NRAS gene, c.1_2insA, and a deletion variant, c.745delT, in the HNF1A gene, both of these mutations produced a termination codon. Other exonic missense variants found in the tumor were CTNNB1, FGFR3, KDR, SMO, HRAS, RAI1, and a TP53 variant (c.430C>G). Moreover, the results of the present study revealed a novel variant, c.430C>G, in TP53 and two missense variants of SND1 (c.1810G>C and c.1814G>C), which were also novel. ALK (rs760315884) and FGFR2 (rs1042522) missense variants were reported previously. Notably, a total of 10 previously reported single-nucleotide polymorphisms (SNPs) were found in this tumor in genes including MLH1 (rs769364808), FGFR3 (rs769364808), two variants (rs1873778 and rs2228230) in PDGFRA, KIT (rs55986963), APC (rs41115), and RET (rs1800861). The results of this study revealed a synonymous mutation (SNP) in c.1104 G>T; p. (Ser368Ser) in the MLH1 gene. In this amino acid (AA) codon, two other variants are also known to cause missense substitutions, c.1103C>G; p. (Ser368Trp); COSM6986674) and c.1103C>T; p., were found in hematopoietic and urinary tract tissue, respectively. However, three SNPs found in genes such as ALK, KDR, and ABL1 in the HBL tumor in this study were not reported in UCSC, COSMIC, and ClinVar databases. Additionally, 19 intronic variants were identified in this tumor. One intronic SNV was present in each of the following genes: EGFR, ERBB4, KDR, SMO, CDKN2B, PTEN, PTPN11, RB1, AKT1, and ERBB2. In PIK3CA and FBXL18 genes, two intronic variants were present, and in the SND1 gene, three intronic variants were detected in the HBL tumor presented in this study. Notably, only one of these was reported in the catalog of somatic mutations in cancer. Only one 3’-untranslated region (UTR) insertion variant in the NRAS gene (c.*2010T>AT) was detected in the tumor of the present study, and this was a splice site acceptor. A TP53 intronic mutation (c.782+1G>T) was the only pathogenic splice_donor_variant found in this HBL tumor. The frequency of variants and Phred scores were markedly high, and the p-values were significant for all of the aforementioned mutations. In summary, a total of 15 missense, 10 synonymous, and 19 intronic variants were identified in the HBL tumor. Results of the present study detected one novel insertion in NRAS and one novel deletion in HNF1A genes, a novel missense variant in the TP53 gene, and two novel missense variants of SND1. Hotspot mutations in other cancer driver genes, such as PTEN, ATM, SMAD4, SMARCB1, STK11, NPM1, CDKN2A, and EGFR, which are frequently affected in gliomas, were not found in the tumor of the present study. Future studies should aim to validate oncogenic mutations that may act as novel targets for the treatment of these tumors.The present study aimed to determine genomic changes in sporadic intracranial hemangioblastoma (HBL), and the mutation patterns were analyzed using next-generation DNA sequencing (NGS). In this NGS analysis of the HBL tumor, 67 variants of 41 genes were identified. Of these, 64 were single-nucleotide variants (SNVs), two were exonic insertions and deletions (INDEL), and one was an intronic INDEL. In total, 15 were missense exonic variants, including an insertion variant in the  VHL tumor suppressor gene , which exhibits an autosomal dominant inheritance pattern. Throughout their lifetime, these patients usually develop multiple tumors within the brain and spinal cord  was also reported (COSM9644514) in the esophagus. Results of a previous study demonstrated a KDR mutation, namely, c.1416A>T; p. (Gln472His) in exon 11, in an anaplastic ependymoma Grade III tumor. In the HBL tumor in this study, c.862T>C; p. (Phe288Leu) was present in exon 7  and c.1103C>T; , were found in hematopoietic and urinary tract tissue, respectively. However, three synonymous variants found in ALK, KDR, and ABL1 were not reported in any of these databases (Table As shown in Table ), KIT rs5986963, ABL1 gene, a mutation c.864C>T; p.  was detected in the skin, and in the HBL tumor in this study, another mutation in c.864A>T was found that caused the same synonymous change p. (Ala288Ala). Results of a previous study reported intronic PIK3CA (rs3729674) and missense KDR (rs1870377) variants in anaplastic ependymoma Grade III [APC (rs41115) variant in MPE [FGFR3 (rs7688609), PDGFRA (rs1873778), and RET (rs1800861) in anaplastic ependymoma Grade III tumor, a-CPP, and myxo-papillary ependymoma (MPE) tumors [In the rade III . Notablyt in MPE , FGFR3  tumors ,22,23.EGFR, ERBB4, KDR, SMO, CDKN2B, PTEN, PTPN11, RB1, AKT1, and ERBB2. In PIK3CA and FBXL18 genes, two intronic variants were detected, and in the SND1 gene, three intronic variants were detected. Notably, two of the SND1 intronic variants  were not reported previously, and only one (c.1780-63 G>A) was reported  in the lung tissue. The intronic mutation of the AKT1 gene detected in the tumor of the present study (in c.175+18C>T) was also previously reported in meningioma tumors (COSN17133117). The intronic TP53 mutation (c.782+1G>T) found in this study is reported in dbSNP (rs1555525429), COSMIC (COSM44640), and the ClinVar miner database as a pathogenic splice_donor_variant. In the same codon, two other variants were also reported previously c.782+1G>C in the lung and pancreas (COSM329756), and c.782+1G>A (COSM43571) in the breast and lung. These two variants are involved in the alteration of a conserved intronic nucleotide and occur as a somatic driver mutation in patients with a wide variety of cancers, such as osteosarcoma, breast cancer, thymic carcinoma, and small-cell lung carcinoma. These mutations have previously been described in the ClinVar miner database (submission accession SCV000923846). The TP53 (c.782+1G>T) variant was not reported in brain tissue so far, and the PolyPhen-2 and SIFT scores predicted that this was likely pathogenic. The intronic variants found in the tumor of the present study were also detected in other tumors; for example, PIK3CA (rs3729674) was present in anaplastic ependymoma Grade III and ACP tumors [ERBB4 variant (rs839541) was reported in anaplastic ependymoma Grade III [KDR variant c.798+54G>A was reported in anaplastic ependymoma Grade III, ACP, and MPE tumors. In the tumor of the present study, another intronic variant of KDR, c.1413-20C>G, was detected [AKT1 (rs3730358), SND1 (rs225241), FBXL18 (rs7799622), PIK3CA (rs3729674), and ERBB4 (rs839541) were reported in SNP and UCSC databases. Notably, Cold Shock Domain Containing E1 (CSDE1) is an NRAS upstream gene protein (CSDE1) untranslated region (UTR) variant that was not reported in the COSMIC database. Other variants were not reported in any databases. Only the CSDE1 gene mutation, (c.*2010T>AT), was a 3'-UTR variant detected in this tumor  was previously reported in this HBL tumor by us; however, no missense mutations were detected in IDH1 and IDH2 genes in this tumor [VHL gene; however, no VHL mutations were detected in this tumor. Previous studies have also reported the absence of VHL gene mutations in sporadic cerebellar HBL, consistent with the findings of the present study [VHL gene are implicated in the development of at least 20% of sporadic CNS-HBL tumors [ALK, KDR, CTNNB1, PIK3CA, FGFR3, FGFR2, HRAS, HNF1A, SMO, and TP53 [Ion AmpliSeq comprehensive cancer panel (cat. no. 4477685) consists of 16,000 primers supplied in four aliquots and targets 409 genes. The Ion AmpliSeq Cancer HotSpot Panel (cat no. 4471262) consists of 50 oncogenes and tumor suppressor genes that are frequently mutated in several types of cancers. The mutation profiling of a-CPP, MPE, and other brain tumors was reported in previous studies using these custom gene panels on the Ion Proton instrument -23,28. Cnt study . HoweverL tumors . Severaland TP53 . These gand TP53 . Ma et aand TP53 . The preand TP53 ,19,35-37CTNNB1, FGFR3, KDR, SMO, HRAS, RAI1, and TP53 variant (c.430C>G). One novel variant in the TP53 gene and two missense variants of SND1 were also detected. The insertion frameshift variant in NRAS and deletion variant in HNF1A genes was also novel. The four synonymous variants detected in the HBL tumor of the present study, in genes such as MLH1, ALK, KDR, and ABL1, were not reported previously. Among the three intronic variants detected in the SND1 gene in the HBL tumor, two of which were novel. Only one 3’-UTR variant in the CSDE1 gene and an intronic splice_donor_variant of the TP53 gene was found in the HBL tumor.In summary, we have presented the mutational spectrum of the sporadic, non-VHL-HBL tumor using NGS. The exonic missense variants were detected in multiple genes, including PTEN, ATM, SMAD4, SMARCB1, STK11, NPM1, CDKN2A, and EGFR, which are frequently affected in gliomas, were not found in the tumor of this study. Notably, these are rarely reported in HBL tumors, which is in accordance with the NGS analysis of the HBL tumor in this study. Currently, no approved targeted therapy for treating VHL-associated and/or sporadic HBLs exists. As these novel mutations have not been previously reported in HBL tumors, the findings of this study may lead to the development of a genetic signature to distinguish between HBL types, such as supratentorial, VHL, and infratentorial. However, further studies using large samples are required. Future investigations using NGS and animal models may determine additional oncogenic mutations that may act as targets for the treatment of these tumors.Mutations in other cancer driver genes, such as


Saccharomyces cerevisiae chitin synthase in the absence and presence of glycosyl donor, acceptor, product, or peptidyl nucleoside inhibitors. Combined with functional analyses, these structures show how the donor and acceptor substrates bind in the active site, how substrate hydrolysis drives self-priming, how a chitin-conducting transmembrane channel opens, and how peptidyl nucleoside inhibitors inhibit chitin synthase. Our work provides a structural basis for understanding the function and inhibition of chitin synthase.Chitin is one of the most abundant natural biopolymers and serves as a critical structural component of extracellular matrices, including fungal cell walls and insect exoskeletons. As a linear polymer of β--linked N-acetylglucosamine, chitin is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. In this study, we determine seven different cryo-electron microscopy structures of a  Chitin, the second most abundant natural polysaccharide in nature, is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. Here the authors determine cryo-EM structures of the chitin synthase, which reveal its activation, catalytic and inhibitory mechanisms Chitin is a primary component of fungal cell walls, the exoskeletons of crustaceans and insects, and it plays an essential role in the reproduction, growth or development of these organisms2. Chitin is a long-chain polymer of β--linked N-acetylglucosamine (GlcNAc) and is synthesized by chitin synthase in the plasma membrane4. Chitin synthase catalyzes the formation of β(1 → 4) glycosidic linkages in chitin by using UDP-activated GlcNAc (UDP-GlcNAc) as the sugar donor and meanwhile transports the polysaccharide product out through the membrane  is a peptidyl nucleoside competitive inhibitor of chitin synthase and has been widely used as an antifungal agent against phytopathogenic fungi and arthropod pests in agriculture and forestry for decades24. Nikkomycin Z (NikkoZ), another peptidyl nucleoside inhibitor of chitin synthase, has shown significant clinical benefits in mammals against pathogenic fungi26. Determining the structures of chitin synthase in complex with these inhibitors will provide insights into the inhibitory mechanism in atomic level of chitin synthase by peptidyl nucleoside inhibitors, and is also very helpful for development of new antifungal drugs.As chitin