bis(monoacylglycero)phosphate (BMP) show r2s of 0.785 and 0.667, respectively . Owing to these complex lipid properties relating to solubility, it is likely that these lipids are (partly) extracted to the polar phase during the two-phase LLE. The final solvents of the metabolomics and lipidomics method are incompatible in the ultra-performance LC (UPLC), and none of these lipid species were measurable using the ZIC-cHILIC platform. However, we performed our two-phase extraction on 2000 control worms and analyzed both phases using the lipidomics solvents and analytical platform. When plotting the abundance of lysolipid internal standards in each of the layers compared to their non-lyso counterparts , we clearly observed that the LPG internal standard partitions almost completely to the polar layer. The internal standard of LPA, LPE and LPC also show partial solubility in the polar phase .At the other end of the lipid polarity spectrum are lysolipid species such as lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE) and lysophosphatidylglycerol (LPG), each containing only a single fatty acid side chain (R numbers K. When iTable S3). To explore these partitioning effects on the detected lipidome in more detail, we made a direct comparison between the detected lipidome of the one-phase extraction . On the other hand, 10% of the lipids were only detected in the two-phase extraction, such as some BMPs and other low-abundant lipids . The first gene targeted was the pyruvate dehydrogenase alpha subunit (pdha-1), which is part of the complex that is responsible for converting pyruvate into acetyl-CoA. Because acetyl-CoA feeds into the tricarboxylic acid (TCA) cycle it links glycolysis to the TCA cycle, and RNAi of this enzyme is expected to affect both of these pathways. Indeed, worms treated with pdha-1 RNAi show many significant changes in metabolite abundance , involved in pyrimidine base degeneration. DPYD-1 is important for nucleic acid metabolism as it catalyzes the reduction of uracil and is involved in the degradation of the chemotherapeutic drug 5-fluoroacil (5-FU). In this dpyd-1 RNAi condition, we also found many significant metabolite changes compared to worms treated with empty vector  . Our data confirm that elongation of carbon chains in fatty acids is inhibited when elo-2 is knocked down in C. elegans, which leads to widespread changes across the lipidome.We next set out to establish biological validation of our lipidomics analysis. With RNAi, we targeted an enzyme involved in fatty acid elongation, elo-2 A. When eis (PCA) B. In ordle bonds C. The sale bonds D,E and omdt-15, a subunit of the Mediator complex. Rather than acting on fatty acid elongation, mdt-15 transcriptionally regulates fatty acid desaturases including fat-2, fat-5, fat-6 and fat-7 . This shift towards saturated TG species in worms treated with mdt-15 RNAi is in line with the previously described regulation of mdt-15 on fatty acid desaturation enzymes . Metabolic profiling revealed the underlying diversity of metabolites present in the different genetic backgrounds (H2) for each metabolite, for both parental and offspring strains  derived from wild-type worm strains N2 and CB4856 . RILs arkgrounds B. To exp strains . H2 servhe trait C.Fig. 5+  and apolar (lipidomics) metabolites have proven essential in these fields. Thus far however, these omics methods required dedicated sample preparation, and therefore a separate sample, for metabolomics and lipidomics. Here, we report a method that uses both the polar (metabolomics) and apolar (lipidomics) layer of a two-phase LLE and analyzes these using high-resolution MS methods, providing an elegant way of exploring a large range of the metabolome and lipidome in a single sample, covering >1100 annotated metabolites of different classes. We show here that this method is robust and sensitive enough to analyze a wide variety of metabolic pathways using both metabolomics and lipidomics, and capable of reliably pinpointing metabolic aberrations in these pathways.pdha-1 inhibition using RNAi was reflected throughout the TCA cycle, and the inhibition caused a decrease in acetyl-CoA and an accumulation of its precursor, pyruvate. Additionally, the pyruvate accumulation was accompanied by a metabolic diversion, leading to higher alanine abundance and reduced levels of TCA intermediates. Together, this comprehensive profiling provides a more detailed picture of the metabolic state. For instance, changes in less directly related metabolites were also seen, such as the increase in histidine abundance. In this way, basic profiling of metabolic changes following pdha-1 inhibition might uncover new (genetic) factors that contribute to metabolic adaptation in these circumstances. Such information is not only valuable for research in C. elegans, but could also be used to study processes in which pyruvate dehydrogenase is involved, such as the aberrant preferential activation of glycolysis in cancer cells ] with a bacterial lawn of either E. coli HT115  or pdha-1, dpyd-1, elo-2 or mdt-15 RNAi bacteria. Similarly, for the parental strains (N2 and CB4856) and eight different offspring , 2000 synchronized eggs were seeded per 10 cm NGM plate with a bacterial lawn of E. coli OP50. After 48 h, the synchronous population at L4 larval stage was washed off the plates in M9 buffer, and the worm pellet was washed with dH2O three times and then collected in a 2 ml Eppendorf tube before being snap frozen and stored at −80°C. Worm pellets were freeze dried overnight and stored at room temperature until extraction.Worm experiments were performed on independent biological replicates unless stated otherwise. N2 worms and RIL strains were cultured at 20°C on nematode growth medium (NGM) agar plates seeded with OP50 strain 15N5-monophosphate (5 nmol), adenosine-15N5-triphosphate (5 nmol), 13C6-fructose-1,6-diphosphate (1 nmol), guanosine-15N5-monophosphate (5 nmol), guanosine-15N5-triphosphate (5 nmol), 13C6-glucose (10 nmol), 13C6-glucose-6-phosphate (1 nmol), 13C3-pyruvate (0.5 nmol). In the same 2 ml tube, the following amounts of internal standards dissolved in 1:1 (v/v) methanol:chloroform were added for lipidomics: BMP(14:0)2 (0.2 nmol), CL(14:0)4 (0.1 nmol), LPA(14:0) (0.1 nmol), LPC(14:0) (0.5 nmol), LPE(14:0) (0.1 nmol), LPG(14:0) (0.02 nmol), PA(14:0)2 (0.5 nmol), PC(14:0)2 (0.2 nmol), PE(14:0)2 (0.5 nmol), PG(14:0)2 (0.1 nmol), PS(14:0)2 (5 nmol), ceramide phosphocholine SM(d18:1/12:0) (2 nmol) .In a 2 ml tube, the following amounts of internal standard dissolved in water were added to each sample of freeze-dried worms for metabolomics: adenosine-g. Of the two-phase system that was now created with protein precipitate in the middle, the top layer containing the polar phase was transferred to a new 1.5 ml Eppendorf tube. The bottom layer, containing the apolar fraction, was transferred to a 4 ml glass vial. The protein pellet in between the two layers was dried and subsequently dissolved in 0.2 M NaOH for quantification using a Pierce™ BCA Protein Assay following the product protocol.After adding internal standard mixes, a 5 mm steel bead and polar phase solvents  were added and samples were homogenized using a TissueLyser II (Qiagen) for 5 min at a frequency of 30 times/s. Chloroform was added for a total of 1 ml to each sample before thorough mixing. Samples were centrifuged for 10 min at 20,000 2 (0.2 nmol), CL(14:0)4 (0.1 nmol), LPA(14:0) (0.1 nmol), LPC(14:0) (0.5 nmol), LPE(14:0) (0.1 nmol), LPG(14:0) (0.02 nmol), PA(14:0)2 (0.5 nmol), PC(14:0)2 (0.2 nmol), PE(14:0)2 (0.5 nmol), PG(14:0)2 (0.1 nmol), PS(14:0)2 (5 nmol), ceramide phosphocholine SM(d18:1/12:0) (2 nmol) (Avanti Polar Lipid). After adding the internal standard mix, a steel bead and 1.5 ml 1:1 (v/v) methanol:chloroform were added to each sample. Samples were homogenized using a TissueLyser II (Qiagen) for 5min at 30 Hz. Each sample was then centrifuged for 10 min at 20,000 g. The supernatant was transferred to a 4 ml glass vial.In a 2 ml tube, the following amounts of internal standards dissolved in 1:1 (v/v) methanol:chloroform were added to each sample: BMP(14:0)Tables S1, S2, S4 and S5, as well as in the MetaboLights online database under the following code: MTBLS2370 (https://www.ebi.ac.uk/metabolights/).After the polar phase was transferred to a new 1.5 ml tube, it was dried using a miVac vacuum concentrator at 60°C and processed as reported previously . The res® LUNA silica, 250×2 mm, 5 µm 100 Å. Injection order for samples was random, with an injection of a pooled sample every ten injections. Column temperature was held at 25°C. Mobile phase consisted of (A) 85:15 (v/v) methanol:water containing 0.0125% formic acid and 3.35 mmol/l ammonia and (B) 97:3 (v/v) chloroform:methanol containing 0.0125% formic acid. Using a flow rate of 0.3 ml/min, the LC gradient consisted of 10% A for 0-1 min, reach 20% A at 4 min, reach 85% A at 12 min, reach 100% A at 12.1 min, 100% A for 12.1-14 min, reach 10% A at 14.1 min, 10% A for 14.1-15 min. For reversed-phase separation, 5 μl of each sample was injected onto a Waters HSS T3 column . Injection order for samples was random, with an injection of a pooled sample every ten injections. Column temperature was held at 60°C. Mobile phase consisted of (A) 4:6 (v/v) methanol:water and B 1:9 (v/v) methanol:isopropanol, both containing 0.1% formic acid and 10 mmol/l ammonia. Using a flow rate of 0.4 ml/min, the LC gradient consisted of 100% A at 0 min, reach 80% A at 1 min, reach 0% A at 16 min, 0% A for 16-20 min, reach 100% A at 20.1 min, 100% A for 20.1-21 min. MS data were acquired using negative and positive ionization using continuous scanning over the range of m/z 150-2000. Data were analyzed using an in-house developed metabolomics pipeline written in the R programming language (https://www.r-project.org/). In brief, it comprises the following five steps: (1) pre-processing using the R package XCMS; (2) identification of metabolites; (3) isotope correction; (4) normalization and scaling; and (5) statistical analysis  retention times and the injection of relevant standards. Lipidomics data from these experiments can be found in Tables S3-S5, as well as in the MetaboLights online database using the code MTBLS2370 (https://www.ebi.ac.uk/metabolights/).After the solvents containing the lipids were transferred to a 4 ml glass vial, they were evaporated under a stream of nitrogen at 45°C. The residue was dissolved in 150 μl of 1:1 (v/v) chloroform:methanol. Lipids were analyzed using a Thermo Fisher Scientific Ultimate 3000 binary UPLC coupled to a Q Exactive Plus Orbitrap mass spectrometer. Nitrogen was used as the nebulizing gas. The spray voltage used was 2500 V, and the capillary temperature was 256°C. Other conditions were as follows: S-lens RF level, 50; auxiliary gas, 11; auxiliary gas temperature, 300°C; sheath gas, 48; sweep cone gas, 2. For normal-phase separation, 2 μl of each sample was injected onto a Phenomenexanalysis . All repH2 was calculated as described previously , where Vg is the variance attributed to genetics and Ve is the variance attributed to other factors . Significance of the heritability was calculated by permutation, where the trait values were randomly assigned to strains. Over these permutated values, the variance captured by strain and the residual variance were calculated. This procedure was repeated 1000 times for each trait. The obtained values were used as the by-chance distribution, and a false discovery rate (FDR) of 0.05 was taken as the 50th highest value. In the parental strains, H2 was calculated as H2=0.5Vg/(0.5Vg+Ve). The factor 0.5 corrects for the overestimation of the additive variation in inbred strains (H2 was applied, taking the FDR=0.05 threshold as significant. An H2 above the FDR value indicates that there is only a 5% chance the result is a false positive.eviously . Briefly strains . The samCross comparisons between metabolites and lipids were performed as follows: individual metabolite abundances were used. Lipid class abundances were calculated by summing the abundances of each lipid species from a given lipid class. These were cross-correlated using the imgCor function from the mixOmics package v6.6.2  in R. As


ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human  These include the secondary structures in the 5′-leader sequence, which can affect ribosomal recognition of the uORF initiation codon [N6-methyladenosine (m6A), or the presence of exon-exon junctions and consequently exon junction complexes, in the mRNA 5′-leader sequence can promote uORF translation [Gene expression is extremely regulated at the level of mRNA translation, especially at its initiation step, where the initiation codon is identified and decoded ,2. Transpressors ,19,20,21pressors ,23,24,25on codon . More renslation ,28. Recenslation ,29,30. Mnslation ,31.Under cellular stress, such as the endoplasmic reticulum (ER) stress, global protein synthesis is inhibited in part by decreasing the amount of available ternary complex through phosphorylation of the alpha-subunit of the initiation factor eIF2 ,33,34. NABCE1 gene is localized in chromosome 4, at position 4q31, and its main ORF encodes a 599 amino-acid protein (~67 kDa), found in the nucleus and in the cytoplasm of mammalian cells [ABCE1 gene is a fragile site already associated with structural rearrangements in cancers [ABCE1 mRNA is ubiquitously expressed both in normal and in cancer colorectal cells [ABCE1 mRNA in a pool of colorectal cancers was shown [The human ATP-binding cassette (ABC) protein family is the largest family of transporters, being ABCE1 protein the only member of the subfamily E (ABCE) ,41. ABCEan cells ,43,44. Aan cells ,46. Addian cells ,48,49,50 cancers . In fact cancers ,53,54, b cancers , esophag cancers , ovarian cancers , and gli cancers . On the as shown .ABCE1 transcript [ABCE1 5′-leader sequence [ABCE1 transcript may itself be subjected to a high translational regulation mediated by uORFs. Taking into account the important functions of the ABCE1 protein and the unknown role of its uORFs, our aim was to study the function of ABCE1 uORFs in its translational control in colorectal cancer cells. Here, we show that the human ABCE1 5′-leader sequence contains five functional AUG uORFs, from which uORF3 (or uORF4 as they are in frame and overlapped) and uORF5 function as repressors of the main ORF translation, while uORF1, and to a less extent uORF2, function as regulators of the downstream repressive uORFs. On the contrary, the five non-AUG uORFs identified do not exhibit significant repression activity. We also show that this uORF-mediated translational regulation of ABCE1 responds to ER stress conditions. However, it works equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation.Several Ribo-Seq studies performed in different cell lines have revealed a wide ribosome occupancy in the 5′-leader sequence of the human anscript ,61,62. Isequence . These dABCE1 transcript . In the first PCR, the ABCE1 5′ untranslated sequence was amplified from a cDNA sample obtained from NCM460 cells  with the primers #1  polymerase chain reaction (PCR) was used to obtain a firefly luciferase (FLuc) reporter plasmid carrying the 515 nt-long 5′-leader sequence of the human The SOEing PCR product and the pGL2-FLuc plasmid were digested with HindIII  and BsrGI , and the two products were ligated with the T4 DNA ligase  as instructed by the manufacturer, forming the ABCE1_5′UTR construct (5′UTR as simplification of 5′-leader sequence). NZY5α competent cells (NZYTech) were transformed with the ABCE1_5′UTR construct to generate clones of this vector. NZYMiniprep kit (NZYTech) was used for plasmid DNA (pDNA) extraction, following the manufacturer’s instructions and the sequence of the reporter plasmid confirmed by sequencing.All the following constructs used in this study were obtained by site-directed mutagenesis following a standard protocol. Briefly, 30 ng of template (pDNA) were amplified in a PCR reaction using lengthened primers containing the genetic alteration of interest and a proof-reading enzyme . The resulting PCR product was digested with DpnI , NZY5α competent cells transformed, and pDNA extracted. All the mutagenesis constructs were again confirmed by sequencing.Thus, by site-directed mutagenesis and using the ABCE1_5′UTR plasmid, each uAUG was altered from an ATG to TTG  for uORF3 and uORF4 constructs, 1 nucleotide (T) was deleted in the intercistronic space between the shared uORF’s stop codon and the beginning of the FLuc ORF (primers #15 and #16 in v/v) fetal bovine serum . The non-tumorigenic colorectal cell line NCM460 and two colorectal adenocarcinoma cell lines CaCo-2 and HT-29 were grown in Roswell Park Memorial Institute 1640  supplemented with 10% (v/v) FBS. Cells were maintained in an incubator at 37 °C, in a humidifier atmosphere with 5% (v/v) CO2.The human colorectal carcinoma cell line HCT116, two human colorectal adenocarcinoma cell lines DLD-1 and SW480, were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% (Renilla luciferase (RLuc), using Lipofectamine 2000 Transfection Reagent  and following the manufacturer’s instructions. Briefly, in a first solution, the amount of each reporter plasmid was diluted in 250 µL of Opti-MEM medium , and in a second solution, 4 µL of Lipofectamine 2000 were also diluted in 250 µL of Opti-MEM. Both solutions were mixed and rest for at least 20 min at room temperature. In the meantime, the culture medium was changed to fresh growth media, where afterward, the mixture above was added dropwise, and cells were incubated for 24 h at 37 °C, in a humidifier atmosphere with 5% (v/v) CO2. In stress studies, HCT116 cells were also transiently co-transfected, and 4 h later, treated with 4 µM of thapsigargin  or dimethyl sulfoxide , the vehicle control of thapsigargin, for 24 h at 37 °C, in a humidifier atmosphere with 5% (v/v) CO2. In either cases, cells were harvested for further analyses.HCT116 and NCM460 cells were seeded in 35 mm plates at 70–80% confluence and 24 h after, were transiently co-transfected with 1.5 µg of each constructed plasmid, and 0.5 µg of pRL-TK vector (Promega), which expresses ABCE1 (5′-UCAUCAAACCUCAAUAUGU-3′), or the siRNA for Luciferase , used as a control condition (Thermo Fisher Scientific), were transiently transfected using Lipofectamine 2000, as above. Twenty-four hours after transfection, the knockdown was reinforced with 40 pmol of each corresponding siRNA. Cells were harvested 48 h after the first siRNA transfection, and cleared cell lysates were used for Western blot analysis. For cell viability assays, cells were kept adherent to the plate.For transfections with siRNAs, HCT116, and NCM460 cells were seeded at 30–40% confluence in 96-well plates and after 24 h, 40 pmol of the siRNA for v/v) phosphate-buffered saline (PBS 1×), lysed in 100 µL of 1× (v/v) passive lysis buffer  and centrifuged at maximum speed to obtain cleared cell lysates. FLuc and RLuc activity was assessed using the Dual-Luciferase® Reporter Assay System (Promega), according to the manufacturer’s instructions, on a GloMax® 96 Microplate Luminometer (Promega). Briefly, 10 µL of the cleared cell lysates were plated in a white 96-well plate. First, we measured the luminescence signal of the FLuc reporter by adding 40 µL of the Luciferase Assay Reagent (containing the firefly luciferase substrate) to each sample. Then, RLuc reporter luminescence was sequentially quantified from the same sample by adding 40 µL of the Stop & Glo® Reagent that first quenches the FLuc luminescence reaction and contains the substrate for the RLuc reaction. The collected data were expressed in arbitrary light units. Luciferase activity of each condition is obtained by normalizing FLuc to RLuc luminescence.Cells were washed with 1 mL of pre-chilled 1× (v/v) sodium dodecyl sulfate (SDS) sample buffer (NZYTech) to the protein sample and heating at 95 °C for 15 min. Afterward, samples were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 10 or 12% acrylamide/bisacrylamide gel and then transferred to methanol pre-activated polyvinyldene difluoride (PVDF) membranes . Depending on the primary antibody, membranes were blocked in 5% (w/v) non-fat dry milk or bovine serum albumin , diluted in 1× (v/v) Tris-buffered saline (TBS 1×) supplemented with 0.05% (v/v) Triton x-100 (Sigma) or Tween 20 (Sigma). Membranes were blotted overnight (o/n), at 4 °C, with shacking, with the following primary antibody solutions: mouse anti-α-tubulin (Sigma) diluted 1:50,000 in 5% (w/v) non-fat milk in 0.05% (v/v) Triton-TBS 1×; rabbit anti-Pospho-eIF2α  diluted 1:250 in 5% (w/v) BSA in 0.05% (v/v) Tween-TBS 1×; goat anti-Firefly Luciferase  diluted 1:250 in the solution 1 for primary antibodies from the SignalBoost™ Immunoreaction Enhancer Kit ; and rabbit anti-ABCE1 (Abcam) diluted 1:1000 in 5% (w/v) non-fat milk in 0.05% (v/v) Tween-TBS 1×. Detection was performed at room temperature, with shaking, using secondary peroxidase-conjugated anti-mouse IgG (Bio-Rad), anti-rabbit IgG (Bio-Rad) or anti-goat IgG (Bio-Rad), diluted 1:4000, 1:3000, or 1:5000, respectively, in 5% (w/v) non-fat milk in the corresponding buffers, followed by enhanced chemiluminescence.From the cleared cell lysates obtained after cells were washed with 1 mL of pre-chilled PBS 1×, lysed in 100 µL of PLB 1× and centrifuged at maximum speed, we used the correspondent volume to 30 µg of total protein for Western blot analysis, previously quantified with NZYBradford reagent (NZYTech), as instructed by the manufacturer, in the NanoDrop 1000 . Denaturation of samples was performed by adding 5× (w/v) non-fat milk in 0.05% (v/v) Triton-TBS 1×, membranes are probed o/n, at 4 °C, with shacking, with rabbit anti-eIF2α  diluted 1:1000 in 5% (w/v) non-fat milk in 0.05% (v/v) Triton-TBS 1×. Detection was performed as described above, followed by enhanced chemiluminescence.As in the case of eIF2α-P and eIF2α proteins (with a similar molecular weight), membranes were stripped off from the previously used antibody and probed again, according to standard protocols. Briefly, membranes were used immediately after chemiluminescence or as dried membranes that require an additional methanol activation. In either case, membranes were washed with bi-distilled water, incubated in a 250 mM solution of sodium hydroxide (NaOH), washed in bi-distilled water, and then in the respective buffer of the antibody to be used. After a blockage