Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms—EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.Some plant  Eucommia ulmoides that synthesize exceptionally high molecular weight trans-1,4-polyisoprene (TPI). Through crystal structure and mutational analyses, they identify key residues required for TPI synthesis and reveal its functional importance in seed development.Kajiura and Yoshizawa et al. identify three new prenyltransferases in the tree  Ultrahigh molecular weight cis-1,4-polyisoprene and trans-1,4-polyisoprene (TPI), which are the principal components of natural rubber and gutta-percha, respectively, are also isoprenoid metabolites that are assumed to be synthesized by specialized prenyltransferases. These prenyltransferases are divided into two major groups, the cis-1,4-prenyltransferases (CPTs) and trans-1,4-prenyltransferases (TPTs), based on the stereochemistry of the double bond formed during IPP condensation. Although both types of prenyltransferases require an allyl substrate and IPP during the common condensation step, they do not share consensus and conserved sequences, indicating that TPTs and CPTs originated from evolutionally distinct proteins3. CPTs form a membrane-associated complex with accessory proteins to generate ultrahigh molecular weight cis-1,4-polyisoprene, whereas the molecular mechanism of TPTs and the physiological functions of TPI remain largely unclear.Prenyltransferases, also known as isoprenyl diphosphate synthases, catalyze the sequential condensation of a basic five-carbon building block  at the fourth and fifth position upstream of the first DDxxD motif are critically involved in chain length determination of the polymeric product7. Typically, FPS produces TPI with a chain length range of C10–40, but enzymes with mutations in Tyr88 and Phe89 generate TPI with a chain length of up to C507. However, some plant species, including Eucommia ulmoides, Palaquium gutta, Manilkara bidentata, and Manilkara zapota, produce ultrahigh molecular weight TPI with a chain length of C100 or much more 10, which indicates the biosynthetic activity of unique TPTs. Especially E. ulmoides has become highly accessible for further research due to the availability of the dataset derived from an expressed sequence tag (EST) library11. Furthermore, a TPT candidate for the synthesis of ultrahigh molecular weight TPI in E. ulmoides was proposed based on the recently sequenced genome of this tree species12.Farnesyl diphosphate synthase (FPS) represents a well-characterized TPT prototype. It is homodimeric, and each monomer has seven highly conserved TPT-specific domains and two Asp-rich motifs (DDxxD), which are essential for the interaction with IPP and the allyl substrateE. ulmoides EST library and identified three potential TPTs, thereafter referred to as EuTPT1, 3, and 5. We aimed to investigate the involvement of these enzymes in TPI production and elucidate the potential molecular mechanism of this biosynthetic pathway. The in vitro analysis of the EuTPTs included a crystallography study of EuTPT3 to further unravel the structural requirements for the synthesis of ultrahigh molecular weight TPI and the formation of the enzyme core of ultralong-chain TPTs. An accompanying in vivo analysis of EuTPT5 had the objective to elucidate the physiological function of this enzyme and the involvement of its product, TPI, in fruit development and maturation in E. ulmoides.In this study, we searched an E. ulmoides EST library11 using FPS amino acid sequences from Arabidopsis thaliana (AtFPS1 and 2) and Saccharomyces cerevisiae  as queries. This search identified five potential EuTPTs, EuTPT1 to EuTPT511  shows inviable phenotype13. If any of the EuTPT could rescue the Δfps lethality, the encoded EuTPT was identified as an FPS and ruled out as an ultralong-chain TPT candidate. In the tetrad analysis, ScFPS, EuTPT2, and EuTPT4 rescued Δfps lethality. It should be noted that we previously characterized EuTPT2 and EuTPT4 as E. ulmoides FPS proteins, referred to as EuFPS1 and EuFPS2, respectively13. In contrast, the attempt to complement the Δfps lethality with EuTPT1, 3, and 5 was unsuccessful , geranyl diphosphate , farnesyl diphosphate , and geranylgeranyl diphosphate . The reaction products were analyzed by reverse-phase thin-layer chromatography . The RP-TLC analysis also detected degraded polyisoprene products, which were presumably generated by the oxidative degradation and/or dephosphorylation during the reaction and the following purification of the reaction product in preparation for the RP-TLC analysis  was characterized by testing their activity using an in vitro