is not available in plants and vertebrates, the biosynthesis of chitin is considered an attractive target for fungicides, insecticides, acaricides, and antifungal drugsS. cerevisiae Chs1 in the apo state and in complex with UDP-GlcNAc, UDP-GlcNAc+GlcNAc, UDP, UDP+GlcNAc, PolyB, and NikkoZ. Our studies provide molecular insights into how donor and acceptor substrates bind in the active site, how chitin synthesis is initiated, how the chitin-conducting transmembrane channel works, and the molecular mechanism of how peptidyl nucleoside inhibitors inhibit chitin synthase.In this report, we determined seven cryo-electron microscopy (cryo-EM) structures of S. cerevisiae with a C-terminal triple FLAG tag and purified with anti-FLAG resins followed by size-exclusion chromatography , and the N-terminal region of Chs1 zymogen likely plays an inhibitory function.To understand how trypsin proteolysis activates Chs1, we incubated purified Chs1 with trypsin in different mass ratios and times, and analyzed the product by SDS-PAGE and tryptic digestion mass spectrometry  at 2.9-Å resolution Fig. . Compari2+; the GlcNAc moiety of UDP-GlcNAc is located right above IF2 in a bent configuration extending outward from the active site. At the bottom of the active site, Y654 in IF1, W760 in IF2, R718, and a conserved VLPGA motif (residues 673–677) form the GlcNAc acceptor binding site  in Chs1  glycosidic bond in the following reaction Fig. . The UDPhs1 Fig. . Such arChs1Δ yeast phenotype. In addition, K578A, D745R, Q756L, and R759A moderately affected Chs1 function, suggesting that they are less important.We mutated key residues in the active site identified above and performed an in vivo growth complementation assay Fig. . We foun16. Therefore, we also named the loop of Chs1 “switch loop”, which closes the chitin transporting path in the apo state and opens it during chitin synthesis  of the Chs1 structure in UDP-GlcNAc loading state. 3DVA is a newly developed software in cryoSPARC to reveal the continuous variability and discrete heterogeneity of a structurened Fig. . This ma16.The cavity has a lateral opening in the middle of the lipid bilayer, which was formed by TMH3, TMH4 and IF3 , and donor+acceptor bound states, we were able to propose a working model of activated Chs1 Fig. . In the 38. However, our structure of Chs1 showed each subunit only has one active site and two active sites in Chs1 dimer are too far to cooperate with each other. Further research is required to fully understand the mechanism of chitin elongation of Chs1, including how to connect GlcNAc residues in alternating orientations. Capturing Chs1 in complex with chitin will undoubtedly provide more insights into this model.The active site and chitin transporting channel of each Chs1 subunit in the dimer are independent, but the domain swapping loop in one subunit probably regulates the other subunit’s function by inhibiting and releasing its GTD. This makes the chitin synthesis of the two subunits simultaneous, which is probably important for forming chitin fibers Fig. . Besides21, although their structural details and oligomeric state are different. Chitin, hyaluronan and cellulose synthases have a similar cytosolic glycosyltransferase domain and a transmembrane polysaccharide transporting channel. Notably, the GlcNAc moiety of the UDP-GlcNAc donor in complex with HAS faces the acceptor binding site  in apo, UDP-GlcNAc bound, nikkomycin Z bound, and polyoxin D bound states34. caChs2 and Chs1 from Saccharomyces cerevisiae (scChs1) in our work share sequence identity of ~50%. Structures of caChs2 and scChs1 in four different states are all superimposable with a root-mean-square-deviation (rmsd) of ~1.2 Å, indicating their high similarity . Besides, the UDP-GlcNAc substrate and two types of inhibitors are all bound in similar positions in structures of caChs2 and scChs1, suggesting their similar catalytic and inhibitory mechanisms. Because of the lack of structures in primed and UDP-GlcNAc+GlcNAc bound states, detailed catalytic cycle of chitin synthase and regulation of the chitin transporting channel by TMH4 were not discovered in the work of caChs2. Inhibitory function of the domain swapping loop was also not defined in that work.During preparation of this manuscript, another group reported the cryo-EM structures of chitin synthase 2 from In conclusion, our systematic structural and biochemical studies of Chs1 have provided deep insights into the molecular mechanisms for proteolysis activation, glycosyltransferase activity and transmembrane chitin transport of a fungal chitin synthase, and the inhibitory mechanisms of two peptidyl nucleoside chitin synthase inhibitors. Given the critical role of chitin synthase in pathogenic fungal infections, our work provides a platform for the development of new small antifungal molecules.g for 30 min at 4 °C and collected the supernatant for another centrifuge cycle at 100,000 × g for 60 min at 4 °C. The membrane pellet was collected and then resuspended in buffer A containing 10% glycerol, 20 mM Tris-HCl (pH 7.4), 1% DDM, 0.1% cholesteryl hydrogen succinate (CHS), 500 mM NaCl, 1 mM MgCl2, 1 mM EDTA, and 1 mM PMSF. After incubation for 30 min at 4 °C, the mixture was centrifuged for 30 min at 100,000 × g to remove the insoluble membrane. We loaded the supernatant into a pre-equilibrated anti-FLAG (M2) affinity column (GenScript) at 4 °C and washed the affinity gel with buffer B , 0.0033% GDN, 0.0013% CHS, and 1 mM MgCl2). The proteins were eluted with buffer B containing 0.15 mg/mL 3 × FLAG peptide and were further purified in a Superose 6 10/300 Increase gel filtration column in buffer C . The purified proteins were assessed by SDS‒PAGE and concentrated for cryo-EM analysis. We screened a range of detergents in the purification, and found LMNG/GDN/CHS is the best detergent for preparing cryo-EM grid, and DDM/CHS is the best for stability and yield of Chs1. To make the experiment simple, DDM/CHS purified sample was used in the activity assays.We tagged Chs1 (Uniprot ID: P08004) with a C-terminal triple-FLAG to the modified pRS423 vector  or inhibitors (5 mM Nikkomycin Z or 5 mM Polyoxin B) on ice. After incubation, 2.5-μL aliquots of Chs1 at a concentration of ~3 mg/mL were placed on glow-discharged holey carbon grids  and flash-frozen in liquid ethane using an FEI Vitrobot Mark IV. Grids were screened in a 300-keV FEI TF30 electron microscope from the School of Basic Medical Sciences, Peking University. Cryo-EM data were collected automatically with SerialEM in a 300-keV FEI Titan Krios electron microscope from the School of Physics, Peking University and Institute of Biophysics Chinese Academy of Sciences with defocus values ranging from −1.2 to −2.5 μm. The microscope was operated with a K3 direct detector at a nominal magnification of 225,000×. The total doses were 50–60 electrons per Å39 for motion correction and CTFFIND-4.140 for calculation of contrast transfer function parameters. We used RELION-3 for particle picking and extraction and used CryoSPARC for all remaining steps41. The resolution of the maps was estimated by the gold-standard Fourier shell correlation at a correlation cutoff value of 0.143.We used the program MotionCorr-2.0For the Nikkomycin Z bound Chs1 structure, we collected 2711 raw movie micrographs. A total of 4,135,734 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 837,015 particles were retained for further refinement, and postprocessing, resulting in a 2.4-Å average resolution 3D map using C2 symmetry.For the Chs1 structure in apo state, we collected 3672 raw movie micrographs. A total of 3,529,578 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 1,342,168 particles were retained for further refinement, and postprocessing, resulting in a 3.0-Å average resolution 3D map using C2 symmetry.For the UDP-GlcNAc+GlcNAc bound Chs1 structure, we collected 1252 raw movie micrographs. A total of 996,080 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 208,320 particles were retained for further refinement, and postprocessing, resulting in a 3.1-Å average resolution 3D map using C2 symmetry.For the UDP-GlcNAc bound Chs1 structure, we collected 1076 raw movie micrographs. A total of 1,036,417 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 330,289 particles were retained for further refinement, and postprocessing, resulting in a 3.1-Å average resolution 3D map using C2 symmetry.For the UDP+GlcNAc bound Chs1 structure (primed state), we collected 2469 raw movie micrographs. A total of 1,059,472 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 184,449 particles were retained for further refinement, and postprocessing, resulting in a 3.1-Å average resolution 3D map using C2 symmetry.For the UDP bound Chs1 structure, we collected 632 raw movie micrographs. A total of 431,546 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 115,773 particles were retained for further refinement, and postprocessing, resulting in a 3.6-Å average resolution 3D map using C2 symmetry.For the Chs1 structure in apo state after incubation with GlcNAc, we collected 1564 raw movie micrographs. A total of 1,077,654 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 218,698 particles were retained for further refinement, and postprocessing, resulting in a 3.5-Å average resolution 3D map using C2 symmetry.For the Polyoxin B bound Chs1 structure, we collected 2311 raw movie micrographs. A total of 2,608,153 particles were picked automatically for 2D classification and heterogeneous refinement. Based on the quality of three heterogeneous refined 3D map, 142,887 particles were retained for further refinement, and postprocessing, resulting in a 3.6-Å average resolution 3D map using C2 symmetry.43, fitted it into our map, and corrected it in COOT44 and Chimera45. The complete Chs1 model was refined by real-space refinement in the PHENIX program46 and subsequently adjusted manually in COOT. Finally, the model was validated using MolProbity47. Structural figures were prepared in Chimera and PyMOL (https://pymol.org/2/).We used the predicted Chs1 structure by Alphafold2 as the initial model2, 10 mM GlcNAc and 10 mM UDP-GlcNAc in a total volume of 5 μL. The mixture was first incubated for 60 min at 30 °C, and then 5 μL of UDP Detection Reagent was added. The mixture was incubated for 60 min at room temperature, and then luminescence was detected by a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek).The activity of Chs1 was measured using the UDP-Glo™ Glycosyltransferase Assay from Promega , which can measure the activity of any glycosyltransferase (GT) that uses a UDP-sugar as a substrate. Each reaction contained 0.125 μg purified protein, 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.025% DDM, 0.0025% CHS and 10 mM MgCl27. The assay mainly includes two steps: binding of synthesized chitin to a WGA-coated surface and detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity can be determined in absorbance at 600 nm, and the values are converted to amounts of chitin using commercial chitin as a standard. Firstly, wells of 96-well plates were precoated with WGA (wheat germ agglutinin). Then, 100 μL reaction solution containing 3.4 μg purified protein, 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.025% DDM, 0.0025% CHS, 1 mM MgCl2, 2 mM GlcNAc and 1 mM UDP-GlcNAc was added and incubated for 60 min at room temperature. After the solution in the wells was removed, 100 μL WGA-HRP (wheat germ agglutinin - horse reddish peroxidase) at a concentration of 1 μg/mL in buffer solution (0.2 mM HEPES at pH 7.4) was added to each well and cultured at 37 °C for 20 min. Then, the solution in the wells was removed, and the wells were washed with buffer solution three times. To each well, 100 μL of 1-Step Ultra TMB-ELISA substrate solution was added, and