of 30 min in 5%  following the manufacturer’s protocol. Total RNA was then treated with RNase-free DNase I (Promega) for approximately 1 h at 37 °C, and the RNA was purified using phenol-chloroform, as described in standard protocols. cDNA was synthesized using the NZY Reverse Transcriptase (NZYTech) and oligo(dT) primers (NZYTech), as instructed by the manufacturer.ABCE1 , as instructed by the manufacturer, in an Applied Biosystems® 7500 Real-time PCR System (Applied Biosystems). The amplification program was the following: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The mRNA levels of ABCE1 were normalized to the mRNA levels of the internal control, GAPDH, and the relative mRNA expression levels were calculated by applying the comparative Ct method (ΔΔCt). For that, amplification efficiencies were calculated for each set of primers using serial dilutions of a cDNA sample.The cDNA synthesized was used as a template for the quantification of the mRNA expression levels of FLuc (primers #1 and #2 in RLuc (primers #3 and #4 in ® DNA polymerase (Promega) following the manufacturer’s protocol. The thermal cycling conditions were the following: initial denaturation at 95 °C for 4 min; 30 cycles of 95 °C for 30 s (denaturation), 55 °C for 30 s , and 72 °C for 1 min (extension); and then a final extension at 72 °C for 10 min. After amplification, 15 µL of the PCR products were resolved by electrophoresis in a 3% (w/v) agarose gel stained with ethidium bromide. The density of the bands was quantified using ImageJ software , and the absolute mRNA amount was determined using a standard calibration curve performed with serial dilutions of a cDNA sample. Luciferase mRNA levels of each condition are obtained by normalizing FLuc to RLuc mRNA levels.The cDNA synthesized was used as a template for the quantification of the mRNA expression levels of p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).The experimental analyses were performed at a minimum number of three independent assays, and the results are expressed as mean ± standard error of the mean (SEM). Student’s t-test, two-tailed and unpaired, was applied for statistical significance: ABCE1 mRNA 5′-leader sequence is a 515 nt-long sequence, as annotated in the Ensembl database (assembly GRCh37) with the transcript ID ENST00000296577. Our in silico analysis, using the ExPASy Translate tool, showed five AUG start codons in frame with stop codons (UGA) located within the ABCE1 5′-leader sequence, constituting five AUG uORFs   a. Next, 1_5′UTR) a. In add1_5′UTR) a. HCT116ABCE1 mRNA 5′-leader sequence are able to repress the translation of the main coding sequence. Regarding the ability of each of the AUG uORFs to repress translation, we observed that uORF1 or uORF2 do not affect the relative luciferase activity levels when comparing to the results obtained from the “no AUG uORFs” construct  using the “no AUG uORFs” construct as a template, obtaining constructs with no functional uORFs , and constructs carrying only one functional non-AUG uORF   a. The ex uORF10) b. Nevert uORF10) b. This rABCE1 non-AUG uORFs is due to their frequent ribosomal bypass as a consequence of the non-canonical nature of their start codons. For that, the start codon of each of the non-AUG uORFs was mutated to an AUG (CTG/GTG→ATG), using as template the construct “no AUG uORFs”, obtaining the constructs named uORF6_AUG, uORF7_AUG, uORF8_AUG, uORF9_AUG, and uORF10_AUG  when comparing to uORF5 expression levels b, indicaTo investigate the potential of translation reinitiation at the main ORF after translation of each one of the AUG uORFs, the context of each uAUG was replaced by an optimal Kozak sequence context (ACCATGG ), obtainABCE1 mRNA in terms of uORFs and knowing the potential of each AUG uORF to be translated or leaky scanned, we next decided to investigate how the five ABCE1 AUG uORFs function when together in the native configuration, to regulate the expression of the main coding sequence. With this aim in mind, we mutated the ABCE1 uAUGs in several possible combinations: (i) only one uAUG mutated (ATG → TTG), originating constructs named “no uORF1”, “no uORF2”, “no uORF3”, “no uORF4”, and “no uORF5”, and (ii) two or three uAUGs mutated, where uORF1 and uORF2 initiation codons are always mutated, originating the constructs named “no uORF1+2”, “no uORF1+2+3”, “no uORF1+2+4”, and “no uORF1+2+5” , and cells were treated with thapsigargin to induce ER stress. The efficiency of the ER stress was monitored by immunoblotting of phosphorylated eIF2α protein levels   a in the uORF1-5) b. As shouORF1-5) a, indicall lines a. These CE1 mRNA b and proCE1 mRNA c in seveABCE1 5′-leader sequence is extremely complex due to the presence of at least 10 uORFs  of unkner cells . Trying er cells . On the er cells , which ser cells . In addited ones , while uted ones . Furtherted ones . Neverther cells . This leer cells . Of noteABCE1 5′-leader sequence  the ribosomes can be dissociated and recycled, or (ii) the ribosomes can be stalled during translation elongation or termination.The inhibitory activity of a uORF is positively correlated with (i) a strong uORF initiation codon Kozak context, (ii) a long distance from the transcript’s 5′-end to the uORF initiation codon, (iii) the presence of other uORFs (additive effect), (iv) a long uORF length, and/or (v) a short intercistronic distance ,13,22,23sequence . Altogetsequence . Thus, isequence . Indeed,sequence . Howeversequence . Thus, iABCE1 5′-leader sequence are deprived of significant repressive functions (EPRS) transcript, there are two non-AUG uORFs , which induce a downregulation of the main coding sequence expression if the initiation codons are mutated to an AUG in an optimal context [On the other hand, we observed that the non-AUG uORFs within the unctions , which aunctions . It was  context . A similABCE1 uORF1 translation, the ribosome will, at least some times, reinitiate translation at one of the subsequent start codons upstream of the main AUG, as for instance, the initiation codons of the repressive uORF3, uORF4, or uORF5. A similar mechanism was shown for the general control protein (GCN4) transcript that, in physiological conditions, its AUG uORF1 is responsible for translation reinitiation at the three downstream inhibitory AUG uORFs, leading to GCN4 downregulation [ABCE1 transcript do not seem to act exactly in the same way, since abolishing only uORF1 translation results in a similar repression level to the one performed by all the AUG uORFs in the intact ABCE1 5′-leader sequence regulation and tumorigenesis ,31, our 16 cells . Support60 cells . These rg HCT116 . ConversABCE1 transcript. This tight regulation contributes to maintaining approximately constant levels of ABCE1 protein in different cell environments.In conclusion, our study identified so far unknown uORFs that tightly regulate translation of the human


Arenobufagin (ArBu), one of the main active bufadienolides of toad venom with cardiotonic effect, analgesic effect, and outstanding anti-tumor potentiality, is also a potential cardiotoxic component. In the present study, the cardiac effect of ArBu and its underlying mechanism were explored by integrating data such as heart rates, toxicokinetics, myocardial enzyme and brain natriuretic peptide (BNP) activity, pathological sections, lipidomics and proteomics. Under different doses, the cardiac effects turned out to be different. The oral dose of 60 mg/kg of ArBu sped up the heart rate. However, 120 mg/kg ArBu mainly reduced the heart rate. Over time, they all returned to normal, consisting of the trend of ArBu concentration-time curve. High concentrations of myocardial enzymes and BNP indicated that ArBu inhibited or impaired the cardiac function of rats. Pathological sections of hearts also showed that ArBu caused myocardial fiber disorder and rupture, in which the high-dose group was more serious. At the same time, serum and heart tissue lipidomics were used to explore the changes in body lipid metabolism under different doses. The data indicated a larger difference in the high-dose ArBu group. There were likewise many significant differences in the proteomics of the heart. Furthermore, a multi-layered network was used to integrate the above information to explore the potential mechanism. Finally, 4 proteins that were shown to be significantly and differentially expressed were validated by targeted proteomics using parallel reaction monitoring (PRM) analysis. Our findings indicated that ArBu behaved as a bidirectional regulation of the heart. The potential mechanism of cardiac action was revealed with the increased dose, which provided a useful reference for the safety of clinical application of ArBu. In addiChinese) , ArBu isChinese) . HoweverChinese) . ResearcChinese) . It has rhythmia . HoweverLipids play an important part in cells. Under the action of phospholipase, they produce cell signaling factors and activate cellular signaling pathways, and are widely involved in and regulate various physiological activities of the human body . ResearcLC-MS/MS in parallel reaction monitoring (PRM) mode was used to verify the differential proteins. It was an antibody-independent targeted quantitative technology. Therefore, for some proteins without antibodies, and for proteins with post-translational modifications, quantitative results could be quickly obtained through PRM. The PRM mode had a high sensitivity and a good specificity. Its quantification range could reach more than 4 orders of magnitude, realizing accurate quantification of the protein in the true sense. In addition, in terms of throughput, an experiment using LC-MS/MS in PRM mode could simultaneously quantify up to dozens of proteins, which was also difficult for western blot to achieve.Cardiotoxicity of ArBu was initially explored by determining the changes in heart rates, myocardial enzymes , brain natriuretic peptide (BNP) and pathological sections. Furtherly, toxicokinetics, lipidomics and proteomics were being used to explore the cardiotoxic mechanism of ArBu. Oral administration of 60 mg/kg-ArBu accelerated heart rates in rats, while 120 mg/kg-ArBu initially accelerated and subsequently decreased the heart rates, consistent with the toxicokinetic results. They all returned to normal after 60 and 110 min, respectively. The levels of cardiac function assessment (CFA) indexes  increased to various degrees, suggesting that ArBu might lead to inhibition or damage of cardiac function. Moreover, the pathological sections of hearts in high-dose (more serious) and low-dose ArBu groups showed certain cardiotoxicity (muscle fiber disorder or rupture). The major differentially-expressed lipids were cholesteryl ester (CE), phosphatidylcholines (PCs), sphingomyelins (SMs) in serum and PCs, phosphatidylethanolamines (PEs), triglyceride (TG) in hearts, respectively. The blood differential lipids in low-dose ArBu group was mainly related to BNP. While, the blood differential lipids in the high-dose ArBu group was associated with a variety of CFA indexes. Similarly, the differential proteins also showed significant differences among the groups. Multi-layered network analysis showed that the main pathways involved in the molecular mechanism of low-dose ArBu group were quite different from those of high-dose ArBu group. PRM verification showed that the regulation trends of ArBu on Trap1, Idh2, Mrps6 and Pcca were consistent with proteomics. In summary, high-dose ArBu caused inhibition or damage of cardiac function in rats. From cardiac excitation to cardiac inhibition, the underlying molecular mechanisms were quite different.ArBu  was purchased from Baoji Herbest Bio-Tech Co, Ltd. . The 0.9% sodium chloride solution was purchased from Hebei Tiancheng Pharmaceutical Co., Ltd. . Chloral hydrate, propylene glycol and heparin sodium were purchased from Sinopharm Chemical Reagent Co., Ltd. . Ammonium acetate was purchased from Sigma-Aldrich . Thermo Scientific (United States) provided MS-grade methanol, formic acid, acetonitrile and protease inhibitor (Pierce) as well as trypsin inhibitor.The equipment and manufacturers used in the study were as follows: EMKA non-invasive electrocardiography telemetry system , AU480 automatic biochemical analyzer , DNM-9602G enzyme micro-plate reader , DNX-9620A computer board washer, ABS2TM automatic blood collection instrument and ABS2 software , Mix-3000 shock mixer , Mikro 220R high-speed refrigerated centrifuge , and vacuum centrifugal concentrator .The animal studies were approved by the Animal Experimentation Committee of the Institute of Chinese Materia Medica, and were in compliance with the Nation Institute of Health guidelines for the Care and Use of Laboratory Animals. Specific pathogen free male Wistar rats (300 ± 30 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.  