assay. Purified recombinant EuTPTx proteins were incubated with IPP was carried out in a 100-μL total reaction volume containing 50 mM cacodylic acid, pH 7.0, or 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.1% Triton X-100, 20 μM [1-14C]IPP, supplemented with 100 μM DMAPP, GPP, FPP, or GGPP, and 1.0 μg of purified protein. Reaction samples were incubated at 25 °C for 20 h and terminated by adding 1 mL chloroform/methanol mixture , followed by vortexing. The radiolabeled products were extracted with a chloroform/methanol/water mixture , and the organic layer was recovered. The radioactivity was measured using a Beckman LS6500 multi-purpose scintillation counter . The radiolabeled reaction products were dephosphorylated using acid phosphatase from potato (Sigma) as previously reported13 with some modifications: the chloroform/methanol/water extracted products were dried and mixed with a final concentration of 10 mM acetate buffer, pH 4.8, 0.1% Triton X-100, 60% methanol, and 1.5 units of acid phosphatase in 100 μL for incubation at 37 °C for 20 h. The hydrolyzed products were extracted with n-hexane, spotted onto reversed-phase thin-layer chromatography (RP-TLC) plates, Silica gel 60 RP-18 F254S , and separated using an acetone/water/hexane mixture (199:1:20) or an acetone/water mixture  as solvent. The position of the standard prenyl alcohols was visualized by exposing the dried plate to iodine vapor. The RP-TLC-separated products were visualized by autoradiography using a Typhoon FLA7000 laser scanner .Assays for purified EuTPTs using radiolabeled [1-Nicotiana tabacum SR-I plants were co-cultivated with Agrobacterium tumefaciens LBA4404 strain carrying pBI121-EuTPTs in the dark for 3 days on callus inducing medium (CIM): Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose and 0.5 g/L MES, pH 5.2, 0.2 mg/L benzylaminopurine, 2 mg/L 1-naphthaleneacetic acid, and 3 g gellan gum. The disks were placed onto CIM containing 50 mg/L kanamycin, 250 mg/L carbenicillin for cultivation at 25 °C with a 16-h photoperiod; the plates were changed every week. Then, an individual shoot was separated and transferred onto hormone-free medium containing 50 mg/L kanamycin, 250 mg/L carbenicillin for selection. The regenerated plantlets (designated the T1 generation) were transferred to soil and maintained under greenhouse conditions. The T2 and T3 generation of transformants were used for real-time PCR analysis and histochemical analysis, respectively.Leaf disks of wild-type GFP-EuTPTx, were generated by PCR methods using full-length EuTPTs and GFP as templates and ligated with the pBI121 vector. The resultant vectors were introduced into Agrobacterium tumefaciens LBA4404 by electroporation. Tobacco BY-2 cells stably expressing Golgi marker XYLTCT-DsRed45 were generated using pGPTV-HPT-XYLTCT-DsRed and by Agrobacterium-mediated transformation45 and supertransformed with the GFP fusion constructs. Fluorescence signals were documented 3–4 days after the sub-cultivation. Cells expressing GFP and DsRed fusion proteins were first stained with 1 μM ER-tracker and analyzed with DIGITAL ECLIPSE C1si  equipped with CFI Plan Apo objectives and EZ-C1 3.40 software (Nikon). Fluorescence was excited with the 408-nm line of a blue diode laser, the 488-nm line of a solid laser, and the 543-nm line of a G-HeNe laser.All fusion constructs, E. ulmoides fruit were prepared using CryoFilm  and a cryostat . The cryosections were washed with 50% ethanol once. In histochemical staining, the sections were stained with 30 μg/mL Nile red in 50% ethanol and 1 mg/mL Fluorescent Brightener 28 in 50% ethanol for 1 min each at room temperature of around 25 °C, then washed three times with 50% ethanol.Approximately 20 μm-thick cryosections of ® Immunostain Immunoreaction Enhancer (TOYOBO), for 12 h at 4 °C. After washing with PBS three times, the cryosections were incubated with Alexa Fluor 647-conjugated secondary antibody, diluted 1:100 in Can Get Signal® Immunostain Immunoreaction Enhancer, for 2 h at room temperature. The immunostained cryosections were further stained with Nile red and Fluorescent Brightener 28. The stained cryosections were placed on a glass slide, mounted in SlowFade® Gold , and then sealed with nail varnish.In immunostaining, the cryosections were washed with PBS three times, and the proteins in the sections were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS three times, followed by blocking with 5% BSA in PBS for 30 min and then washed three times. The sections were incubated with the primary antibody, anti-EuTPTs, diluted 1:100 in Can Get Signaltrans-polyisoprene in chloroform at a final concentration