the mixtures were lucifugely reacted for 10 min at room temperature. Finally, all reactions were stopped by 100 μL 0.5 M H2SO4, and UV–Vis absorption spectra were recorded on a microplate reader at 450 nm by a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek).Chitin synthase activity was evaluated using the method described by Lucero with some modification4HCO3 overnight at 37 °C. The trypisn-digested peptides were then eluted from the gel, and dried by vacuum. The nano-LC-MS/MS experiments were then performed using a LTQ orbitrap velos pro mass spectrometer . Each sample was loaded into a C18 reverse phase column . The peptide mixtures were eluted with a 0-40% gradient  over 60 min and were detected in mass spectrometer using a data-dependent TOP10 method. The general mass spectrometric conditions were: spray voltage, 2.2 kV; no sheath and auxiliary gas flow; ion transfer tube temperature at 250 °C; 35% normalized collision energy using for MS/MS(MS2). Ion selection thresholds were 1000 counts for MS2. An activation q = 0.25 and activation time of 30 ms were applied in MS2 acquisitions. The mass spectrometer was operated in positive ion mode and a data-dependent automatic switch was employed between MS and MS/MS acquisition modes. For each cycle, one full MS scan in the Orbitap followed by ten MS2 in the LTQ on the ten most intense ions. Selected ions were excluded from further selection for 30 s. Final LC-MS/MS data were submitted to database searching against the S. cerevisiae sequence library in the Uniprot protein sequence database using Sequest HT algorithm in the Proteome Discoverer 1.4 software package (Thermo Scientific).The protein bands in SDS-PAGE gel were firstly cutted and processed by in-gel digestion. After distained and reductive alkylated, the gel pieces were incubated with 500 ng trypsin in 100 μL of 50 mM NH2PO4, pH 6.0). The chitinase  was added and incubated at 37°C for 24 h.Insoluble chitin was synthesized following above chitin synthesis assay and was collected by centrifugation. The chitin was washed and resuspended with buffer (50 mM NaHWe prepared the Chs1 knockout strain (ΔChs1) in the W303 strain  is a non-specific fluorochrome stain for 1,3-β and 1,4-β-linked polysaccharides and displays fluorescence when exposed to long wavelength ultraviolet. The staining of synthesized chitin by CFW was performed by a modification of the previous procedureBlue Native PAGE was carried out under nondenaturing conditions in gels (1.0 mm thickness) containing 4–15% polyacrylamide. It was carried out in Blue Native PAGE electrophoresis running buffer (Sangon Biotech (Shanghai) Co., Ltd.) at 100 V for 15 h.SDS‒PAGE was first performed in gels (1.0 mm thickness) containing 4–12% polyacrylamide. Proteins in SDS‒PAGE gel were then transferred to a polyvinylidene fluoride membrane. Anti-Flag mouse monoclonal antibody  and HRP conjugated goat anti-mouse IgG (H + L)  were used for blotting at 1:1000 and 1:5000 dilution ratio respectively.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting SummarySource Data


However, the specific epigenetic mechanisms governing human CNS development remain poorly elucidated. Here, we generated small−molecule−assisted shut−off (SMASh)−tagged hESC lines to reduce METTL3 protein levels, and found that METTL3 is not required for human neural progenitor cell (hNPC) formation and neuron differentiation. However, METTL3 deficiency inhibited hNPC proliferation by reducing SLIT2 expression. Mechanistic studies revealed that METTL3 degradation in hNPCs significantly decreased the enrichment of m6A in SLIT2 mRNA, consequently reducing its expression. Our findings reveal a novel functional target (SLIT2) for METTL3 in hNPCs and contribute to a better understanding of m6A−dependent mechanisms in hNPC proliferation.METTL3, a methyltransferase responsible for N6−methyladenosine (m NPCs shl stages . Using telopment . HoweverNeural specification of human pluripotent stem cells (hPSCs) provides a valuable model to investigate human CNS development at the molecular and cellular levels ,21,22,23SLIT2) expression. Mechanistic studies revealed that METTL3 degradation in hNPCs significantly decreased the enrichment of m6A in SLIT2 mRNA, consequently reducing its expression. Our findings identified SLIT2 as a functional target of METTL3 in hNPCs.Here, we generated small−molecule−assisted shut−off (SMASh)−tagged hESC lines to diminish METTL3 protein, and found that METTL3 is not required for human neural progenitor cell (hNPC) formation and neuron differentiation, but the proliferation of hNPCs decreases with METTL3 deficiency by reducing slit guidance ligand 2 (s/s) lines  lines b. To tess) lines c. Thus, s/s after ASV administration  staining. We noted that the cells maintained a typical undifferentiated state and the number of AP positive colonies was similar for H9 hESCs and H9−METTL3stration d, e. Furstration f. Togeths/s hESCs using a well−defined protocol involving dual inhibition of TGFβ/BMP signaling and the EB method [OCT4 and NANOG, alongside an upregulation of the NPC genes PAX6 and SOX1 at day 16 of differentiation (−/− hESCs (used as a negative control) [s/s hESCs, both treated and untreated with ASV, exhibited a rosette-like morphology, the typical NPC phenotype in hESC neural specification, while PAX6 positive cells and PAX6 protein levels remained consistently in both ASV−treated and untreated METTL3s/s hESCs (s/s hESCs (with or without ASV) exhibited high expression of the canonical NPC marker SOX1  , METTL3s/s hESCs . Additioker SOX1 . Togethe+ hNPCs and MAP2+ neurons or the percentage of NEUN+/TUJ1+ neurons   and transcription end site (TES)  consensus motif   . Furtherte (TES) . Consistus motif c. Additilopment) . Consistr 3′ UTR e. To draS/S hNPCs treated with or without ASV. A total of 451 and 228 genes were found to be downregulated or upregulated, respectively, in hNPCs treated with ASV  in METTL3with ASV a. Intrigwith ASV . Further6A peaks . Moreove6A peaks e.S/S hNPCs (treated with ASV) with m6A marking genes, and found that 56 downregulated genes were regulated by m6A methylation  , tumor progression, stem cell regulation, and cell proliferation ,37,38,39out ASV) i. The reegulated j–l. In awith ASV m. OveralMettl3 in mESCs fails to adequately terminate their naïve state, and blocks differentiation [METTL3 knockout hESCs, the effect of METTL3 knockout on hESCs remains unclear. Here, we successfully construct METTL3-SMASh tagged hESCs. ASV treatment decreases the expression of METTL3 in METTL3-SMASh tagged hESCs, but does not affect the stem cell morphology and the expression levels of OCT4 and NANOG. OCT4 and NANOG are recognized as pivotal components of the core pluripotency circuitry [OCT4 expression leads to differentiation into primitive endoderm and mesoderm [OCT4 results in loss of pluripotency and dedifferentiation to trophectoderm [OCT4 expression remain normal. NANOG is a classic pluripotency-maintaining factor [NANOG deficiency in ESCs leads to exit pluripotency and differentiated into extraembryonic endoderm [NANOG in ESCs is sufficient for colonization and expansion of ESCs [METTL3 knockdown in hESCs [METTL3 plays essential roles in the cell differentiation, reprogramming, embryonic development and tumor progression ,40,41,42ntiation ,45. Howeircuitry ,47,48. Oircuitry ,51,52,53g factor ,56,57,58in hESCs . These pMETTL3 in mice leads to an elevated population of intermediate progenitor cells (iPCs) and inhibits neuronal production [METTL3 alters neurite enrichment of a subset of mRNAs and reduces the localization of select methylated RNAs in mouse hippocampal neurons [Xenopus laevis, knockdown of METTL3 results in anteriorization of neurulae and tailbud embryos, accompanied by the depletion of neural crest and neuronal cells [Xenopus laevis.METTL3 has important roles in the mammal CNS system ,59,60. Roduction . The abs neurons . In Xenoal cells . Accumulal cells . Our stu6A modification, and thousands of studies have found that depletion of METTL3 apparently diminishes m6A signals [6A peaks, while these m6A peaks exhibit a distinct RRACH consensus motif  and the SMASh−tagged hESCs under feeder−free conditions on Matrigel  −coated plates in mTeSR1 medium .https://cctop.cos.uni-heidelberg.de:8043 (accessed on 19 August 2020).The guide RNAs (gRNAs) for METTL3 were designed from To create the SMASh donor vector, we PCR amplified the “PGK−Puro” sequence, the homologous recombination arms (HR arms) of METTL3, and the HA×3−SMASh DNA sequence. These fragments were then cloned into the pZERO vector  using the Hieff Clone Plus Multi One−Step Cloning Kit .To construct SMASh tagged hESCs, electroporation was performed using the LONZA 4D−Nucleofector. The day before nucleofection, we added 10 μM ROCK inhibitor Y27632  to the medium. Subsequently, hESCs were digested using Accutase . A single nucleofection event utilized 10 μg pX330−sgRNA plasmid and 15 μg donor plasmid. All primer sequences are listed in −1) was added between day 4 and day 8 after nucleofection, followed by an increase to 1 μg mL−1 for an additional 4 days. Two weeks after nucleofection, we selected 11 colonies for PCR and Western blot analysis.Puromycin  in accordance with the manufacturer’s guidelines. The cells were rinsed twice with PBST, followed by fixation in fix solution at room temperature for 2–5 min. After washing three times with PBST, the cells were stained with staining buffer (Buffer A: Buffer B: Buffer C = 1:1:1) at room temperature for 5–15 min. Following three PBS washes, the cells were captured with a microscope .ASV  was initially dissolved in DMSO to create a 100 mM stock solution. Before use, this stock solution was further diluted to 10 mM with DMEM/F12 and subsequently incorporated into the culture medium to reach a final concentration of 2 μM. In control groups, an equivalent concentration of DMSO (0.002%) was added.−1 insulin , and 1 μg mL−1 heparin , supplemented with 5 μM SB431542  and 5 μM dorsomorphin  [−1 bFGF and 20 ng mL−1 EGF, with regular medium changes every 2 days. After 5 days of culture, these neurospheres were dissociated into single cells using Accutase and designated as P0. A total of 2.5 × 106 NPCs from P0 were subjected to a proliferation assay. NPCs were passaged and counted every 6 days, resulting in the identification of P1 and P2.The monolayer neural specification of hESCs was conducted as described previously ,67. Brie, S5900) . After 8−1 heparin. On day 7, the EBs were transferred to 10 cm plates in N2 medium plus 20% FBS, with subsequent medium changes every 2 days. After 14 days, rosette-like NPCs appeared and were resuspended in N2 medium.EB neural specification was executed as described previously . Briefly5 hNPCs were plated onto Matrigel-coated 24-well plates and maintained in N2B27 medium containing 20 ng mL−1 BDNF , 20 ng mL−1 GDNF , and 1 mM cAMP . The medium was refreshed every 2 days. After a 14-day period, typical neuronal morphology become evident, and the neurons were subjected to immunostaining analysis.Neuron spontaneous differentiation: hNPCs were dissociated into single cells and utilized for spontaneous differentiation. A total of 1 × 10GAPDH served as the internal reference for normalizing the qRT−PCR outcomes from human samples. Data analysis involved the utilization of three replicates. All primer sequences are listed in We extracted total cellular RNA using RNAiso  and performed reverse transcription using the PrimeScript RT reagent Kit  . Subsequently, qRT−PCR was conducted using SYBR qPCR Master Mix  and a CFX96 machine . Western blot was conducted following previously established procedures . For theImmunostaining was performed as described previously . Cells w6 METTL3s/s hNPCs (with or without ASV) on 6−well plates in N2B27 medium containing 10 μM EdU for approximately 