2016-0006). During the study, rats were exposed to suitable temperature (20°C–24°C) and humidity (40% RH–60% RH). The standard chow and tap water was adlibitum. They all fasted 12 h (h) before the experiment and had free access to water. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate  solution. Rats were randomly divided into three groups: a control group (propanediol + sodium chloride solution), a 60 mg/kg ArBu-administrated group and a 120 mg/kg ArBu-administrated group. ArBu was dissolved in propanediol with sodium chloride solution. The gavage volume was 2 ml.ArBu suspension was freshly prepared for intragastric administration. Namely, ArBu was dissolved in propylene glycol and diluted with 0.9% sodium chloride solution. The stock solution of ArBu (1.000 mg/ml) for the toxicokinetic study was prepared by dissolving ArBu in methanol and stored at −20°C before analysis. The ArBu calibration curve was obtained by serial dilution of the highest concentration (20000 ng/ml) calibration standard to the lowest concentration (100 ng/ml) calibration standard. The internal standard diazepam was prepared with a concentration of 1 μg/ml, 10 μg/ml, 5,000 ng/ml and 1,000 ng/ml of ArBu solutions were dried with nitrogen and then blank blood was added. Eventually, 1,000 ng/ml, 500 ng/ml and 100 ng/ml of ArBu solutions were obtained as high concentration quality control (HQC), medium concentration quality control (MQC) and low concentration quality control (LQC), respectively.n = 8), randomly after acclimatization. Following 120 min (min) of surveillance, the rats were anaesthetized with chloral hydrate. Blood was collected from the abdominal aorta and centrifuged at 21 g for 15 min at 7°C to obtain serum. After the sacrifice of the rats, hearts and livers were removed, frozen in liquid nitrogen and stored at −80°C before analysis. Serum levels of BNP and myocardial enzymes were determined.The abdominal hair was shaved with the electrodes attached. Rats were administered intragastrally with and without ArBu suspension . The BNP level was detected according to the instruction of ELISA Kit . The main instruments used were Beijing Perlong DNM-9602G enzyme label analyzer and DNX-9620A computerized plate washer.n = 3, at random) were analyzed as follows. Hearts and livers were fixed with formaldehyde, dehydrated, embedded in paraffin, sectioned, dewaxed and hematoxylin-eosin stained, which were made into pathological sections and detected.In order to explore the effect of ArBu on the organs and tissues of rats, the pathological sections for 2 h after oral administration : a control group (a mixture of propanediol and sodium chloride solution), a low-dose ArBu group (ArBu suspension 60 mg/kg) and a high-dose ArBu group (ArBu suspension 120 mg/kg). They all received a normal diet 4 h later. Plasma samples were collected from the common carotid vein at the following time points: 0, 2, 5, 15, 20, 30 min, 1, 1.5, 2, 4, 6, 8, 12, 24 and 48 h. The parameters for collecting plasma were as follows: sample volume, 225 μL, sample withdrawal rate, 375 μL/min, drug infusion rate, 375 μL/min, keep vein open rate, 100 μL/h, model, low loss, sample collection chamber temperature, 4–6°C. Plasma samples were collected into heparin tubes, in which 5 µL of 100 U heparin solution was added and volatilized overnight. Samples were mixed with heparin and centrifuged . Then, 90 µL of supernatant was transferred to a 1.5 ml Eppendorf tube and vortexed for 10 s 270 µL of acetonitrile and 9 µL of 1 μg/ml internal standard were added. The sample was vortexed for 2 min and centrifuged at 100 g for 10 min. The supernatant was transferred to a clean Eppendorf tube, dried under a nitrogen dryer and stored at −80°C before analysis. The dried residue was reconstituted with 90 µL of methanol, vortexed and centrifuged for MS analysis.18 column  was used for the gradient elution of the chromatographic separation. The mobile phases were 0.1% formic acid in water (A) and acetonitrile (B). The gradient program used was as follows: 0–2 min, 20% B, 2–4 min, 20%–90% B, 4–4.5 min, 90% B, 4.5–8 min, balance to 20% B. The flow rate was 0.3 ml/min. The injection volume was 1 µL.Analyses were conducted on an Agilent 1290 HPLC  equipped with AB SCIEX QTRAP 6500 system . An Agilent Extend-Cm/z) ranged from 417.2 to 399.2 for ArBu at 5.26 min and 285.3 to 193 for diazepam at 6.44 min, respectively.ArBu was determined by monitoring the product-precursor transition in multiple reaction monitoring (MRM) mode using the positive ion acquisition mode. Other optimized mass parameters were as follows: curtain gas, 20, ion spray voltage, 5500 V, ion source gas 1, 55, ion source gas 2, 55, source temperature, 555°C, DP value of ArBu and diazepam, 140 V, collision energy of ArBu, 35 V, collision energy of diazepam, 41 V. The optimized quantification transition ion pairs (n = 3), exposed at room temperature for 0, 6 and 12 h, were used for the evaluation of the stability of ArBu in plasma. The samples were considered stable if the test values were within the acceptable limits of ±15% relative standard deviation  in comparison with freshly prepared quality control samples.Selectivity was assessed by running plasma blank, ArBu-administrated plasma and plasma blank added with ArBu. Then, potential interferences from endogenous compounds were investigated. The calibration curve consisted of 8 blank plasma samples with an ArBu concentration of 5, 10, 20, 50, 100, 200, 500 and 1,000 ng/ml. The preparation method was in agreement with the previous plasma sample preparation. The lower limit of quantification was the concentration of ArBu with a signal-to-noise ratio greater than 10. The detection limit was at least triple the response  compared to blank, which was calculated by serial dilutions of the lower limit of quantification. QC samples  by comparing the areas of extracted ArBu samples with those of the post-extraction spiked ArBu samples. The area of the extracted ArBu sample was compared with the area of the extracted ArBu sample by LQC, MQC and HQC (n = 6). Then, the extraction recovery rate of ArBu was determined. The matrix effect was determined with LQC, MQC and HQC, comparing peak areas of post-extracted ArBu samples with peak areas of pure samples . During the analysis process, QC samples along with study samples were processed and distributed among the study samples. WinNonlin  software and NCA model were used to calculate and analyze the data obtained, respectively.The toxicokinetics of ArBu in rats was investigated by intragastric administration at doses of 0, 60 and 120 mg/kg (n = 8) at 2 h post-dose were used for lipidomics analysis. The serum samples were thawed on ice for 45 min before the pretreatment. 100 µL of serum and 300 µL of methanol were put into a 1.5 ml Eppendorf tube and vortexed for 15 s to precipitate proteins. The suspension was centrifugated at 100 g for 10 min (4°C). Additionally, the heart was ground with 1 ml of methanol, mixed for 30 min, followed by centrifugation at 100 g for 10 min (4°C). The supernatant taken from serum and hearts was used for lipidomics analysis.The serum and hearts obtained from each group   on UltiMate™ 3000 Rapid Separation LC system  with a flow rate of 300 μL/min. Mobile phase A consisted of 10 mM ammonium acetate and 0.1% formic acid in acetonitrile-water . Mobile phase B consisted of 10 mM ammonium acetate and 0.1% formic acid in isopropanol-acetonitrile . The elution gradient was as follows: 0–1 min, 20% B, 1–16 min, 20%–100% B, 16–18 min, 100% B, 18–19 min, 100%–20% B, 19–19.5 min, 20% B. The injection volume was 1 µL. A Thermo Scientific™ Q Exactive hybrid quadrupole-orbitrap MS equipped with a HESI-II probe was employed. The MS parameters were as follows: positive HESI-II spray voltages, 3.7 kV, negative HESI-II spray voltages, –3.5 kV, heated capillary temperature, 320°C, m/z range, 50–1500, source temperature, 400°C, sheath gas pressure, 30 psi, auxiliary gas pressure, 10 psi, desolation temperature, 300°C, collision gas, nitrogen, collision gas pressure, 1.5 mTorr, resolution, 70000.Lipids was separated at 45°C using an UPLC CSH Cn = 3, at random).Consistent with lipidomics, the hearts used for proteomics analysis were obtained from rats intragastrically administered with or without ArBu suspension for 2 h . A 4 μL protease inhibitor was added to protect proteins from degradation. Protein concentrations were measured using the Bradford method (Eppendorf Biospectrometer).4HCO3. Subsequently, the samples were digested with trypsin at an enzyme-to-protein mass ratio of 1:25 overnight at 37°C. The peptides were extracted and dried .Whole tissue proteins (100 μg) were digested by a FASP procedure. The protein samples were supplemented with 1 M dithiothreitol to a final concentration of 5 mM and incubated for 30 min at 56°C. Then iodoacetamide was added to a final concentration of 20 mM, and incubated in the darkness at room temperature. After half an hour of incubation, samples were added at final concentrations of 5 mM of dithiothreitol and retained in darkness for an additional 15 min. Following these procedures, protein samples were loaded into 10 kD Microcon filtration devices (Millipore) and centrifuged at 12,000 g for 20 min and washed twice with Urea lysis buffer , twice with 50 mM NHvia a nano-electrospray ion source (Thermo Fisher Scientific). The peptide samples were dissolved with the loading buffer (5% methanol and 0.2% formic acid), and loaded onto a C18 trap column (360 μm i.d × 2 cm) at a maximum pressure 280 bar with 12 μL solvent A (0.1% formic acid in water). The peptides were separated on another C18 column  with a series of adjusted linear gradients according to the hydrophobicity of fractions with a flow rate of 600 nL/min. A mobile phase was composed of solvent A (0.2% formic acid-water) and solvent B (0.2% formic acid-acetonitrile) with gradient elution . The MS analysis was performed in a data-dependent manner with full scans  acquired using an Orbitrap mass analyzer at a mass resolution of 120,000 at m/z 200. The highest speed data-dependent mode was selected for fragmentation at normalized collision energy of 32%. The fragment ions were then transferred into the ion trap analyzer with the automatic gain control AGC target at 5e3 and maximum injection time at 35 ms. Dynamically excluding previously acquired precursor ions was activated at 20 s.Orbitrap Fusion LC-MS/MS analyses were conducted on an Easy-nLC 1000 liquid chromatography system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos /z. Targeted MS2 spectra were acquired at 15000 resolution within auto scan range, after HCD with 30% NCE, and using an automatic gain control target value of 5e4 charges, a max IT of 22 millisecond and an isolation window of 1.6 m/z.The sample preparation method was consistent with proteomics analysis with three biological replicates. For LC-MS/MS analysis, peptides were separated by a 120 min gradient elution at a flow rate 0.300 μL/min with a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with the Thermo Orbitrap Fusion mass spectrometer. The analytical column was a homemade fused silica capillary column  packed with C-18 resin . Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The Orbitrap Fusion mass spectrometer was operated in PRM mode using Xcalibur 4.2 software. The MS scans with a resolution of 120,000 were collected using a mass range of 300 and 1 400 mhttp://skyline.gs.washington.edu/) was used to import the LC-MS raw data for peak matching and to create the analytical template. Once the data table was exported and normalized, a multivariate analysis was performed using SIMCA-P 14.0 software , including principal components analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA). Differential lipids were screened based on t-test (p < 0.05), variable influence in the projection  and fold change (FCmax (maximum), FCmax > 1.30 or FCmin (minimum), FCmin < 0.77).Based on retention time, precise mass-to-charge ratio and fragmentation information, a targeted database containing 193 lipids  was established. Skyline .Progenesis QI data analysis  was used to process the LC-MS raw data. Deconvolution, alignment and peak preparation produced a list of mass-to-charge ratio and retention time pairs with peak intensities. QC data was analysed to ensure the reliability of the sample data. The coefficient of variation for all samples above 20% was removed and multivariate analysis was performed. The basis of differential lipid identification was a precise mass-to-charge ratio (error less than 0.01). The Lipid Metabolites and Pathways Strategy (LIPID MAPS) Consortium databases and the Human Metabolome Database (HMDB) were used to identify differential lipids. Metabolic pathways were analysed through the MetaboAnalyst website  was used, which was downloaded from the National Center for Biotechnology Information Search database. A reverse database for the decoy search was generated automatically in MaxQuant. Enzyme specificity was set to “Trypsin.” At least 7 amino acids were required for peptide identification. The default settings were used for variable and fixed modifications [variable modification: acetylation (Protein-N terminus) and oxidation methionine], fixed modification: carbamidomethylation. MaxQuant label-free quantification algorithm was used to quantitate MS signals. Protein intensities were represented in intensity-based absolute-protein-quantification (iBAQ). The iBAQ from each sample was transferred into a fraction of total protein iBAQ amount per experiment (FOT). In addition, the kyoto encyclopedia of genes and genomes (KEGG), universal protein (UniProt) and gene ontology consortium (GO) databases were applied to perform functional annotation and identification of the protein biological process.The raw MS files were handled with MaxQuant software (version 1.5.3.30). The identification of MS/MS-based peptides was carried out using the Andromeda search engine in MaxQuant. Andromeda used a target-decoy approach to identify peptides and proteins at an FDR <1%. As a forward database, the Raw files and target protein panel were loaded into SpectroDive 10.3 and analyzed with the default settings. SpectroDive would determine the ideal extraction window and perform quantification automatically. Qvalue cutoff on precursor was applied 1%. Peptides were manually inspected to verify matching spectra and peak integration. The area under curve (AUC) intensities of all fragments per precursor were summed to calculate the endogenous peptide signals.p < 0.05). 60 mg/kg ArBu accelerated the heart rates, while 120 mg/kg ArBu slightly increased the heart rates in the beginning and then slowed them down. However, the heart rates for the two groups returned to normal after approximately 60 and 110 min, respectively , but high doses of toad venom could inhibit the hearts (toxic effect) . In the p = 0.097)  .The results suggested that ArBu increased serum levels of myocardial enzymes. Myocardial enzymes belonged to cytoplasmic enzymes, mainly existing in myocardium, brain tissue and skeletal muscle. When the myocardium was damaged, impaired cellular integrity led to the release and activity changes of enzymes. Remarkably, CK-MB was a specific myocardial enzyme, primarily distributed in the cytoplasm of myocardial cells. It was a key enzyme for monitoring heart function. BNP was a sensitive indicator of early heart impairment. In summary, overexpressed myocardial enzymes and BNP indicated that ArBu inhibited and damaged of heart function in rats.In order to further determine the toxicity of ArBu to the organs, the hearts and livers of rats in each group were studied by pathological sections. Pathological sections of livers showed that ArBu had no hepatotoxicity (data not shown), which would be helpful for the development of ArBu anti-tumor drugs. In accordance with the results of myocardial enzyme assay, the pathological sections of hearts in high- and low-dose ArBu groups showed certain cardiotoxicity. As shown in −4 X + 0.00122. The limit of detection (S/N = 3/1) and the limit of quantitation (S/N = 10/1) was 0.1 and 0.01 ng/ml, respectively. The RSDs for the stability experiment were less than 15% after 0, 6 and 12 h. It ensured that biomonitoring and toxicokinetic studies could be carried out without unforeseen stability issues. Meanwhile, the intraday and inter-day precision showed RSD values less than 15%. The accuracy values of the intraday and inter-day validation were 88.9–110.4% (n = 6). It indicated that the instrument was stable and the detection method was feasible. The mean extraction recoveries of LQC, MQC and LQC were 93.6, 86.7 and 90.3% (n = 6), respectively. The method showed good matrix effect (80.2–97.5%). The results of extraction recoveries and matrix effect demonstrated that the pretreatment method was reasonable and feasible.Analysis of the blank plasma, the plasma administrated by ArBu and the blank plasma added with ArBu showed that the shape and chromatographic separation of the ArBu MRM peak were presented well . No peakmax) [time of Cmax (Tmax)] of the 60 mg/kg ArBu- and 120 mg/kg ArBu-administrated groups were 0.61 and 0.64 h, respectively, which were very close. The Cmax and mean residence time increased in an ArBu-dependent manner. As a result, apparent plasma clearance of the high-dose ArBu group was significantly lower than that of the low-dose ArBu group.Toxicokinetic samples collected from ArBu-treated rats at 0, 60 and 120 mg/kg were analyzed using the validated method. Toxicokinetic parameters were obtained by using the Winnonlin  software and the NCA model . The meamax. However, in the 120 mg/kg ArBu-administrated group, the heart rates began to increase when the ArBu blood concentration increased to a certain amount. When it increased further, the heart rates became a decrease. With the rapid elimination of ArBu, heart rates returned to normal in ArBu administration groups when it dropped to a certain level. The time was 60 and 110 min for the low- and high-dose ArBu groups, respectively.The results demonstrated that ArBu entered the blood rapidly and was detected only after 2 min CYP3A4 was the major metabolic enzyme of bufogenins. Since bufalin was a strong inhibitor of CYP3A4 , ArBu win = 8) for lipidomics research. The targeted lipidomics analysis was conducted using a targeted lipid database, including 97 PCs, 27 PEs, 24 SMs and 45 Cers. On the basis of accurate mass values and retention time, all the lipids were extracted and analyzed. In order to capture more potential biomarkers, an untargeted lipidomics method was combined with the targeted lipid analysis to systematically study the interference of ArBu on lipid metabolism. All the differentially-expressed lipids in serum and hearts were filtered out based on p < 0.05, VIP >1, and FCmax > 1.30 or FCmin < 0.77.Serum and hearts from rats treated with ArBu for 2 h were obtained from each group (max > 1.30 or FCmin < 0.77) in the low-dose ArBu group, including CEs, Cer, diacyl glycerols (DGs), SMs, phosphatidylserines (PSs), PE and lyso-phosphatic acid (lyso-PA)  . Howeverlyso-PA) .In hearts, a total of 83 differential lipids were obtained in the low-dose ArBu group compared with the control group. The subclasses of down-regulated lipids were mainly TGs (19/83), PEs (17/83), PCs (11/83), PSs (4/83), DGs (4/83) and PAs (2/83). While the subclasses of up-regulated lipids were mainly PEs (9/83), PCs (6/83), PSs (2/83), phosphatidylglycerols (PGs) (2/83) . There wS-plot loadings plot demonstrated the existence of differential lipids between the control and high dosage ArBu groups /18:1(9Z)], PC[20:4/20:3], PC [20:4/16:1(9Z)] and PC [20:4/18:2]. PCs and PEs were the main sources of arachidonic acid (C20:4) in mammals. With respect to metabolism, arachidonic acid was mainly derived from PCs containing the eicosatetraenoic acid chain. PEs also served as a transient source which could be quickly replenished. Arachidonic acid was an important inflammatory mediator . Lyso-PC+-ATPase . Lyso-PC+-ATPase . In thisienolide . TherefoPC[20:3/16:0], PC[18:2/20:5], SM(d18:2/20:0), SM(d18:1/20:0) and lysoPC(20:0), as potential biomarkers, could be broken down into C20 fatty acids. C20 fatty acids could further synthesize important inflammatory mediators and signaling substances, such as prostaglandins, tranexamic acids, leukotrienes and peroxyarachidonic acids . These sWhen the different dose ArBu group was compared with the control group respectively, for the hearts, the changes in PEs, PCs and PSs were significantly different. Remarkably, there were significant differences in TGs, DGs, PAs and SMs between high and low dose ArBu groups. Among the differential lipids in serum, Cer and SM were regulated upward in the low-dose ArBu group and regulated downward in the high-dose ArBu group. PSs was only found in the low-dose ArBu