of 1 mg/mL, and thin films were prepared on glass slides. The films were stained with Nile red, and fluorescence spectra from each of 50 locations (regions of interest) were measured and averaged. The fluorescence spectra of the stained samples were obtained from 10 to 25 locations and averaged. Images were acquired and averaged from five successive scans to improve the signal-to-noise ratio. Image processing, including spectral unmixing, was performed using EZ-C1 3.40 software and Adobe Photoshop CS4.The spectral confocal laser scanning microscopy (SCLSM) analysis of the stained samples was performed at 25 °C using the SCLSM system  equipped with CFI S Fluor 4×, CFI Plan Apo10×, 20×, 40×, and VC60×H lenses and the EZ-C1 3.40 software (Nikon). Fluorescence was excited with the 408-nm line of a blue diode laser, the 488-nm line of a solid laser, and the 640-nm line of a diode laser. The emission spectra in the ranges of 460–470 nm, 500–650 nm, and 640–750 nm with 5 nm bandwidths were recorded for detecting the cell wall, TPI and lipid, and Alexa Fluor 647, respectively, with TPI and lipid simultaneously detected in the rage of 500–650 nm. Reference samples were prepared by dissolving commercial E. ulmoides fruit was isolated as described above, and total RNA from transgenic tobacco plants was isolated using the Maxwell® 16 LEV Plant RNA Kit and Maxwell® 16 Automated Purification System . Quantitative real-time PCR with the GoTaq qPCR Master Mix (Promega) was performed using the prepared cDNA preparations as templates for the StepOne Real-Time PCR . The primer sets used in this analysis are listed in Supplementary Data ACT gene was used as a control to normalize the amount of cDNA template. The relative expression levels were calculated using the lowest EuTPTs transcript levels as the expression of 1.0.Total RNA from E. ulmoides fruit was measured using Image J. The E. ulmoides seed was removed from the fruit. Then, the seed and the residual pericarp were completely dried at 55 °C for 2 days, and their weights were measured. Dried pericarp was ground into a fine powder using CryoMil MM400  for 1 min at 30 rpm with cooling using liquid nitrogen. The powder was suspended in ethanol, followed by centrifugation at 20,000 × g for 1 min, and the supernatant was discarded. This step was repeated until the supernatant was colorless. Five mg of the pericarp powder was dissolved in 1 mL chloroform with polybutadiene rubber dissolved at a concentration of 1 mg/mL as an internal standard and mixed gently for 20 h. In all, 5 μL of supernatant was analyzed by pyrolysis gas chromatography/mass spectrometry (PyGC/MS)46. PyGC/MS analysis was performed with ISQ7000, TRACE 1310 (Thermo Fisher Scientific), and Multi-Shot Pyrolyzer EGA/PY-3030D  using a Ultra ALLOY® Capillary Column . TPI was pyrolyzed at 600 °C. The temperatures of the injector, GC/MS transfer line, and ion source were 200 °C, 250 °C, and 200 °C, respectively. The column temperature program was set at 5 min at 40 °C, and then increased by 8 °C/ min up to 300 °C, and kept at 300 °C for 5 min. The split ratio was set at 100:1. The mass range was set from 29 to 550 m/z.The area of each g for 5 min, and the supernatant was collected and dried using an evaporator.The fine powder of pericarp was dissolved in toluene and heated at 60 °C with gently agitating for 6 h. The toluene extracted product was centrifuged at 1000 × TM PS standard  .Two mg of the TPI was dissolved in 2 mL of tetrahydrofuran (THF) and gently mixed at room temperature for 2 days. The sample was filtrated using a 0.45-µm membrane before size exclusion chromatography analysis. The size exclusion chromatography was performed with a Tosoh HLC-8320GPC EcoSEC chromatograph  using a guard column, TSKgel GMHhr-H (S) , and two TSKgel GMHhr-H (S)  columns with a molecular weight exclusion limit of 400,000,000. THF was used as the mobile phase at a flow rate of 0.5 mL/min at 40 °C. TPI was monitored by a Viscotek 270 Dual Light Scattering Detector . The system was calibrated with the PolyCALE. ulmoides were sampled weekly from March to September in 2013 and 2015. The fresh samples were scanned using the CanoScan LiDE 220 , and the area of each sample was calculated by ImageJ47. Because the dry weight of fruit, pericarp and seed, fruit area and TPI content might converge to the equilibrium points, the growth curves of each of these phenotypes were fitted with a logistic equation. The parameter estimations for the five logistic equations were conducted using the least squares method, and the accuracy of the estimated equations was confirmed by the r2 value for the comparison between the