16 h. A negative control group was cultured without EdU. Subsequently, we dissociated these cells into single cells and fixed them in fixation buffer at room temperature for 20 min. Afterward, the cells were washed with PBS and permeabilized in perm/wash buffer at 4 °C for 10–15 min. After washing, we incubated the cells in PBS containing CuSO4, the fluorescent dye picolyl azide, and reaction buffer additive for 1 h at room temperature. Lastly, we analyzed the samples using a Beckman flow cytometer.We conducted an EdU assay following the manufacturer’s recommendations, utilizing the Click−iT EdU Pacific Blue flow cytometry assay kit . We cultured 1 × 10s/s hNPCs, with and without ASV treatment, were digested to obtain single cells. Subsequently, 1 × 106 cells were fixed in 70% ethanol at 4 °C overnight. After washing, the cells were incubated with binding buffer, RNase and propidium iodide (PI) at 37 °C for 30 min. Then, these cells were analyzed with Beckman flow cytometer.We performed cell cycle assays using a cell cycle detection kit , according to the manufacturer’s recommendations. METTL35 cells were incubated with binding buffer containing Annexin V-FITC and PI at room temperature for 5–15 min. Subsequently, we analyzed the samples utilizing the Beckman flow cytometer.We conducted apoptosis analysis utilizing an Annexin V−FITC/PI cell apoptosis detection kit , following the manufacturer’s instructions. Briefly, 2 × 10s/s hNPCs (treated with or without ASV) using TRIzol . DNA digestion was performed after RNA extraction using DNaseI. The quality of the RNA was evaluated by measuring the A260/A280 ratio using a Nanodrop One C Spectrophotometer . The integrity of the RNA was verified through 1.5% agarose gel electrophoresis. Quantification of the qualified RNAs were performed using Qubit3.0 with the Qubit RNA Broad Range Assay kit .Total RNAs were extracted from METTL3For RNA−seq, 2 μg of total RNAs were used for the preparation of a stranded RNA-seq library using a KC Stranded mRNA Library Prep Kit for Illumina , following the manufacturer’s instructions. PCR products with the range of 200 to 500 bps were enriched, quantified and subsequently sequenced using the Novaseq 6000 sequencer  with a PE150 model.2 was added to mRNAs and incubated at 95 ℃ for 5min to 10min, allowing the RNA fragments of 100–200 nt. Subsequently, 10% of the RNA fragments was saved as “input”, while the remainder was employed for m6A immunoprecipitation (IP). The specific anti-m6A antibody  was applied for m6A IP. RNA samples from both the “input” and “IP” were prepared using TRIzol. The stranded RNA sequencing (MeRIP−seq) library was constructed using the KC−Digital Stranded mRNA Library Prep Kit for Illumina , following the manufacturer’s instructions. The kit minimized duplication bias in the PCR and sequencing steps, by employing the unique molecular identifier (UMI) of 8 random bases to label the preamplified cDNA molecules. Library products in the range of 200–500 bps were enriched, quantified and ultimately sequenced on the Novaseq 6000 sequencer  with the PE150 model.For MeRIP−seq, 50 μg total RNAs were utilized for polyadenylated (polyA) RNA enrichment by VAHTS mRNA Capture Beads . 20mM ZnCl2 to the mRNA and incubated it at 95 °C for 5 min to 10 min, allowing the RNA fragments of 100–200 nt. Subsequently, we preserved 10% RNA fragments as “input”, allocating 40% for m6A IP , which we designated as “IP”. The remaining 40% underwent incubation with rabbit IgG as a negative control, labeled as “IgG”. We then processed the RNA samples from “input”, “IP” and “IgG” using RNAiso. The subsequent steps followed the same procedures outlined in the qRT−PCR protocol.For MeRIP−qPCR, we initiated the procedure by employing total RNAs for polyA RNA enrichment using the Dynabeads mRNA Purification Kit . Following this, we introduced 20 mM ZnCls/s hNPCs by SeqHealth . The cells were treated with cell lysis buffer. A 10% portion of the sample was preserved and labeled as “input”, while 80% was used in immunoprecipitation reactions with METTL3 antibody , designated as “IP”, and the remaining 10% was incubated with rabbit IgG  as a negative control, referred to as “IgG”. RNA from the input and IP samples was extracted using TRIzol reagent . The remaining steps were the same as those in the MeRIP-seq protocol.The RIP assay was conducted on METTL32, 10 mM Hepes (pH 7.0), 0.5% Nonidet P−40, 1 mM DTT, 2 μL protease inhibitor , and 0.5 μL RNase Out Recombinant Ribonuclease Inhibitor . The 10% lysis sample was stored as “input”, while 40% was employed in immunoprecipitation reactions with METTL3 antibody  and designated as “IP”, and another 40% was subjected to incubation with rabbit IgG  as a negative control, and referred to as “IgG”. The subsequent steps followed the same procedures outlined in the MeRIP−qPCR protocol.For RIP−qPCR, the cells were treated with cell lysis buffer containing 100 mM KCl, 5 mM MgClInitially, we employed cutadapt (V1.18) to eliminate adaptor sequences, low−quality bases and reads shorter than 50 bases. Subsequently, the processed clean data were aligned to the human reference genome (hg38) using HISAT2 (V2.1.0) with the parameters described in our previous study . Followi6A/METTL3 enrichment were identified using the Macs2 [Initially, RIP−seq data (METTL3 and MeRIP) were processed using the same filtering steps as RNA−seq. Afterward, the sequenced reads were subjected to base calling and demultiplexing using standard Illumina software (Illumina NovaSeq 6000), following quality control. Bowtie2  was emplhe Macs2 , with thhe Macs2 . Gene ont tests, and one−way ANOVA or two−way ANOVA. Statistically significant differences were defined as those with a p value < 0.05.Typically, the results were expressed as the mean ± SD (standard deviation), computed with Microsoft Excel and GraphPad Prism 10.0 , based on a minimum of three biological repeats. Significance levels among samples were assessed using unpaired


Life on Earth has evolved in the presence of a gravity constraint. Any change in the value of such a constraint has important physiological effects. Gravity reduction (microgravity) alters the performance of muscle, bone and, immune systems among others. Therefore, countermeasures to limit such deleterious effects of microgravity are needed considering future Lunar and Martian missions. Our study aims to demonstrate that the activation of mitochondrial Sirtuin 3 (SIRT3) can be exploited to reduce muscle damage and to maintain muscle differentiation following microgravity exposure. To this effect, we used a RCCS machine to simulate microgravity on ground on a muscle and cardiac cell line. During microgravity, cells were treated with a newly synthesized SIRT3 activator, called MC2791 and vitality, differentiation, ROS and, autophagy/mitophagy were measured. Our results indicate that SIRT3 activation reduces microgravity-induced cell death while maintaining the expression of muscle cell differentiation markers. In conclusion, our study demonstrates that SIRT3 activation could represent a targeted molecular strategy to reduce muscle tissue damage caused by microgravity. Microgravity is a singular condition in which normal gravity 1 g) is altered  g is alt. Random −3 g when vessels rotate at 10 rpm compared with normogravity conditions (1 g) [6) were injected into a 10 mL chamber and all air bubbles were carefully removed to prevent fluid shear stress. Subsequently, the effect of microgravity was achieved by rotation around the horizontal axis at 10 rpm for 24 and 48 h according to the treatments protocol described in this section and using as control the same conditions not subjected to rotation.To simulate microgravity conditions, a RCCS machine has been used. This device is characterized by a continuous rotation of the vessels that simulate microgravity on the principle that continuous rotation constantly changes cell orientation with respect to gravity reproducing the free fall occurring in Space or on International Space Station (ISS). RCCS is a rotating machine with bubble free vessels and a membrane for diffusion gas exchange and characterized by the absence of shear stress among spheroids and cellular aggregates and the culture medium. The clinostat used in these experiments was purchased by Synthecon Incorporated  and provides a vector-averaged reduction on gravity force acting on cells of 10ns (1 g) . Once di2 and 95% air. Differentiation into myotubes was achieved by culturing C2C12 myoblasts in complete medium and then switching into DMEM medium supplemented with 2% FBS for 72 h when cells were 90% confluent. H9C2 rat cardiomyoblast cells were purchased from ATCC and were grown in Dulbecco’s Modified Eagle’s Medium with high glucose. This culture medium was supplemented with 10% Fetal Bovine Serum, 2 mMol/L-glutamine, 100 units/mL penicillin and 0.1 mg/mL streptomycin. Adherent cells were detached by Trypsin-EDTA solution. H9C2 cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. H9C2 cells were then differentiated in low-serum (1% FBS), retinoic acid (RA)-supplemented (10 nM) DMEM for 7 days beginning 24 h after seeding [Mus musculus skeletal muscle cells (C2C12) were purchased from LGC Standards  and were grown in Dulbecco’s Modified Eagle’s Medium with high glucose  supplemented with 10% Fetal Bovine Serum , 2 mMol/L-glutamine , 100 units/mL penicillin and 0.1 mg/mL streptomycin . Adherent cells were detached by Trypsin-EDTA solution . Cell line was maintained at 37 °C in a humidified atmosphere of 5% COSIRT3 activator (MC2791), synthesized and validated as previously described , was disg for 10 min at 4 °C and pellets were resuspended in a solution containing 50 mMol/L Tris-Cl , 250 mMol/L sodium chloride , 0.1% Triton® X-100, 5 mMol/L ethylenediaminetetraacetic acid  and 0.1 mMol/L Dithiothreitol  plus 1 mMol/L phenylmethylsulfonyl fluoride , 1 mMol/L sodium orthovanadate  and 10 mMol/L sodium fluoride , Protease inhibitor cocktail . After 30 min on ice, samples were centrifuged at 14,000× g for 10 min at 4 °C and the supernatants collected. Protein concentration was determined by Bradford assay . Clarified protein lysates (50 μg) were boiled for 5 min, electrophoresed onto denaturating SDS-PAGE gel and transferred onto a 0.45 µm nitrocellulose membrane . The blotting membranes were blocked with 5% nonfat dry milk  for 1 h at room temperature (rt) and then incubated with primary antibody overnight at 4 °C. The following day, membranes were washed three times with 0.1% Tween® 20 in PBS (PBST) for 30 min rt and incubated with the appropriate secondary antibody for 1 h rt. After another three washes in PBST, the detection of the relevant protein was assessed by enhanced chemiluminescence . Densitometric analysis of the bands, relative to β-Actin, was determined by ImageJ Software . The following primary antibodies were used for Western Blot analysis: LC3 , β-ACTIN , SIRT1 , MyHβ (A4840) , MyHα (K13) , MHC , Pax-7 , SIRT3 (D22A3) , BNIP3 , YAP . Horseradish peroxidase-linked anti-mouse (NA931V) and anti-rabbit (NA934V) antibodies were purchased from GE Healthcare.Cells for whole cell lysate were centrifuged at 555× ®-Multi Detection System  and fluorescence intensity at 625/660-720 nm was graphed as shown in Cells were seeded onto RCCS vessels and incubated in microgravity conditions for 24 and 48 h with SIRT3 activator added every 8 h and with the other treatments described in this section. The same protocol was used for cells seeded into 100 mm dishes in normogravity conditions. After 24 and 48 h of treatment in normogravity and microgravity, cells were incubated with MitoSOX Red  dissolved in DMSO at the final concentration of 5 µMol/L for 10 min, according to manufacturer’s instructions. Subsequently cells were collected and washed twice in HBSS/Ca/Mg . Fluorescence was read by CytoFlex flow cytometer (Beckman/Coulter) and Median Fluorescence Intensity (MFI) has been considered for graphic analysis. ROS production in H9C2 was evaluated using Glomaxg for 5 min at 4 °C and, finally fixed with pre-cold 70% ethanol overnight at 4 °C. Samples were stained with 50 μg/mL Propidium Iodide  in PBS for 2 h at 4 °C cover light. Fluorescence was read by BD FACSDiva 8.0.2 flow cytometer . The sub-G1 fraction, which represents the total amount of apoptotic cells, was determined and analyzed through CellQuest™ software.To evaluate cell death, cells were analyzed by flow cytometry. Cells were seeded in normogravity conditions into 100 mm dishes and in microgravity conditions into RCCS vessels and treated as described above. Cells were subsequently harvested with trypsin-EDTA, washed twice with ice cold PBS, centrifuged at 800× 2) were plated in 100 mm dishes in culture medium with 10% FBS to perform the clonogenic assay. At the end of treatments, plates were washed twice with a phosphate buffered saline solution  and fixed with 4% formaldehyde solution in PBS  at rt. After 20 min, dishes were washed twice in PBS and stained for 5 min with 0.5% crystal violet . Finally, cells were washed with distilled water and air-dried. The colonies were counted the following day. In trypan blue exclusion assay, cells were harvested and stained with 0.4% trypan blue . The cell suspension was applied to a haemocytometer and counted with a phase contrast microscopy .Cell viability after normogravity and microgravity treatments was assessed with Clonogenic Assay and Trypan Blue Exclusion Assay. In the colony assay, at the end of treatments, cells (5 × 104) were seeded in 10% FBS onto coverslip placed inside 35 mm dishes. The day after cells were placed in 2% FBS conditions for 48 h during which they were either left untreated or treated with MC2791, NAC and NAC+MC2791. Alternatively, differentiated cells were first exposed to 24 h of microgravity in the presence or absence of indicated treatments and then counted and seeded onto coverslip placed inside 35 mm dishes in differentiating medium in normogravity for 48 h. Subsequently, cells were fixed with 4% formaldehyde solution in PBS for 10 min, washed three times with PBS and permeabilized for 5 min in 0.1 M Tris pH 7.4 and 0.2% Triton® X-100. After three washes in PBS, samples were incubated for 1 h at rt with 0.2 mg/mL BSA followed by 2 h at rt with anti-MHC antibody at 1:500 dilution in BSA . Cells were then washed three times with PBS and incubated for 1 h with the secondary antibody goat anti-rabbit IgG Alexa Fluor® 555 at 1:200 dilution in BSA . Samples were washed again for three times in PBS and nuclei were labeled with SYTO™ green  at 1:20,000 dilution in PBS. Finally, after three washes in PBS, samples were mounted using ProLong® Diamond Antifade Mountant  and analyzed with a LSM510 confocal microscopy . Fusion Index was measured manually according to Hinkle et al. [C2C12 cells  and a digital camera , we determined the dimensions of the cells in control conditions after treatment with DMSO, MC2791, NAC and NAC plus MC2791. Dimensions are expressed in μm.C2C12 cells were cultured in 10 mm dishes in normogravity conditions and in RCCS vessels in microgravity conditions and treated as described for 48 h. Cells were washed with warm PBS and fixed with 2.5% glutaraldehyde solution  in 0.1 M sodium cacodylate buffer pH 7.3  at 4 °C overnight. The following day, samples were collected, washed three times with cacodylate buffer and fixed for 2 h rt with 2% osmium tetroxide  using the same buffer. Subsequently, samples were dehydrated with two washes of 10 min each in several solutions of different ethanol concentrations  and finally embedded in Epon resin. Ultrathin sections obtained through a Reighert-Jung Ultra cut E ultramicrotome  were stained with uranyl-acetate replacement stain  and lead hydroxide. A Jeol-1400-plus TEM was used for observation and photographic analysis.t-test. The level of significance was set as * p < 0.05, ** p <0.01 and *** p < 0.001.Data are presented as the mean of standard deviation, determined from three or more experiments per condition. Differences between pairs of groups were analyzed by Student’s −3 g). Compared to normogravity, control cells in microgravity show a higher percentage of cell death , MyHα, MyHβ, YAP, and Pax-7.The MyHβ/MyHα ratio can be used to determine the level of differentiation of muscle cells with a higher ratio indicating a higher stemness state . Our resFurthermore, we also determined the expression of YAP, a transcriptional co-activator considered a stemness factor blocking the differentiation of skeletal muscle cells . As showIt is well known that SIRT3 is responsible of ROS reduction with protective role against oxidative stress-dependent disorders ,52. We aSeveral studies demonstrate that autophagy plays an important role in myoblast differentiation . In factUltrastructural analysis of C2C12 cells under microgravity revealed an increased number and electron density of mitochondria after MC2791 treatment  suggesti2O2 induced damage, etc. [In the present study, we propose SIRT3 activation as a potential mechanism to revert and limit the damage to skeletal muscle tissue due to microgravity exposure. As it is known, microgravity causes extensive damage to muscle tissue, bone tissue, immune system, circulation system, etc. In this complex scenario, our purpose was to identify a possible molecular mechanism that acts to interfere with microgravity-induced muscle cells damage, characterized by myocytes dedifferentiation, and able to restore cell homeostasis. Several works have shown that cells exposed to microgravity display changes in morphology, biochemical pathways and gene expression related not only to the gravity sensing but also to altered cytoskeleton architecture and unbalance with the microenvironment . Furtherge, etc. . Microgrge, etc. ,66,67, tge, etc. . Myoblasge, etc. . Our resge, etc. . Our obsge, etc. . Althougge, etc. ,72,73. Ige, etc. ,75. Autoge, etc. ,20. Ther


Matrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear.We investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16–20 weeks.en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs.DKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic We conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.The online version contains supplementary material available at 10.1186/s12933-023-02064-3. Cardiometabolic diseases (CMDs) represent a group of interrelated conditions that include visceral obesity, type 2 diabetes (T2D), dyslipidemia, hypertension, non-alcoholic fatty liver disease, and cardiovascular disease (CVD) . The risIn an unbiased bioinformatic approach combining published human data, microarray analyses of mouse white adipose tissue (WAT) and aorta, and a meta-analysis, we identified MMP12 as a massively upregulated pro-inflammatory factor in metabolic and vascular tissues in mice and humans suffering from CMDs . MMP12 bWe therefore investigated the consequences of genetic deletion of MMP12 on the Ldlr-deficient background (DKO) in mice fed a high-fat sucrose- and cholesterol-enriched (HFSC) diet that mirror human disease by simultaneously developing the complications associated with CMDs, such as WAT inflammation, insulin resistance, and cardiovascular dysfunction . We demoMmp12KO were crossed with LdlrKO mice  to generate LdlrMmp12-DKO mice. Age- and sex-matched LdlrKO and DKO mice were housed in a clean and temperature controlled-environment  with unlimited access to food and water on a regular 12-h/12-h light–dark cycle. At 6 weeks of age, female and male mice were fed a HFSC diet , 28 kcal% carbohydrates ; D09071704, Research Diets Inc, New Brunswick, NJ) for 20 and 16 weeks, respectively. Body weight was monitored weekly during dietary treatment. Female mice were used for atherosclerosis, whereas male mice were used for metabolic studies. All experiments were performed in accordance with the European Directive 2010/63/EU and approved by the Austrian Federal Ministry of Education, Science and Research .Blood was collected by facial vein puncture from 12-h fasted mice. Lipoprotein fractions were separated using 200 µl of pooled plasma per genotype. Plasma lipid parameters (triglycerides (TG), total cholesterol (TC), free cholesterol (FC), cholesteryl esters (CE)) and lipoprotein profiles were determined as previously described .Blood glucose concentrations were measured in 12-h fasted mice using Accu-Chek® Active glucometer and glucose strips For the GTT, mice were fasted for 6 h and then gavaged with 2 g glucose/kg body weight. For the ITT, mice were fasted for 5 h and then injected i.p. with 0.5 IU insulin/kg body weight . In both tests, blood glucose concentrations were determined 0, 15, 30, 60, 90, and 120 min after glucose or insulin administration using Accu-Chek® Active glucometer and glucose strips .−ΔΔCT method. Primer sequences are listed in Additional file Total RNA was isolated using TRISure™ reagent  according to the manufacturer’s protocol. Two µg of total RNA were reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit . Quantitative real-time PCR was performed on a CFX96 Real-Time PCR detection system  using the GoTaq® qPCR Master Mix . Samples were normalized to RPLP0 mRNA expression as reference gene. Expression profiles and associated statistical parameters were determined by the 2g and 4 °C for 30 min, the middle fat-free layer was isolated, and protein concentrations were estimated . Fifty µg of protein were separated by SDS-PAGE and transferred to a PVDF membrane. The blot was incubated with rabbit polyclonal anti-BAX  and anti-calnexin (CNX) antibodies  . HRP-conjugated goat anti-rabbit antibody  was visualized by enhanced chemiluminescence detection on a ChemiDoc™ MP imaging system . BAX was quantified by densitometry  and normalized to the expression of CNX.One hundred mg of epididymal WAT (eWAT) were lysed in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (PIC) . Subsequently, the samples were centrifuged at 16,000 x eWAT samples were fixed in 4% neutral-buffered formalin for 24 h and subsequently embedded in paraffin. Section (7 μm) were deparaffinized and stained with hematoxylin and eosin (H&E) as described elsewhere . ImmunohTo quantify F4/80-positive regions, images were analyzed using Visiopharm (version 2021.09). Tissue regions were manually labeled, and color deconvolution was performed for F4/80 staining, resulting in an image of staining intensity to which a threshold for F4/80 positivity was applied. The cumulative F4/80 staining intensities were used to calculate the mean F4/80 intensity on the tissue regions within one image.x g and 4 °C for 30 min. Thereafter, proteins were precipitated with four times the volume of acetone per sample and incubated for 15 min at -80 °C and 120 min at -20 °C. Proteins were pelleted by centrifugation at 16,000 x g and 4 °C for 15 min, then air-dried and resuspended in buffered urea . The protein content in eWAT and the aorta was determined for all tissues using a Lowry assay. A volume corresponding to 10 µg of proteins from aorta and 50 µg of proteins from eWAT was completely dried in a vacuum concentrator at 45 °C for 45 min. The dried protein pellet was resuspended in 10 µl of water with the addition of 10 µl of ammonium bicarbonate , followed by protein reduction with 100 mM DTT for 30 min at 55 °C. Proteins were then alkylated at RT by incubation with 150 mM iodoacetamide in 50 mM ammonium bicarbonate solution for 30 min in the dark. Trypsin digestion  with a 1:20 enzyme to protein ratio was performed overnight at 37 °C and terminated by acidification with 1% trifluoroacetic acid. LC-MS/MS analyses were performed as previously described [Aortic arch and