group. However, PCs, TGs and CL only existed in the high-dose ArBu group. In summary, the differential lipids in the hearts were significantly more numerous than in the serum. The major lipid subclasses were PCs, PEs and TGs in the hearts. Meanwhile, the differential lipids obtained from different dose ArBu groups were more different than the same in quantity and trend in both hearts and serum.p < 0.05 and an absolute value of r > 0.5. The results showed that only PC(0:0/22:6) was positively correlated with multiple CFA indexes among the 12 differential lipids in the low-dose ArBu group. The remaining 11 differential lipids all showed a negative correlation and only a significant correlation with BNP. However, these lipids were significantly correlated with multiple indexes of CFA in the high-dose ArBu group. In addition, there were 15 differential lipids in the high-dose ArBu group, negatively correlated with a number of CFA indexes, which could be used as potential biomarkers of ArBu cardiotoxicity. Overall, the differential lipids in the low-dose ArBu group were mainly related to BNP. However, they were CK > BNP = CK-MB > AST in the high-dose ArBu group. This was also an important basis for distinguishing the cardiotoxicity from ArBu.Spearman correlation analysis was carried out the correlation between the targeted differential lipids of different ArBu dose groups with CFA indexes respectively . These dp < 0.05 and FCmax > 1.3 (or FCmin < 0.77), the differential proteins of the ArBu-administrated groups were screened. The results showed that 31 of 3,073 proteins were up-regulated and 47 of 91 proteins were down-regulated in the low-dose ArBu group. However, 49 of 3,073 proteins were up-regulated and 55 of 97 proteins were down-regulated in the high-dose ArBu group. When three groups were analysed simultaneously, 95 ArBu-dependent differential proteins were selected from 3,073 proteins  network of differential proteins  was analAs we know, mitochondria are the most important energy supply site for cell life activities, providing about 95% of the energy of cells . MeanwhiIn previous mechanism studies of cardiotoxicity for bufadienolide revealed that calcium ion disorder in cardiomyocytes was related to heart rate disorders , suggestSome important differential proteins  were alsThe conclusion of the above literature search, heatmap and network of proteins and CFA indexes based on spearman analysis was that important differential proteins were closely linked to blood coagulation, calcium ions, mitochondrial energy metabolism, lipids and heart-related diseases.Based on the above analysis, differential lipids, differential proteins and CFA indexes were correlated, and further related to the metabolic pathways involved . It coulIn view of proteomics data analysis results, the relationships and status in the above two network diagrams, biological implications and references, important differential proteins were selected for protein validation using PRM mode. In PRM technology, all ions produced were detected by a high-resolution detector following the fragmentation of the precursor ions. This was the difference between the PRM and MRM. Due to the different detection modes of secondary MS, PRM technology was more accurate than MRM technology in terms of qualitative and quantitative accuracy.After screening, 23 differential proteins were used as verification targets, including Idh2, Fermt3, S100A1, Mrps6, mrpl9, Plg, Trap1, Prdx3, CPB2, Snx6, Golga4, Xirp2, CMA1, Ddx17, Atp8b2, Dnm2, Cbr4, Nebl, Mrpl16, F13a1, Popdc2, TMEM16F and Pcca. In order to ensure the reliability of the verification results, the differential protein with more than two characteristic peptides was determined as the further verification object. The abundance of proteins of interest was confirmed by targeted proteomics using PRM analysis. The proteomics results showed that mitochondrial proteins , cell cycle-related proteins (Trap1), heart disease related proteins , and important network node proteins  were enriched in administration groups . In the Tumor necrosis factor receptor-associated protein 1 (TRAP1), a molecular chaperone, was a major member of the mitochondrial heat shock protein 90 family. It was closely related to mitochondrial function and cell cycle. Mitochondrial dynamics played vital roles in calcium homeostasis and cell cycle regulation . The altIdh2 was known as Isocitrate dehydrogenase [NADP], mitochondrial. In the lipid-protein network map (high dose), Idh2 was closely related to coenzyme binding. It was closely associated with mitochondrial dysfunction. Reactive oxygen species induced oxidative stress was an important component of cardiovascular diseases such as hypertension, cardiac hypertrophy and atherosclerosis . Idh2 waThe full name of Mrps6 was mitochondrial ribosomal protein S6, which was an important nodal protein in PPI. Mrps6 showed an increasing trend in mitochondrial diseases, cardiovascular diseases, myocardial infarction and coronary artery diseases. In addition, it was involved in cell apoptosis, signal transduction and cell cycle. MRPS6 encoded a subunit of the mitochondrial ribosome . It was 2+ homeostasis. The increased expression of Pcca in the ArBu administration groups affected the impairment of systolic Ca2+ release and to irregular diastolic Ca2+ release, leading to cardiac and cellular contractility abnormalities. These changes were also associated with increased levels of oxidative stress in cardiomyocytes  in the heart could be decreased by nanostructured lipid carrier drug delivery . AlbuminIn the present investigation, a verified method was successfully applied to characterize the toxicokinetics of ArBu in rats. The cardiotoxicity mechanism of ArBu was studied by lipidomics and proteomics using the LC-MS/MS technique. Toxicity studies demonstrated that heart rates varied with the dosage of ArBu. Low-dose ArBu accelerated heart rates. However, the heart rates increased slightly and then decreased after high-dose ArBu treatment. Over a period of time, they all went back to normal, which was in line with the trend of ArBu concentration-time curve. Increased myocardial enzymes and BNP indicated that ArBu inhibited or impaired cardiac function. Pathological sections of livers showed that ArBu had no hepatotoxicity. However, the pathological sections of hearts in high-dose (more serious) and low-dose ArBu groups showed certain cardiotoxicity (muscle fiber disorder or rupture). Lipidomics analysis revealed the difference between serum and heart differential lipids. The difference was positively correlated with the dosage. Furthermore, cardiotoxicity was related to a variety of CFA indexes. The cardiotoxicity of ArBu was also reflected in proteomics research. Proteomics demonstrated that the differential proteins affected the calcium ion levels, mitochondrial energy metabolism, blood coagulation, lipid metabolism and heart-related diseases. Mrpl16, Mrps6, Prdx3, F13a1, mrpl9 and Ddx17 were significant nodal proteins in the PPI diagram. The multi-layer network analysis revealed the difference of the pathways involved in different ArBu doses. In the low-dose ArBu group, they were mainly chaperone binding, Alzheimer disease and Huntington disease. However, in high-dose ArBu group, these were coenzyme binding, oxidoreductase activity (acting on peroxide as acceptor), peroxidase activity, and complement and coagulation cascades. PRM verification showed that the regulation trends of ArBu on Trap1 (low-dose group), Idh2 (low- and high-dose group), Mrps6 (low- and high-dose group) and Pcca (low-dose group) were consistent with proteomics. Trap1, Idh2, Mrps6 and Pcca were potential biomarkers for the cardiotoxicity of ArBu. In conclusion, high-dose ArBu caused inhibition or damage of cardiac function in rats. From cardiac excitation to cardiac inhibition, the underlying molecular mechanisms were quite different. These results provided a theoretical basis for a comprehensive regulation of the microenvironment on the cardiotoxicity effects of ArBu.


Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an oligodendrocyte marker, in mixed glial cultures under pro- and anti-inflammatory conditions. Then, we characterized TRPV2 time-course expression in experimental animal models of hypomyelination (jimpy mice) and de-/remyelination ). TRPV2 showed upregulation associated with remyelination, inflammation in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expression was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methionines in TRPV2, which we found increased in inflammatory conditions. These results suggest that TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that it may become an interesting clinical target in the treatment of demyelination disorders. Transient potential receptor vanilloid 2 (TRPV2) is a thermo-, mechano- and lipid sensor non-selective cation channel, and is a member of the superfamily of TRP channels . ActivatPrevious work from our lab  showed tIn this study, we characterized the TRPV2–Opalin interaction in vitro and in mixed glial cell culture to assess if TRPV2 is expressed in oligodendrocytes. TRPV2 presence in oligodendrocyte-lineage cells, in addition to neurons, astrocytes and microglia, may provide support for the importance of TRPV2 in neuron-glial cells’  cross-taSince previous results from our group indicated a possible interaction between TRPV2 and NTM, PLP1 and Opalin , we studNext, to assess the possibility that TRPV2 and Opalin co-express in the same cell type, we determined if they were expressed in vitro in mixed glial cell cultures containing microglia, astrocytes and oligodendrocytes . Co-exprCellular expression of TRPV2 was determined with double immunocytochemical staining performed in glial mixed cultures. These cultures were composedof approximately 25% microglia, 30% astrocytes and 35% oligodendrocytes in basal conditions, and their populations varied slightly after pro-inflammatory treatment . In basap < 0.01) compared to controls. After an anti-inflammatory IL-4 treatment, TRPV2 expression tended to increase (p = 0.05) when compared to controls. Oxidative stress is known to induce oxidation of specific Met residues in TRPV2 (Met528 and Met607 in rat TRPV2) [Quantification of total TRPV2 expression in mixed glial cultures showed a significant decrease of TRPV2 after LPS pro-inflammatory treatment (t TRPV2) , endogene p = 0.0 when comp < 0.01). Determination of NO in the cell media of LPC-treated cells, as a measure of oxidative stress, demonstrated similar levels compared to the control condition -positive mature oligodendrocytes (see cell populations in ondition M.p < 0.0001).TRPV2, Opalin and MSRA protein expression was explored in spinal cord sections of control and jimpy mutant mice by immunohistochemistry. First, we determined the degree of hypomyelination in jimpy mice by staining the MBP, one of the most abundant proteins in myelin. As expected, MBP was highly expressed and homogenously distributed in the white matter (WM) of spinal cord sections of control mice A,a and sp = 0.0062).In basal conditions, TRPV2 expression in GM was mainly located in the cell body of spinal cord neurons, while in WM, it was in the soma of cells, putatively