estimated and measured scores. The analyses were conducted using R ver. 3.2.3.The 15 reproductive organs of E. ulmoides pericarps stored at −80 °C were ground into a fine powder using CryoMil MM400 for 3 min at 30 rpm with cooling using liquid nitrogen. The fine powder was suspended in the equal volume of ice-cold 100 mM phosphate buffer pH 7.5, 5 mM MgSO4, 5 mM 2-mercaptoethanol, Complete EDTA-free protease inhibitor cocktail , and 0.9 g/mL polyvinylpyrrolidone, followed by vigorous vortexing and centrifugation at 2400 × g for 10 min. The unwashed, creamy, and floating fraction was transferred to a new tube, washed with 100 mM phosphate buffer pH 7.5, 5 mM MgSO4, and 10 mM dithiothreitol, and centrifuged at 2400 × g for 10 min. Washed TPI was resuspended in 100 mM phosphate buffer, pH 7.5, 5 mM MgSO4, 10 mM dithiothreitol, 0.5% CAHPS, and washed TPI protein was solubilized on ice by sonicating ten times for 30 s. Then, the sample was centrifuged at 20,000 × g for 15 min at 4 °C, and the aqueous phase was filtered using a 0.22-μm filter to remove remaining traces of TPI from the washed TPI protein solution. The TPI pellet was dried completely, and the weight was measured. Depending on the total weight of TPI, the washed TPI protein solution  was desalted by methanol/chloroform extraction and acetone precipitation and dissolved in sample buffer for SDS-PAGE.Fresh TM Ace 10–20% gradient gels  and analyzed by silver staining or western blotting with the anti-EuTPT5 antibody.Purified TPI proteins were separated using SuperSep2, for a 20-min incubation on ice. Crystals were obtained by hanging drop vapor diffusion at 20 °C (1.0 μL protein + 0.3 μL reservoir solution). EuTPT3 crystals (form1 and form2) were obtained in a reservoir solution of 9% PEG 3350 and 0.1 M dl-malic acid. To crystallize EuTPT3 (C94Y/A95F) mutant, a truncated version of EuTPT3 (residues 66–70 of EuFPS1 were replaced with GG) was used. The crystal of EuTPT3 (C94Y/A95F) was obtained in a reservoir solution of 0.1 M CHES, pH 9.5, and 20% PEG 8000. EuFPS1 crystal was obtained in a reservoir solution of 0.1 M HEPES, pH 7.0, and 25% PEG1000, 8% ethylene glycol, and 1 mM MgCl2. Crystals were cryoprotected by the addition of ~30% ethylene glycol and flash-cooled in a 100-K nitrogen stream.Purified EuTPT3 and EuFPS1 were concentrated to 10 mg/mL in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM DTT. Prior to crystallization, aliquots of two more buffer components were added to the EuTPT3 preparation at the following final concentrations, 10 mM risedronate and 5 mM MgClArtemisia spiciformis FPP synthase (Chain A from PDB ID 4KK2). The EuTPT3 (C94Y/A95F) structure was determined by molecular replacement using Molrep with a search model of the EuFPS1 dimer. Several rounds of refinement using phenix.refine or REFMAC5 and manual model building with Coot were performed. Stereochemical parameters were checked using the Molprobity suite in PHENIX. The final coordinates of EuTPT3 WT form 1, EuTPT3 WT form 2, EuTPT(C94Y/A95F), and EuFPS1 have been deposited in protein data bank with accession codes 7BUU, 7BUV, 7BUW and 7BUX, respectively. Structures contained 0.90%, 1.45%, 1.31%, and 0.15% Ramachandran outliers, respectively. Clash scores of the structures were 12.81, 11.23, 6.56, and 9.01, respectively. The figures were prepared using PyMol (http://www.pymol.org).X-ray diffraction data were collected at 1.0 or 0.9 Å wavelength on the SPring-8 beamline 26B1 and 44XU . Diffraction data sets were indexed, integrated, and scaled using XDS. The EuTPT3 and EuFPS1 structures were determined by molecular replacement using Molrep with a search model of All statistical analyses were performed using Microsoft Office Excel. Data are expressed as means ± standard deviation. All the experiments were evaluated at least three biological replicates with similar results.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Duchenne muscular dystrophy (DMD) is a common and severe X-linked myopathy, characterized by muscle degeneration due to altered or absent dystrophin. DMD has no effective cure, and the underlying molecular mechanisms remain incompletely understood. The aim of this study is to investigate the metabolic changes in DMD using mass spectrometry-based imaging. Nine human muscle biopsies from DMD patients and nine muscle biopsies from control individuals were subjected to untargeted MSI using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry. Both univariate and pattern recognition techniques have been used for data analysis. This study revealed significant changes in 34 keys metabolites. Seven metabolites were decreased in the Duchenne