descending thoracic segments from LdlrKO and DKO mice (n = 6/group) were pooled up to 20 mg and homogenized in in the presence of RIPA buffer  supplemented with protease inhibitors at a ratio of 1:100  as previously described . eWAT frescribed . In brieData processing and analysis were performed as previously described , 41. Brihttps://huygens.science.uva.nl/VolcaNoseR2/) [Volcano plots were generated using VolcaNoseR (NoseR2/) . To labePlasma MCP-1 was analyzed using a commercially available ELISA kit following the manufacturer’s instructions . Samples were diluted 1:4.Peripheral blood was collected by facial vein puncture into EDTA-coated collection tubes. Samples were analyzed using a fully automated hematology analyzer . For flow cytometry, 50 µl of blood were treated with ACK buffer to lyse red blood cells, and Fc receptors were blocked with anti-mouse CD16/CD32 . Subsequently, samples were stained with CD45-FITC, CD11b-AlexaFluor647, and Ly6G-APC-Cy7 antibodies (BD Biosciences) and analyzed using a Cytoflex LX . Data were acquired using CytExpert software (Beckman Coulter Life Sciences) and the analysis was performed using FlowJo .Relaxation to cumulatively increasing concentrations of acetylcholine (Ach) and the nitric oxide (NO) donor sodium nitroprusside (SNP) was recorded in vessels preconstricted to 80% of the maximal KCl (60 mmol/l)-induced contraction using U-46,619  as a stable analog of thromboxane A2 as described . Relaxaten face analysis. The upper two-thirds of the heart were fixed in formalin for 24 h and stored in 30% sucrose until aortic valves sectioning. Staining, imagining, and quantification of the aortas and aortic valve sections were performed as previously described [Mice were anesthetized by i.v. injection of Na-pentobarbital (200 mg/kg body weight), perfused with PBS/EDTA for 15 min, and then with 10% neutral buffered formalin for 15 min. The thoracic part of the aorta was excised and stored in formalin until oil red O (ORO) staining was performed to quantify the atherosclerotic lesions by escribed .4 for 2 h at RT. After dehydration in graded series of ethanol, tissues were infiltrated  and placed in TAAB epoxy resin (8 h), transferred into embedding moulds, and polymerized . Ultrathin Sect. (70 nm) were cut with a UC 7 Ultramicrotome  and stained with lead citrate for 5 min and platin blue for 15 min. Electron micrographs were taken at 120 kV using a Tecnai G2 transmission electron microscope  with a Gatan ultrascan 1000 CCD camera .Aortic tissue was fixed in 2.5% (wt/vol) glutaraldehyde and 2% (wt/vol) paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h and post-fixed in 2% (wt/vol) OsOImmediately after isolation, aortic specimens were frozen in Ringer’s solution containing 10% DMSO. For the extension-inflation test, the samples were slowly thawed overnight and a segment with a length of 5 mm that was straight as possible. Two disposable blunt cannulas (Sterican 0.40 × 25 mm) were then inserted into both ends of the aortic sample with tweezers and fixed by knotting with surgical sutures. Cyanoacrylate adhesive glue  was used to achieve a stronger fixation between the samples and the cannulas. Two black dots, each one-third the length of the sample from the cannulas, served as markers for the length measurements with the video extensometer (VE). The specimen (with the cannulas) was then inserted load-free into the extension-inflation test setup as previously described .The preconditioning ensured a reproducible behavior of the aorta under cyclic loading. In this procedure, the aortas were first stretched axially to 1.3 and then subjected to a pressure of 100 mmHg for 10 min. The aortas were then subjected to three pressure cycles from 0 to 100 mmHg. After preconditioning, the unloaded length L and the unloaded outer diameter D of the aorta were measured with the VE.The aortas were axially stretched in increments of 0.1 from 1.2 to 1.7, while the aorta was pressurized five times at 70, 90, and 110 mmHg at each axial stretch step. Extension-inflation tests were performed with continuous recording of axial force, transmural pressures, outer diameter, and axial gauge length. The axial inversion stretch (IVS), where axial force is independent of inflation pressure, is assumed to be consistent with the in vivo axial stretch of the aorta and was therefore determined for each sample. To determine the IVS, we used the relationships between axial force and length as well as between pressure and axial stretch, similar to methods described by others –51. For Serum samples were obtained from a cross-sectional study involving a total of 128 individuals  aged 45 to 65 years. These samples and all corresponding biochemical parameters of the patients were collected as part of a previous study . Serum Mp < 0.05, **p ≤ 0.01, ***p ≤ 0.001.Statistical analyses were performed using GraphPad Prism 9.3.1 software. Data are presented as mean ± SD or SEM (for myography) and differences between two groups were assessed using the unpaired t test. For the human samples, Mann-Whitney-U test was used instead because of skewed distributions. Correlations were calculated using Spearman’s method. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic ability of MMP12. The following levels of statistical significance were used: *We first investigated whether constitutive deletion of MMP12 affected body or tissue weights upon a HFSC diet feeding for 16 weeks. DKO mice had slightly reduced body weight particularly before the dietary treatment, while tissue weights remained unchanged , ELAVL1, PADI1, BAX, PSAP, COTL1, NCK1, NPM1, USP14, and ACSL1 , HPX, PCLO, AFDN, CES1D, MYDGF, PLIN1, CAVIN1, AOC3, PPM1A, CREG1, FGFR1, SOD3, LRP6, and MFN2  in eWAT from LdlrKO but not from DKO mice Fig. A-B. In lsed Fig. , 274 werSL1 Fig.  as well FN2 Fig.  were assFN2 Fig. D. The ca+ cells to exclude non-hematopoietic cells, we separated CD45+ cells based on their CD11b and Ly6G expression. We detected a lower number of CD11b+Ly6G− cells in the peripheral blood of DKO mice  population. Concentrations of monocyte chemoattractant protein 1 (MCP-1) were reduced in the plasma of DKO mice Fig. A. In linice Fig. C-E. ThisSystemic inflammation and immune cell dysregulation also play a central role in the development of CVD, including atherosclerosis. Since endothelial dysfunction represents the first step in atherogenesis, we investigated whether genetic loss of MMP12 impacts vasorelaxation by myography of U-46,619 preconstricted aortic rings in response to cumulatively increasing concentrations of Ach and SNP, which mediate endothelium and direct smooth muscle cell relaxation, respectively. We observed an improved relaxation pattern in aortic rings from DKO mice after exposure to Ach , COL11A1, C2, ACTR10, COL1A1, TFRC, XDH, CNN1, DIAPH1, IDH1, CYB5R3, and LMOD1  included DBN1, LONP1, SWAP70, MBL2, CSAD, LRP8, CTSB, CYB5A, DDX3X, PARK7, ITIH4, CALR, ARPC1B, FN1, and ANXA11 associated with the selected tags     Fig. . Accordi07) Fig. . Since fTo determine the relevance of MMP12 in human disease, we measured its levels in serum from patients with metabolic syndrome (MS), who had 41% higher concentrations of circulating MMP12 compared to healthy volunteers (HV) Fig. A. ROC cuMMP12 is a macrophage-secreted protein that has been previously associated with pro-inflammatory functions in a variety of pathological conditions in humans and in animal models , 23, 54.DKO mice displayed improved glucose tolerance and insulin sensitivity despite comparable body and tissue weights to control animals. This observation is consistent with a previous study showing that Mmp12KO mice are more insulin sensitive than wild type mice after 10 weeks of high-fat diet feeding . HoweverProteomic profiling of eWAT and aortas as key tissues involved in the onset of CMDs helped to identify proteins and signaling pathways affected under genetic Mmp12-deficient conditions. In eWAT from DKO mice, we detected protein upregulation of a plethora of local protective factors  against metabolic dysregulation, inflammation, reprogramming of the diabetic gene signature in WAT and other tissues including non-alcoholic fatty liver disease, impairment of brown adipose tissue thermogenesis, and in atherosclerosis –66. SeveSeveral studies have shown that obesity and prolonged high-fat diet feeding enhance systemic inflammation, hematopoiesis, and lymphopoiesis –77. The Published data from humans, rabbits, or other mouse models have already described detrimental effects of MMP12 on atherosclerosis development , 30, 93.Taken together, our data in mice suggest that MMP12 is an attractive target for controlling and even ameliorating CMDs. MMP12-selective inhibitors have already been successfully tested in mouse models of allergic asthma, progression of osteoarthritis, and atherosclerosis , 94, 95.In the present study, circulating MMP12 was only weakly associated with indicators of CMDs in humans. Of note, serum MMP12 levels partially reflect MMP12 bioavailability in tissues, where MMP12 exerts its biological functions. Despite the poor capacity of circulating MMP12 concentrations for serving as a marker for CMDs, it does not exclude its (patho)physiological role in humans.We conclude that the genetic deletion of MMP12 in a mouse model mimicking CMDs in humans leads to altered immune cell composition and improvements in systemic inflammation, insulin sensitivity, and atherosclerotic development, supported by beneficial changes in the proteome signature of WAT and aortas. In humans, circulating MMP12 levels are weakly linked to MS and indicators of CMDs. Further studies are needed to establish the association between tissue MMP12 bioavailability and CMDs.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


In twenty-one healthy participants, CVR responses to orthostatic stress  were assessed using two methods: (1) the traditional CO2 inhalation method, and (2) transfer function analysis (TFA) between middle cerebral artery blood velocity (MCA V) and predicted arterial partial pressure of CO2 (PaCO2) during spontaneous respiration. During HUT, MCA V steady-state  and MCA V onset  responses to CO2 inhalation were decreased (p < 0.001) and increased (p = 0.001), respectively, indicative of attenuated CVR. In contrast, TFA gain in the very low-frequency range  was unchanged, while the TFA phase in the VLF approached zero during HUT , indicative of a shorter time  response of CVR. These findings indicate that CVR metrics determined by TFA without a CO2 inhalation do not track HUT-evoked reductions in CVR identified using CO2 inhalation, suggesting that enhanced cerebral blood flow response to a change in CO2 using CO2 inhalation is necessary to assess CVR adequately.The present study aimed to examine the validity of a novel method to assess cerebrovascular carbon dioxide (CO This mechanism of cerebral blood flow (CBF) regulation is termed cerebrovascular CO2 reactivity (CVR) and functions alongside the central respiratory chemoreflex to help maintain brain pH homeostasis , baseline value, gain term (G), and time constant (τ): MCA Vmean = G × {1 − exp[− (t − t0)/τ]} + MCA Vmean 0, where t is time, and MCA Vmean 0 is a baseline value. Time 0 reflects the start of CO2 inhalation  (mmHg) and beat-to-beat (MCA Vmean) (cm/s) data without (5 min steady-state data) CO2 inhalation were resampled at 4 Hz for spectral analysis /[Sxx(f)y], and the normalized gain was calculated as 20 log H′(f) to provide values in decibels. The TFA coherence, phase, and normalized gain were calculated in the very low , low , and high frequency  ranges, respectively. This frequency range was determined by previous studies [2 are more prominent in the VLF range [2 and MCA Vmean as an index of dynamic CVR gradually decreased with an increase in frequency over a cut-off