oligodendrocytes D,d,d’. Ip = 0.0092) in comparison with control animals  , we explp < 0.01) (p < 0.01) , was observed compared to basal conditions A,B, and  < 0.01) L. In the < 0.01) H,K, sinc < 0.01) I,K.p = 0.0014) compared to control mice. Later, at 21 and 28 days post-immunization, TRPV2 expression levels decreased and were maintained constant  and MS subjects (n = 6). In MS subjects, a non-significant tendency to decrease of TRPV2 mRNA levels was observed when compared to healthy subjects (p = 0.0014) when compared to healthy subjects (p = 0.0232) (p = 0.0124)  C, MSRA p 0.0124) C.In this study we aimed to explore the role and expression of TRPV2 in the context of myelination disorders of the CNS, taking advantage of previous work indicating the potential role of TRPV2 and its interactors in myelination processes . Opalin The role of TRPV2 in microglia and phagocytosis under oxidative stress is slightly better understood so far. NO promotes TRPV2 translocation to the membrane which results in a higher microglia phagocytic capacity  and atteNGF-dependent activation of endosomal TRPV2 in neurite outgrowth showed that TRPV2 can be active both at the plasma membrane and in intracellular compartments . The neuThe jimpy mouse model, a pathophysiological model for PMD ,18,19, sIn the experimental models of demyelination, the loss of myelin integrity was produced by oligodendrocyte cell death in the cuprizone model , or by aIndeed, the positive results in the reduction of demyelination in the cuprizone model and the amelioration of the EAE clinical symptoms after probenecid administration ,15,16 maEvaluation of MSRA levels in our experimental models showed an expression pattern linked to inflammation and resolution. Oxidative stress is known to be elevated in all three models ,43,44 anTo focus on TRPV2 and human myelin pathophysiology, we evaluated mRNA and protein expression of TRPV2 and MSRA in frontal cortex samples from MS patients compared to control subjects. In this case, a decrease in TRPV2 protein, which was non-significant in TRPV2 mRNA, was observed for MS patients compared to controls, while MSRA mRNA and protein expression were significantly increased in MS patients. Owing to the fact that ROS involvement has been described in MS pathogenesis ,49, the n = 6/group) were used. Animals were maintained with food and water ad libitum, in a 12 h light/dark cycle, at 22 ± 2 °C and 50–60% humidity. All experimental work was conducted according to Spanish regulations  in agreement with European Union directives  and was approved by the Ethical Committee of the Autonomous University of Barcelona, Spain.C57BL/6 males aged 8–10 weeks and females aged 2–4 months were used. Jimpy mutant male mice (jp/Y) were obtained and distinguished from normal control animals (+/Y) as described in Vela et al. . Male jiw/w) cuprizone , as described in Petkovic et al. [n = 6), a demyelination group fed for 5 weeks with cuprizone (n = 7) and a remyelination group fed for one additional week with standard powdered chow (n = 6).C57BL/6 males were fed with 0.2%  and 1 mg/mouse of H37R M. tuberculosis  in 200 μL of CFA (Sigma-Aldrich). Control sham-treated mice were injected with a similar emulsion without MOG35-55. All mice were injected intraperitoneally with toxin from B. pertussis  at 1 and 48 h after immunization. Clinical EAE symptoms were evaluated daily according to the following score: 0 = no symptoms; 0.5 = tail weakness; 1 = tail completely flaccid; 1.5 = mild difficulty in righting; 2 = great difficulty in righting; 2.5 = unsteady gait and paraparesis ; 3 = complete paralysis of one hind limb; 3.5 = complete paralysis of one hind limb and mild paralysis of the other hind limb; 4 = paraplegia  and incontinence; 4.5 = paraplegia and mild paralysis of one or two forelimbs; and 5 = moribund or dead. CFA and MOG-EAE mice were killed at the following timepoints: 9 DPI (presymptomatic phase), 14 DPI (symptomatic phase), 21 DPI (EAE peak), 28 DPI and 40 DPI (chronic phase) and processed for immunohistochemistry (n = 2–3/group) or western blot (n = 4–5/group).C57BL/6 female mice were immunized with a subcutaneous injection emulsion containing 100 μg/mouse of myelin oligodendrocyte glycoprotein (2) peptide 35-55 . The whole procedure was performed in accordance with the Helsinki Declaration, the Convention of the Council of Europe on Human Rights and Biomedicine, and approved by the Ethical Committee of the University of Barcelona. Postmortem histological frontal cortex samples were obtained from control subjects and MS patients, as shown in Pichia pastoris following our lab protocol as described in Suades et al. [Rat TRPV2 FL tagged with a C-terminal GFP and a 8xHis tag was overexpressed in yeast s et al. . Yeast cEscherichia coli BL-21 strain, as described previously [ARD of rat TRPV2 tagged with GFP and 8xHis tag in the C-terminus were heterologously expressed in eviously ,54. Exprg and supernatant was collected and purified using Ni-NTA resin . See All lysates were centrifuged for 45 min at 25,000× FL rat TRPV2 and ARD TRPV2 domain were purified and placed at specific protein concentrations (see n = 5), following the protocol described by Marignier et al. [g and the pellet obtained was resuspended in complete medium described above and brought to a single cell suspension by repeated pipetting followed by passage through a 100 μm pore mesh. Glial cells were seeded at a density of 2.0 × 105 cells/mL in T25 cell culture flasks and were kept at 37 °C in humidified 5% CO2/95% air. After three days, a first medium replacement was performed with DMEM-High glucose-20% FBS. The medium was replaced every 5–6 days until the confluence of the cell culture was reached (after 14–16 days in vitro (DIV)). Once primary cultures were confluent, cells were washed for 5 min with 0.1 M PBS (pH 7.4—Gibco #14190094) and 2 mL of 0.125% trypsin-EDTA solution was added. After 5 min at 37 °C in humidified 5% CO2/95% air, trypsinization was stopped by adding 2 mL of complete medium. Cells were centrifuged for 7 min at 200× g, and the pellet was resuspended with a 1000 μL pipette for 3–5 steps. For secondary glial cultures, the cells were seeded at a density of 1.5 × 105 cells/mL in T25 cell culture flasks and incubated at 37 °C in humidified 5% CO2/95% air. The medium was replaced every 5–6 days until cell confluence was reached (after 28–32 DIV). Quaternary cell cultures were obtained by repeating this process twice. For treatments, cells were seeded in 96- or 48-well culture plates  coated with poly-L-lysine (Sigma-Aldrich). Secondary and quaternary cortical mixed glial cultures enriched in oligodendrocytes were obtained from 0- to 3-day-old WT C57BL/6 pups , 48 h with LPS+IFNγ (100 ng/mL LPS + 4 ng/mL IFNγ—Sigma-Aldrich #I4777), or 24 h with 20 ng/mL IL-4 (BioLegend #57430). LPC (4 ng/mL) was assayed for 24 h with quaternary cell cultures. Control cells were treated with an equivalent volume of culture medium. After treatment, conditioned media were removed and frozen. For immunocytochemistry, cells were fixed with 4% paraformaldehyde  in 0.1 M PBS for 20 min at RT. For ELISA, cells were removed and frozen.Cultures were treated for 24 h with 100 ng/mL of LPS from Nitric oxide (NO) production in the conditioned medium of cultures was assessed by the colorimetric Griess reaction. Twenty-five μL of conditioned medium from each culture and treatment were mixed with 50 μL of a 1% sulfanilamide solution (Sigma-Aldrich # S9251) and 5% phosphoric acid (PanReac Applichem #131032) for 5 min. Then, 50 µL of 0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride solution (Sigma-Aldrich #222488) was added and incubated for 5 min at RT. Sodium nitrite (PanReac AppliChem #131703) was used to generate a standard curve (1.56–100 µM). Optical density at 530 nm was determined using a Varioskan™ LUX microplate reader . Triplicates were performed for each sample and treatment.Cells were permeated with 25% methanol solution for 1 min, and washed afterwards with TBS. To block inspecificities, cells were treated for 15 min with TBS + 5% FBS  and then incubated overnight at 4 °C with primary antibodies diluted in TBS + 5% FBS . After sOpalin) coefficient estimates the degree of red signal overlapping green, the M2 (MTRPV2) coefficient and vice versa.At least four images were taken from cell cultures stained for TRPV2 (green) and Opalin (red) with and without LPS + IFNγ. Colocalization was evaluated using ImageJ and the JACoP plugin , which pTM Microplate Reader , using Microplate Manager Software 6 (Bio-Rad).After washing with 0.1 M PBS, proteins were extracted using a RIPA buffer  and quantified using BCA Assay . Samples were stored at −20 °C until use. TRPV2 was quantified using the Mouse Transient Receptor Potential Cation Channel Subfamily V, Member 2 (TRPV2) ELISA Kit  following the manufacturer’s protocol. Briefly, 100 μL of blank, standards (0.16 ng/mL–10.00 ng/mL) and samples (0.3 μg protein/μL) were added to wells and incubated for 2 h at 37 °C. After incubating the detection reagents and washing, the Substrate Solution was added for 15 min at 37 °C. Finally, 50 μL of Stop Solution was added and measurements were performed at 450 nm with an iMarkFollowing anesthesia of ketamine (80 mg/kg) and xylazine (20 mg/kg), animals were perfused intracardially for 10 min with 4% PFA in 0.1 M phosphate buffer . Brain samples of cuprizone-treated mice and spinal cords of jimpy mice were dissected and included in paraffin blocks, as previously described . Ten-μm-Brain and spinal cord paraffin sections and cryostat spinal cord sections were processed for immunohistochemistry. Paraffin sections were first deparaffined with xylol and hydrated with decreasing graded ethanol solutions, followed by an antigen retrieval step with sodium citrate buffer with 0.1% Triton . After washing with 0.1 M TBS, paraffin or cryostat sections were inactivated for endogenous peroxidase for either 5 or 10 min, respectively, with 2% H2O2 in a 70% methanol solution. Sections were washed with TBS+0.1% Triton (TBS-0.1%T) or TBS-1%T for cryostat sections, and incubated for 1 h with blocking buffer solution . Sections were incubated with the primary antibody diluted in BB overnight at 4 °C followed by 1 h at RT . After wn = 3–4/experimental group), one photograph of the CC was taken at 10× magnification per section. All images were obtained using a DXM 1200F Nikon digital camera joined to a brightfield Nikon Eclipse 80i microscope and the corresponding ACT-1 2.20 software . Quantification was performed using AnalySIS software , manually setting the threshold for the staining. Analysis resulted in the percentage of area (% Area) and the intensity of the immunoreaction . AI index was calculated by multiplying the percentage of the immunolabeled area and the Mean Gray Value Mean [Quantitative analysis was performed on sections stained for MBP, TRPV2, Opalin and MSRA. Two to four sections per animal were analyzed. In spinal cord sections of jimpy mice, two photographs per section were taken at 10× magnification for MBP, Opalin and MSRA analysis, and at 20× magnification for TRPV2 analysis. In brain sections of cuprizone-treated mice (lue Mean . Resultsn = 3/group). After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2  