biopsies compared to control biopsies including adenosine triphosphate, and glycerophosphocholine. The other 27 metabolites were increased in the Duchenne biopsies, including sphingomyelin, phosphatidylcholines, phosphatidic acids and phosphatidylserines. Most of these dysregulated metabolites are tightly related to energy and phospholipid metabolism. This study revealed a deep metabolic remodelling in phospholipids and energy metabolism in DMD. This systems-based approach enabled exploring the metabolism in DMD in an unprecedented holistic and unbiased manner with hypothesis-free strategies. This lethal X-linked recessive neuromuscular disorder is caused by mutations in the DMD gene resulting in absent or reduced functional dystrophin (DYS). As in many genetic diseases, DMD exhibits a continuum of disease severity, which may be correlated to the presence or absence of a functional protein. Becker muscular dystrophy is a milder form of dystrophinopathy in which DYS levels are reduced, but a residual partially functional protein remains3. The dystrophin-associated protein complex underlies the link between the extracellular matrix and the cytoskeleton, thus ensuring the skeletal muscle strength. Dystrophin dysfunction results in severe architectural muscle changes with subsequent degeneration and impaired regeneration5. Dystrophin has been shown to be temporo-spatially expressed by discrete neuronal populations, mainly in the pyramidal cells of the hippocampus, in the amygdala, in Purkinje cells and granular neurons of the cerebellum. Thus, DYS is involved in several cognitive functions and human brain development, although the precise mechanisms remain unclear6. Muscle weakness and motor delay are usually early symptoms. In the absence of steroid treatment, the ambulation loss occurs at about 10 years of age and near paralysis by age 20, with scoliosis, loss of upper limb function, cardiac involvement, and respiratory insufficiency. Brain dysfunction also occurs at the start of various neurobehavioral developmental disorders, such as intellectual disability, or more specific cognitive disorders, delayed speech and coordination acquisition, executive dysfunction, attention deficit disorder and autism spectrum disorders7. To date, there is no cure for DMD, but the life expectancy of DMD patients has improved owing to steroid treatments and better care. DMD patients lose the ability to walk around 13–14 years of age and eventually die in their 30 s due to cardiopulmonary complications3. However, numerous clinical trials and experimental approaches are ongoing, such as exon skipping, gene therapy, myostatin inhibitors, utrophin modulation, CRISPR/Cas9 suppression of stop codons and stem cell therapy8. Validated outcome measures are required to assess the potential benefits of these treatments. Currently, several functional tests are used such as the 6 min walk test, the North Star ambulatory assessment, and the performance of upper limb test or time items in addition to muscular imaging. However, these tests are often ineffective, and their results do not necessarily correlate with the patient’s condition9. Owing to wide phenotypic variability, reliable outcome measurements that would enable assessing treatment efficacy remain tentative, and new biomarkers must be identified to better understand the DMD pathophysiology and more accurately evaluate patient prognosis and follow-up1. Metabolic impairments have been associated with DMD since the 1970s13. However, the advent of new omics technologies allows exploring the metabolism and its components in an unprecedented holistic and unbiased manner with hypothesis-free strategies14. Metabolites are small organic molecules involved in enzymatic reactions to form the metabolism. The “metabolome” refers to all metabolites present in a given biological system, fluid, cell, or tissue. Metabolomics is the omics technology that enables exploring the metabolome by comprehensively measuring metabolite levels in a given biological sample15. Its main goal is to parse the biochemical changes in a biological system by probing the metabolite variations related to genetic, environmental, drug, dietary and other factors or interventions. Mass spectrometry is a widely used metabolomics technology because of its high detection sensitivity, metabolome coverage and rapid data acquisition turnover16. The spatial distribution of metabolites can be tracked holistically and systematically, which is highly valuable for gaining a mechanistic understanding of biological processes. Mass spectrometry imaging (MSI) achieves this by simultaneously revealing the spatial distribution