frequency (0.024 Hz) [2 fraction) from the PETCO2 to mean MCA V or minute ventilation, respectively. The authors demonstrated that the dynamic characteristics of the relationship between CO2 inhalation-induced binary white noise of PETCO2 and MCA V were maintained below a cut-off frequency of 0.024 Hz but gradually decreased with an increase in frequency over 0.024 Hz. In the present study, therefore, we focused on the TFA parameters within the VLF range below 0.024 Hz to examine the dynamic CVR during spontaneous respiration.TFA-determined CVR (TFA gain and phase): TFA analysis between predicted PaCOanalysis . The tim studies ,39. AddiLF range ,39 and Tn = 6) were used to perform a prospective power analysis. The critical sample size was estimated to be 19 participants to detect differences in τ assessed by MCA Vmean between supine and 50° HUT conditions, with an assumed type 1 error of 0.05 and statistical power of 80%. Thus, the sample size of the present study was sufficient to achieve the desired statistical assurance. A linear mixed-effects model with fixed-effects of “Condition (supine vs. 50° HUT)” and/or “Time ” was used to compare steady-state data. Before the analysis for step-change in CO2- and TFA-determined CVR during supine and 50° HUT without (baseline) or with CO2 inhalation, the Shapiro-Wilk’s test was applied to verify the normal distribution for each variable. A normal distribution was confirmed in all CVR indices . To compare normally distributed outcomes between supine and 50° HUT, and between without and with CO2 inhalation, we incorporated paired samples t-tests. The Wilcoxon matched-pairs signed-ranks test was employed where appropriate as a non-parametric equivalent. All data were analyzed using SPSS (IBM SPSS Statistics Version 27.0) and expressed as mean ± standard deviation (SD). Statistical significance was set at p < 0.05.Data from our pilot study , predicted PaCO2 (p = 0.007), and PETCO2 decreased (p = 0.009). MAP, RR, VE, and Vt were unchanged during HUT .As expected, 50° HUT caused an increase in HR  to 50° HUT  during HUT, and it was not matched with the time response of CVR determined from the step-change in CO2. Furthermore, LF and HF TFA phases without CO2 inhalation were not different between supine and 50° HUT conditions , that may be advantageous for patients with respiratory disease or the elderly, etc. As expected, we observed that CVR, determined with the traditional method, using a CO2 inhalation challenge was impaired during 50° HUT. However, TFA gain and phase did not represent a similar trend to these traditional CVR indexes during 50° HUT. These findings suggest that TFA (gain and phase) without CO2 inhalation under spontaneous respiration does not adequately assess CVR.In the present study, we assessed whether CVR can be reliably determined using TFA between the predicted PaCOτ) determined by CO2 inhalation, the traditional index of CVR, were attenuated during 50° HUT  without CO2 inhalation was not different between the supine and 50° HUT conditions  as a traditional CVR index. On the other hand, the value of the VLF TFA phase as a dynamic time response was increased but approached zero from supine during 50° HUT to reach a positive value without CO2 inhalation  calculated from the MCA Vmean onset response to step-change in CO2  as a traditional CVR index. Collectively, these findings suggest that the TFA phase and gain without CO2 inhalation may not be adequate to assess changes in CVR as the traditional index during HUT. As in previous studies ,39, the nditions . This fi2 stimuli provide a more sensitive means of identifying reductions in the vasodilatory reserve. This suggests that without a sufficiently large stimulus, such as CO2 inhalation, the CBF response to CO2 may be underestimated. BOLD imaging has the benefit that it gives semi-quantitative information on CVR, and it provides high spatial resolution, which allows the identification of smaller regions of impaired reactivity. However, the BOLD signal itself provides an indirect rather than a direct measure of cerebral perfusion, as provided by newer MRI techniques such as arterial spin labeling. In addition, MRI measurement is expensive and many previous studies used CBF velocity measured by the Doppler method to assess CVR [2 exposures can identify CVR using the Doppler method and TFA technique to minimize the potentially confounding effects of CO2 inhalation. The main findings of our study are supported by a previous study  using blsess CVR ,22,23,28sess CVR ,42; thersess CVR . Thus, fETCO2, as well as arterial blood pressure, can contribute significantly to the observed CBF velocity variability. In addition, Mitsis et al. [ETCO2 fluctuations, as well as nonlinear interactions between arterial pressure and CO2, have a considerable effect in the lower frequencies (below 0.04 Hz). These findings indicate that the CBF response to a change in CO2 is slower than that of a response to a change in arterial pressure. However, the independent contribution of change in PETCO2 on cerebrovasculature in the dynamic analysis below 0.04 Hz is unclear because this previous study used a multiple input model. More recently, Ogoh et al. [2 inhalation-induced binary white noise of PETCO2, and MCA V gradually decreased with an increase in frequency over a cut-off frequency (0.024 Hz). This finding suggests that the reflection of the dynamic relationship between PETCO2 and MCA V to TFA data reduces in the range of frequency above 0.024 Hz. Indeed, the coherence was highest in the VLF range and gradually decreased above 0.024 Hz  and MCA V. Taken together, CO2 inhalation, which enhanced MCA V response to changes in PETCO2, appears necessary to assess CVR, even using TFA. Previous studies ,38,43 pr0.024 Hz . Thus, t2 inhalation [2 [2 inhalation did not increase TFA coherence, further studies using techniques to increase TFA coherence  could modify blood velocity measured using transcranial Doppler independently of flow. MCA diameter is changed during severe COhalation ,45. Howeation [2  and duriation [2 . Second,ion rate ) may be 2 inhalation challenge to increase the CBF response range to accurately identify CVR, but the changes produced in PaCO2 are much larger than the spontaneous changes in PaCO2 that occur in everyday life. Along with increasing ventilation via a central chemoreflex [2 inhalation causes activation of sympathetic nerve activity [2 inhalation can cause acute stress [2 inhalation to assess CVR is that it may affect ventilation via a central chemoreflex [2, and thus CBF, via the closed-loop feedback system [2 inhalation, is attenuated in patients with respiratory disease, e.g., chronic obstructive pulmonary disease (COPD) [2 inhalation modifies the CBF regulatory system, as well as cerebral vasculature, and, consequently, it may alter the CBF response to change in PaCO2 per se. Therefore, determining CVR without CO2 inhalation may be better to assess CVR accurately, especially in patients with respiratory disease. Unfortunately, the findings of the present study indicate that the TFA phase and gain cannot be used to assess CVR during spontaneous respiration without CO2 inhalation. As such, we may need to establish other methods which do not affect respiratory function, such as spontaneous respiration with low concentration CO2 inhalation, to accurately identify CVR.The traditional methods for determining CVR employ a COmoreflex ,3,28, COactivity ,49. Moree stress  and anxie stress , which amoreflex ,3,28, ank system ,2. Thus,e (COPD) ,51,52,53e (COPD) ,55,56 de2 inhalation using TFA and assessed whether these TFA data without CO2 inhalation are representative of CVR identified using the time domain CO2 challenge method (the magnitude and time responses of CVR). These findings indicate that CVR metrics determined by TFA without a CO2 inhalation do not track HUT-evoked reductions in CVR identified using CO2 inhalation, suggesting that CO2 inhalation which enhances CBF response to changes in CO2 is necessary to assess CVR adequately. Future research is needed to investigate whether low-concentration CO2 inhalation can be used to identify cerebral CO2 reactivity in those for whom pronounced hypercapnia is unsuitable.The present study, for the first time, identified CVR during spontaneous respiration without CO


Liver cancer is a prevalent form of cancer worldwide. While research has shown that increasing sphingomyelin (SM) hydrolysis by activating the cell surface membrane-associated neutral sphingomyelinase 2 (nSMase2) can control cell proliferation and apoptosis, the role of total glutathione depletion in inducing tumor cell apoptosis via nSMase2 activation is still under investigation. Conversely, glutathione-mediated inhibition of reactive oxygen species (ROS) accumulation is necessary for the enzymatic activity of nSMase1 and nSMase3, increased ceramide levels, and cell apoptosis. This study evaluated the effects of depleting total glutathione in HepG2 cells using buthionine sulfoximine (BSO). The study assessed nSMases RNA levels and activities, intracellular ceramide levels, and cell proliferation using RT-qPCR, Amplex red neutral sphingomyelinase fluorescence assay, and colorimetric assays, respectively. The results indicated a lack of nSMase2 mRNA expression in treated and untreated HepG2 cells. Depletion of total glutathione resulted in a significant increase in mRNA levels but a dramatic reduction in the enzymatic activity of nSMase1 and nSMase3, a rise in ROS levels, a decrease in intracellular levels of ceramide, and an increase in cell proliferation. These findings suggest that total glutathione depletion may exacerbate liver cancer (HCC) and not support using total glutathione-depleting agents in HCC management. It is important to note that these results are limited to HepG2 cells, and further studies are necessary to determine if these effects will also occur in other cell lines. Additional research is necessary to explore the role of total glutathione depletion in inducing tumor cell apoptosis. The incidence of hepatocellular carcinoma (HCC) dramatically increased over the past two decades and is presently the fourth most common cause of cancer-related death worldwide ,2. MultinSMase2 gene hypermethylation and loss of expression were detected in the majority of the examined primary HCC [++-dependent nSMases, notably nSMase1 (SMPD2) and nSMase3 (SMPD4), also catalyze the cleavage of the phosphodiester bond in SM to ceramide and phosphocholine, but unlike nSMase2, are not associated with the cell surface membrane, are located in the endoplasmic reticulum, Golgi apparatus, and nuclear matrix of multiple tissues [Evidence is now mounting for the role of cell surface membrane sphingomyelin (SM) in tumor initiation, immune escape, growth, and metastasis . Notablymary HCC . The HCCmary HCC ,12. Incrmary HCC . Other M tissues ,15,16. C tissues  or consi tissues  in HCC cThe nSMase1 enzymatic activity requires reducing agents and is reversibly inhibited by reactive oxygen species (ROS) and oxidized glutathione (GSSG) but irreversibly inhibited by reactive nitrogen species such as peroxynitrite ,20. ConvTo resolve this controversy, we investigated the specific role of total glutathione in HepG2 cells survival via its depletion with its biosynthesis inhibitor, buthionine sulfoximine (BSO)  Figure , and ass2DCFDA dye (5935), were purchased from Tocris Bioscience . DMEM  was purchased from Lonza-Bioscience . QiAzol lysis buffer (79306) and RNAse/DNAse free water (129114) were purchased from Qiagen . Ethylenediamine tetraacetic acid (39760.01) and Tris-HCl (37192.01) were purchased from Serva . L-BSO 97% TLC grade (B2515), quantification kit for oxidized and reduced glutathione (38185), cambinol ≥97% (HPLC) (C0494), myriocin ≥98% (HPLC) (M1177), hydrochloric acid (szbe1990v) was purchased from Merk . The ceramide kit (MBS7254089) was purchased from MyBioSource, . 