diluted in BB at 4 °C and 1 h at RT  [g for 10 min at RT and the supernatant was collected. Thoracic spinal cords from control (CFA) and EAE (MOG) mice were dissected and quickly frozen in dry ice. Tissues were first sonicated at 4 °C in RIPA buffer  containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). One mL of ice-cold RIPA buffer was used per gram of tissue. After 30 min of incubation at 4 °C, samples were centrifuged at 5000× ethanol) ,59 usingProtein quantification was determined by Bradford assay . Samples were stored at −20 °C to be used for western blot analysis.2. Membranes were incubated overnight at 4 °C with primary antibodies . One microgram of RNA was reverse-transcribed with random primers using Transcriptor Reverse Transcriptase . Then, cDNA was diluted 1/10 to perform quantitative real-time polymerase chain reaction (qRTPCR) of MSRA, TRPV2 and GAPDH (housekeeping) mRNA with iTaq™ Universal SYBR® Green Supermix (Bio-Rad #1725121) by qPCR in a Bio-Rad CFX384 RT-PCR System. Reaction mixtures totaling 15 μL contained 5 ng of cDNA, 7.5 μL of iTaq™ Universal SYBR® Green Supermix, 500 nM of primer forward and 500 nM of primer reverse  and results were expressed as mean ± Standard Error of the Mean (SEM). The statistical tests used were: for cell culture treatments, one-way ANOVA followed by Dunnett’s post hoc test (for LPS/IL-4 treatments) and paired Student’s t-test (for LPC treatment); for time-course experiments , one-way ANOVA with either Tukey’s or Newman–Keuls post hoc test, respectively; and for TRPV2–Opalin colocalization, jimpy mouse and MS human samples compared to controls, unpaired Student’s t-test.Statistics was performed using Graph Pad PrismPrevious studies have shown that TRPV2 is expressed mainly in neurons at developmental stages . In this


The natural compound Hydroxysafflor yellow A (HSYA) has been demonstrated to exert anti-cancer effect on multiple cancers. This study aimed to clarify the role of HSYA in inhibiting colorectal cancer (CRC) in vitro and the underlying mechanisms. Different concentrations of HSYA  was exposed to HCT116 CRC cells, then cell proliferation, apoptosis, migration, and invasion were estimated by colony formation assay, TUNEL staining, wound-healing, and transwell assays, respectively. Western blotting assay was utilized to observe the expression of proteins involved in cell apoptosis, migration, and peroxisome proliferator-activated receptor γ (PPARγ)/PTEN/Akt signaling, including PCNA, Bax, Bcl-2, cleaved-caspase3, E-cadherin, N-cadherin, vimentin, PPARγ, and phosphorylated (p)-Akt. HCT116 cells that treated with 100 μM HSYA were also pre-treated with PPARγ antagonist, GW9662, or knockdown with PPARγ using short hairpin (sh)-RNA, to down-regulate PPARγ expression. Then, the above functional analysis was repeated. Results demonstrated that HSYA  significantly reduced HCT116 cell viability, but had no effect on the cell viability of human normal intestinal epithelial cell HIEC. HSYA also inhibited colony formation, migration, and invasion but promoted apoptosis of HCT116 cell in a concentration-dependent manner. Besides, the PPARγ/PTEN/Akt signaling was activated upon HSYA treatment. Finally, GW9662 and PPARγ knockdown blocked all the effects of HSYA on HCT116 cells. In conclusion, HSYA could exhibit anti-cancer effect on CRC via activating PPARγ/PTEN/Akt signaling, thereby inhibiting cells proliferation, migration, and invasion in vitro. Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, and it has become the second leading cause of cancer-related deaths all over the world. The latest data released by the World Health Organization indicate that the incidence and mortality of CRC in 2020 are approximately 24.8 and 12.0 per 100,000 .Hydroxysafflor yellow A (HSYA) is the major active ingredient in traditional Chinese medicine safflower extract, and it is also the water-soluble component with the highest content and the strongest pharmacological effect in safflor yellow . StudiesPeroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor of the nuclear receptor superfamily. PPARγ has been reported to play an important role controlling gene expression that related to a variety of physiological processes, thereby regulating cell proliferation, apoptosis and inflammation . In addiTherefore, we speculated that HSYA may exert anti-cancer effect on CRC via activating PPARγ/PTEN/Akt signaling. This study aimed to evaluate the effect of HSYA on CRC cells proliferation, migration, invasion, and apoptosis alongside the potential mechanisms.HSYA  was obtained from Shanghai Yuanye BioTech  and dissolved in sterile pyrogen-free water at a concentration of 100 mM as a working solution. The PPARγ antagonist, GW9662 (cat. no. SC9123) was purchased from Beyotime Biotechnology Research Institute . To inhibit PPARγ expression, cells were pre-treated with 2 μM GW9662 for 6 h .2 [The human intestinal epithelial cell HIEC and CRC cell line HCT116 were purchased from the cell bank of the Chinese Academy of Sciences . All cells were cultured in DMEM medium  with 10% fetal bovine serum , maintained at 37°C with 5% CO2 .The PPARγ-targeting short hairpin RNA (shRNA) sequences were designed and synthesized by GenScript Biotech , then cloned into pSuper-retro-puro, named sh-PPARγ#1 and sh-PPARγ#2. Transfection of shRNAs was carried out using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction as previously reported . At 48 h3 cells per well, in 96-well plates, then incubated at 37°C and 5% CO2 overnight for confluence. Subsequently, cells were treated with 0, 25, 50 and 100 μM HSYA for 48 h, with or without 2 μM GW9662 pretreatment for 6 h. Then, the culture solution was discarded and 10 μL of CCK-8 solution was added. After incubation for 1 h, the cell viability was estimated using a microplate reader  based on the absorbance at 450 nm.The CCK-8 kit (Beyotime) was used to measure HIEC and HCT116 cell viability as previously reported . Cells w3 cells /well) were seeded in a 6-cm plate and cultured overnight for confluence, then cells were exposed to 0, 25, 50, and 100 μM HSYA for 48 h, with or without 2 μM GW9662 pretreatment for 6 h. Subsequently, the culture medium was replaced with fresh medium and cultured for another 14 days. Colonies were infiltrated with methanol for 10 min, stained with crystal violet for 10 min, counted and photographed (magnification x100).Colony formation was performed as previously reported . HCT116 Apoptosis of HCT116 cells was observed using a TUNEL apoptosis detection kit (Alexa Fluor 488)  according to the manufacturer’s instruction . BrieflyWestern blotting was performed as previously reported . The HCT4 cells/well and grown overnight for confluence. Then, the cells were scraped with pipette tips, washed with PBS and photographed. After that, fresh medium that supplemented with indicated drugs were added. After incubation for 48 h, three random areas of cells were photographed under a microscope (magnification ×200).To assess cell migration, HCT116 cells were seeded in 6-well plates at a density of 1 × 105 cells per chamber. The lower chamber was filled with 500 μL DMEM containing 10% FBS. After treatment, the invaded cells on the lower-chamber were fixed with formalin for 0.5 h and stained with crystal violet for 15 min, and finally photographed and counted by a light microscope (magnification x200).Transwell assay was employed to detect cell invasion capacity . HCT116 −ΔΔCT method [Total RNA of treated HCT116 cells was extracted using TRIzol reagent. PrimeScript RT reagent Kit (Takara) was employed to perform reverse transcription according to the protocol. RT-qPCR was carried out using RT-PCR Kits (Takara) on a StepOnePlus Real-Time PCR system . Gene expression was analyzed via the 2T method  and primAll data represented three independent experiments and shown as mean ± standard deviation using GraphPad Prism 8.0. A one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to calculate the statistical differences. P < 0.05 was considered statistically significant.Firstly, we assessed the effect of HSYA on CRC cells viability. The chemical structure of HSYA was presented in Subsequently, the effects of HSYA on CRC cells migration and invasion were estimated. Results from wound-healing assay revealed that the cell migration rate was remarkably reduced upon HSYA  treatment ). SimilaTo uncover the possible mechanisms involving in HSYA, we investigated the effects of HSYA on PPARγ/PTEN/Akt signaling expression in CRC cells. We found that HCT116 cells that treatment with HSYA  showed higher expression of PPARγ and PTEN, but lower expression of p-Akt/Akt, when compared with control cells, suggesting the activation of PPARγ/PTEN/Akt signaling caused by HSYA ). We theTo further verify the above speculation, we knockdown PPARγ expression in HCT116 cells by shRNAs transfection, and sh-PPARγ#2 was selected for the following experiments based on the better transfection efficiency ). GW9662Finally, the alterations in HCT116 cells proliferation, apoptosis, migration and invasion, in the presence of GW9662 and sh-PPARγ were evaluated. In this study, we illustrated the promising anticancer function of HSYA in CRC in vitro, along with the molecular mechanisms underlying the concentration-dependent inhibitory effect of HSYA on cell proliferation, migration, and invasion and inducible effect on apoptosis in CRC cells. Importantly, we reported the activation of HSYA on PPARγ/PTEN/Akt signaling and PPARγ down-regulation reversed the anti-cancer effect of HSYA on CRC. Our results implied HSYA as a promising candidate for CRC therapy.HSYA, a natural compound, produces anti-cancer effect via inhibiting tumor cells proliferation, migration, invasion, and angiogenesis, and inducing cells apoptosis ,11,12. IWe next intended to uncover the potential signaling pathways involving in the function of HSYA in CRC. Previous studies have indicated the activation of HSYA on PPARγ ,17,18. HTo further validate the involvement of PPARγ/PTEN/Akt signaling in the effect of HSYA on CRC. In addition to 100 μM HSYA treatment, HCT116 cells were also exposed to PPARγ antagonist GW9662, or knockdown with PPARγ by shRNA transfection, to down-regulate PPARγ expression in HSYA-treated HCT116 cells. Then the changes in cells development processes were investigated. We found that GW9662 pre-treatment or PPARγ shRNA transfection effectively down-regulated PPARγ and PTEN expression, but up-regulated p-Akt expression, indicating the activation of PPARγ/PTEN/ Akt signaling induced by HSYA, was inhibited by PPARγ down-regulation. Importantly, all the effects of HSYA on cell proliferation, apoptosis, migration and invasion were reversed by both GW9662 treatment and PPARγ knockdown. Akt signaling is an important event in colorectal carcinogenesis, activation of it upon phosphorylation can contribute to cell proliferation and tumor progression by modulating cellular events, such as cell growth, adhesion, migration and survival ,37. As aTo conclude, HYSA suppressed proliferation, migration, invasion, and EMT in HCT116 CRC cells by activating the PPARγ/PTEN/Akt signaling pathways, implying that HYSA may be a promising candidate for CRC therapy.