of multiple molecules in a single experiment from various biological samples, particularly tissue Sects.17. Thus, this technology shows promising translational potential in the biomedical field19. MSI has been applied to study DMD muscle biopsies using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and TOF secondary ion mass spectrometry. Previous investigations highlighted lipid compositional changes21 or Krebs cycle intermediates22 in muscles from DMD mouse models and DMD patients23. However, the relatively low mass resolution of the TOF–MS instruments makes peak annotation very challenging. Thus, powerful new mass spectrometry instruments based on Fourier-transform ion cyclotron resonance (FTICR) offer better analytical performance in terms of resolution, sensitivity and specificity26. Thus, this technology offers unprecedented exhaustive metabolome coverage and non-ambiguous molecular formula assignments26.Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy in children, with an incidence of 1 in 5000 live-born boys annually and an estimated prevalence of 15.9 per 100,000 boys in the USA and 19.5 per 100,000 boys in the UKThis work presents the first study ever published using MSI based on MALDI coupled with FTICR to explore muscle metabolic remodelling in DMD patients. Given the scope of the study and the high analytical superiority of the platform, this investigation allowed holistically parsing the complex metabolic remodelling in DMD tissues, revealing the DMD-associated molecular metabolic impairments and deregulations, aiming to open new avenues for deeper biological investigations to uncover new therapeutic targets, biomarkers and diagnostic tools.Morphological lesions meeting DMD diagnostic criteria were observed in muscle biopsies including fiber necrosis with inflammatory response, diffuse variation in fiber size with rounded and hypercontracted fibers, basophilic regenerative fibers, and endomysial and perimysial fibrosis. Immunohistochemical studies revealed that six patients lacked DYS1, and three had severely decreased and irregular DYS1 immunoreactivity, all patients but one having no DYS2 and DYS3  and phosphocholine (183.066045 Da). Supplementary Fig. 28H50NO7P at m/z 566.32166 [M + Na]+. Given the FTICR high mass accuracy, a loss of m/z 59.07351 and m/z 183.06608 corresponded to trimethylamine and phosphocholine with mass errors of 186 ppb and 191 ppb, respectively. Therefore, the ion at m/z 566.32166 with the molecular formula, C28H50NO7P, was attributed to lysoPC(20:4) [M + Na]+. Other PC and SM lipids were similarly unambiguously assigned. In negative-ion mode, other lipids included phosphatidic acid and phosphatidylserine. Adenosine triphosphate (ATP) at m/z 505.98833 was annotated using MS/MS analysis by the loss of an adenine group at m/z 272.95698 , phosphatidic acid (PA), PS and SM classes, as well as triacylglycerols, are increased, while glycerophosphocholine (GPC) is decreased in DMD muscles compared with control muscles  patients and controls. These authors observed that GPC levels were lower or near absent in DMD muscles compared with control muscles and dramatically increased (15-fold) in one Werdnig-Hoffman patient30. Increased triacylglycerol and SM in DMD mouse muscles have been interpreted as resulting from increased phospholipase C activity13. High phospholipid levels and high phospholipid-to-cholesterol ratios have been documented using1H-NMR spectroscopy, which helps distinguish DMD patients from healthy subjects34. Phospholipase D activity is reported to be increased in DMD muscles. Calcium homeostasis has been shown to be altered in DMD patients, with calcium concentration being increased in dystrophin-deficient muscle. Phospholipase D activity is enhanced under high concentrations of free calcium, resulting in increased PA levels36. In summary, profound phospholipid metabolism remodelling is associated with DYS deficiency; thus, DMD has been considered a glycerophosphocholine deficiency37. Morphological alterations of the mitochondria have been reported in DMD muscles with higher rates of swollen mitochondria compared with that of normal muscle38. PL alterations in DMD and a dysregulation of Ca2+ homeostasis may induce mitochondrial membrane fragility and associated morphological changes with subsequent energy and oxidative metabolism disruptions42.Phospholipids (PLs) are major components of all cell membranes, and glycerophospholipids (GLs) are the most abundant membrane PLs. GLs are composed of a glycerol backbone linked to a phosphate group in position 3. Acyl groups may be attached at positions 1 and 2 of the glycerol. Different