5-sulphosalicylic acid dihydrate extra pure (S-08755) was purchased from Oxford Lab Fine Chem LLP . Protease inhibitor cocktail (100×) (5871) was purchased from Cell Signaling Technology .The human hepatoma HepG2 cell line was obtained from ATCC (HB-8065),  in Dulbecco’s Modified Eagle Medium, high glucose DMEM supplemented with 10% fetal bovine serum was purchased from Gibco™ . Amplex red sphingomyelinase assay kit (A12220), dimethyl sulfoxide (DMSO) (67-68-5), chloroform , isopropanol , ethanol , revertaid cDNA kit (K1621), maxima SYBR green qPCR (K0251), mRNA primers (designed by NCBI primer blast tool), BCA assay kit , were all purchased from Thermo Fisher Scientific . Penicillin-streptomycin mixture, 100× (09-757F), and phosphate-buffered saline (10×) (PBS) (17-516Q), trypsin (CC-5012), 3--2, 5- diphenyltetrazolium bromide , H2 flasks, using DMEM containing 10% FBS and 1% penicillin-streptomycin mixture (complete medium) until 80% confluence at 37 °C and 5% CO2 humidified incubator. The old medium was discarded, and the cells were washed with 5 mL of 1× PBS. Then, the PBS was discarded, and 4 mL of 0.025% trypsin in PBS was added. The flask was shaken for 1 min, trypsin was discarded, and the cells were incubated for 4 min in a 37 °C and 5% CO2 humidified incubator. In the flask, 4 mL of DMEM/10% FBS were added and pipetted up and down for 1 min to ensure that all the cells were evenly suspended before seeding in different plate formats based on the experiment performed.HepG2 cells were grown in 75 cm2 tissue culture flasks or 96-well plates (5 × 103 HepG2 cells/well) and left to incubate at 37 °C and 5% CO2 humidified atmosphere for 24 h. Cells were then exposed for 24 h to BSO at 0.00, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 µM in a complete medium. The media were discarded, the cells were washed with PBS, which was discarded, and cells were collected via trypsin treatment and washed with PBS. The supernatant was discarded, and 80 μL of 10 mM HCl was added. Cells were lysed by freezing and thawing twice, then 20 μL of 5% sulfosalicylic acid dihydrate (SSA) was added. The mixture was centrifuged at 8000× g for 10 min. Finally, the supernatant was transferred to a new tube, and deionized water was added to reduce the SSA concentration to 0.5% for the assay. Ten μL of the cell lysates were added to each well in a 96-well plate; then the assay was performed according to the manufacturer’s instructions. Briefly, 30 μL of 5% SSA and 120 μL buffer were added to each well. After one h incubation period at 37 °C, 20 μL each of substrate, coenzyme, and enzyme working solutions were added to each well. Finally, the plate was incubated for 10 min at 37 °C and 5% CO2. The absorbance was read immediately at 405 nm. The total glutathione content was calculated using a standard curve according to the manufacturer’s instructions [HepG2 cells were seeded in 75 cmructions ,30. 2DCFDA for two h (in 100 μL medium) before the addition of 0.1, 0.5, and 1 μM BSO (in another 100 μL), a total of 200 μL. Pilot studies indicated a lack of need for higher BSO concentrations to achieve highly significant (p < 0.0001) increases in ROS levels. The plate was incubated for 24 h in the dark at 37 °C and 5% CO2. Then, fluorescence was measured at excitation and emission wavelengths of 485 and 535 nm, respectively. The change in ROS was calculated as a percentage of the control where the control is set at 100%. The ROS level was calculated using the following equation: ROS level (%) = (A sample − A blank/A control − A blank) × 100.To determine the degree of ROS generation induced by the BSO treatment on HepG2 cells, a fluorometric assay was used, utilizing the unique intracellular oxidation of 2′,7′-dichlorofluorescein diacetate (DCF-DA) ,32. Cellg at 4 °C. The precipitated pellet was washed with 70% ethanol, vortexed briefly, and then centrifuged at 7500× g at 4 °C for 5 min. The supernatant was discarded, and the micro-centrifuge tubes were left to air dry for 5–10 min. Finally, the pellet was resuspended in 25 µL nuclease-free water [After the 6 h or 24 h incubation period with BSO, total RNA was extracted using QIAzol lysis reagent according to the manufacturer’s instructions. The aqueous phase containing RNA was transferred to a new micro-centrifuge tube. After that, 150 µL isopropanol were added, incubated for 10 min, and then centrifuged for another 10 min at 12,000× ee water . The RNAThe RNA first-strand complementary DNA (cDNA) was synthesized using the Revertaid cDNA synthesis kit according to the manufacturer’s guidelines. Briefly, 1 μg of the total RNA of each sample was diluted with nuclease-free water up to 10 µL. Then 10 µL from the cDNA reaction was added to each sample for a final volume reaction of 20 µL. The cDNA reaction master mix is composed of 4 μL 5× reverse transcription (RT) buffer, 2 μL 10 mM dNTP mix (100 mM), 1 μL RT random Hexamer primers, and 1 μL Oligo (dt)18 primer, 1 μL RevertAid M-MuLV RT (200 U/μL) reverse transcriptase, 1 μL RiboLock RNase Inhibitor (20 U/µL). The final reaction mixture was subjected to the following thermal cycle conditions: 25 °C for 5 min, followed by 42 °C for 60 min, and then 70 °C for 5 min to terminate the reaction, and finally cooled to 4 °C in 96 well Thermal cycler . The completed reaction was stored at −80 °C until further analysis. Before real-time polymerase chain reaction (qPCR) quantification, the newly synthesized cDNA was diluted 1:3 with nuclease-free water by adding 40 µL nuclease-free water to the 20 µL cDNA, from which 3 µL were taken for the real-time polymerase chain reaction (PCR) .https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 27 March 2023) and synthesized at Thermo Fischer (−Δ(ΔCt), where ΔCt = Ct(target) − Ct(GAPDH) and Δ(ΔCt) = ΔCt(treated) − ΔCt(untreated).The primers used for real-time PCR were generated using the online NCBI primer designing tool  = (A sample − A blank/A control − A blank) × 100. The change in nSMase activity was calculated as a percentage of the control where the control is set at 100% .Cell lysate samples were diluted 1:1 with PBS. To the enzyme-linked immunosorbent assay strip wells (MyBioSource) coated with polyclonal anti-ceramide antibody, 100 μL of each sample was added, ten μL balance solution was dispensed, and 50 μL of the conjugate was added to each well. Then, the plate was covered and incubated for one h at 37 °C. After incubation, the plate was washed five times with the 1× wash solution. The plate was tap-dried, and 50 μL of substrate A and B were added to each well, followed by a 15 min incubation period at 37 °C. After the incubation period, 50 μL of stop solution was added, and the absorbance was measured at 450 nm. The ceramide content was calculated according to a standard curve .After BSO treatment and washing, HepG2 cells were incubated with the MTT solution. After that, 200 µL 99.5% isopropyl alcohol was added per well. After one h shaking, the absorbance was measured at 540 nm, and cell proliferative capacity was calculated according to the following equation: Cell proliferation (%) = (A sample − A blank/A control − A blank) × 100 ,39.t-test was used to identify the statistical significance between multiple groups. A p-value < 0.05 was considered statistically significant. The comparative analysis of results between different experimental groups respective to their corresponding controls was conducted using Graph Pad Prism . Data are presented as means ± SE for the indicated number of independently performed experiments. Two-way Analysis of Variance (ANOVA) and Student p < 0.0001) reduced the total glutathione content by 53, 88.1 and 89.5%, respectively, while BSO at 0.1, and 0.5 µM non significantly decreased total glutathione content by 22.6 and 21.2%, respectively, compared to vehicle treated control cells  increasing ROS production in HepG2 cells by 663.5, 682.6, and 721.7%, respectively, compared to 0 μM BSO-treated cells, hereafter referred to as control cells. A slight decrease in total glutathione content induced by BSO concentrations of 0.1 and 0.5 μM translated in such a ROS burst, indicating that cell glutathione thoroughly performs its anti-oxidative task provided its intracellular content is not impaired, as in aging [The effect of different BSO concentrations on ROS production in HepG2 cells is shown in in aging .p < 0.005) increased nSMase1 mRNA levels by 134.5, 145.7, 222.4, 552.9, and 1502.8% respectively, compared to control cells (p < 0.05) increased in response to BSO at 0.1, 0.5, 1, 5, and 10 μM by115.3, 237, 324.1, 596.9 and 657%, respectively, compared to control cells  in HepG2 cells before or after BSO treatment. nSMase1 RNA level was ten times higher than nSMase3 A. Furtheol cells B. Similaol cells C.p < 0.05) nSMase activity by 18.75, 25, and 38.5%, respectively, compared to control cells lysate. In contrast, BSO at 0.1 and 0.5 µM decreased nSMase activity by 10.4 and 11.4%; respectively; however, this did not reach statistical significance. Treatment with 10 µM myriocin significantly decreased nSMase activity in HepG2 cell lysate by 40% compared to control. In contrast, 30 µM cambinol showed an insignificant (9%) decrease in HepG2 cell lysate nSMase activity compared to control cell lysate  reduced the ceramide content by 9, 11, and 12%, respectively, compared to control cell lysate, while BSO at 0.1 and 0.5 µM reduced the ceramide content by 2 and 7%, respectively. Treatment with 10 µM myriocin elicited a significant (p < 0.05) reduction of the ceramide content by 15% compared to control cell lysate. In contrast, treatment with 30 µM cambinol showed only a 7% non-significant decrease in ceramide content compared to the control cell lysate.p < 0.05) increased cell proliferation when compared to the cells still containing significant amounts of glutathione (tathione .Gamma-glutamyl-cysteinyl-glycine (glutathione) is a tripeptide α-amino acid that is an antioxidant ,27,28,29The present work investigated the effects of BSO-mediated glutathione depletion in HepG2 cells. The obtained results fully confirmed the glutathione inhibitory action of BSO ,29,40,41Our results regarding the lack of nSMase2 mRNA detection in untreated HepG2 cells were in full accord with the findings of Revill et al.  and KaraReduced nSMase activity following BSO treatment was associated with a decrease in the content of the pro-apoptotic ceramide ,8 in accUnimpaired content of the pro-apoptotic ceramide ,8 in 0.0The data support the role of high ROS concentrations in maintaining tumor cells’ vigorous proliferation rate ,28,52,53The most salient outcome of this study is the lack of ready detection of nSMase2 mRNA in HepG2 cells before or after BSO treatment. Otherwise, HepG2 cell treatment with BSO did provoke a dramatic reduction in glutathione content and an increase in ROS levels, in addition to a considerable increase in nSMase1 and nSMase3 mRNA levels. However, almost complete inhibition of cell lysate nSMase activity and significantly lower ceramide levels were not observed, likely due to antagonistic effects on putative nSMase2 protein activity. Nevertheless, BSO treatment, especially at high doses, led to the potentiation of HepG2 cell proliferative capacity. The results indicate that the combined activity of nSMase1, nSMase2, and nSMase3 is required to control HepG2 cell proliferation. Extensive experiments are planned to define nSMase2 mRNA and protein content and activity in several cancer cell lines, biopsies, and normal para-cancer cells. After that, it is possible to examine whether changes in nSMase2 content predict the impact of glutathione depletion-mediated ROS levels on tumor cell growth or apoptosis  and dete