moieties, such as choline, inositol, ethanolamine and serine, are linked to the phosphate group, contributing to GL diversity , PS, and phosphatidylglycerol). PCs are the most abundant GL in mammalian cells. Other lipid classes, such as cholesterol and glycosphingolipids, play structural roles in cell membranesles Fig. . Lysophorol Fig. . It has 43 as illustrated by mitochondrial function improvement upon the partial restoration of DYS expression44. Accordingly, we showed a significant increase in ADP and decreased ATP levels in DMD muscles compared with those of the control muscles  , existing cardiac complications , digestive complications  and iatrogenic complications. Inclusion in therapeutic protocols, hospitalization frequency, cognitive delays and behavioral problems, including autism and school attendance, were also recorded.The mean patient age at disease onset was 4 ± 1.5 years, at last visit was 10.5 ± 2.2 years, and at time of biopsy was 5.5 ± 1.9 years. All patients are presently alive, and six  were ambulant at the last visit. One non-ambulant patient (P2) lost the ability to walk at age 7; he was not given steroids, nor included in any therapeutic protocol. The other two patients  lost the ability to walk at 11 and 13 years of age. CK levels ranged from 5418–52,000. Symptoms at disease onset were muscle weakness with difficulty walking and climbing stairs and frequent falls. Eight patients had calf hypertrophy. Six patients presented no motor features at onset but had speech delays  and/or behavioral/autistic disturbances  that had likely led to delayed diagnosis. Four patients had cognitive delays . Two patients  developed behavioral abnormalities. Six patients had learning difficulties affecting speech, writing, attention and memory . Three patients  had nutritional and feeding problems, with anorexia in two and obesity in one (P9). All but two patients were given steroids ; one of these two patients were lost to follow up one year after diagnosis, and the other received no treatment owing to poor family compliance. Four patients  could run, the others could walk without help. Two patients had mild respiratory insufficiency that did not require ventilation. Eight patients had no significant cardiac arrhythmia or functional abnormalities. Patient P4 had rhythm abnormalities consisting of supraventricular extrasystoles 3.9% of the time, which did not require specific treatment. All but one patient received a prophylactic antiarrhythmic drug (perindopril), and one (P1) was included in a perindopril protocol (2-year double-blind treatment protocol). Three patients  were included in exon-skipping protocols, and one (P6) was included in a givinostat (histone deacetylase inhibitor) protocol. Clinical data overview is presented in Fig. 48. For immunohistochemical studies, 6-μm frozen sections were immunolabeled with antibodies against β-spectrin , dystrophin , and α-, β-, γ- and δ sarcoglycans . Dystrophinopathy was morphologically diagnosed per the criteria of Dubowitz et al.48.Each patient underwent a muscle biopsy at a mean age of 5.5 ± 1.9 years, which was assessed according to standardized histochemical and histoenzymological methodsAs age-matched control muscles were not available, nine male adults aged 28–58 years were selected as controls for comparative analyses. These patients presented myalgias, muscle weakness, and/or muscle fatigability. In one case, a polymyositis was suspected; Buschke scleroderma was suspected in another. In all control patients, routine biological tests, CK levels, EMGs, cardiac ultrasonography, pulmonary testing and muscle MRI were considered normal. Their muscle biopsies were also normal, and they were concluded to have probable fibromyalgia. One obvious potential limit of this work that needs to be highlighted consists in absence of age-matched control muscles could hamper interpretation of the results, as biochemical properties of muscles change over the lifetime. Even though paediatric muscle samples could be obtained during orthopaedic surgery, the location of the biopsy may change and may not entirely match with that of Duchenne patients.-1 (ACN/H2O 50:50 [v/v]) was used as a calibrant before each analysis.Methanol, acetonitrile, ethanol and water of LC–MS grade were purchased from Fisher Scientific ; 2.5-dihydroxybenzoic acid (DHB) and 9-aminoacridine hydrochloride monohydrate (9-AA) MALDI matrices were purchased from Sigma-Aldrich . Sodium trifluoroacetate  at 0.1 mg mL49. In the positive-ion mode, DHB matrix (30 mg mL-1 in MeOH/H2O 50:50 [v/v]) was deposited with the following parameters: nozzle temperature 80 °C, nozzle velocity 1200 mm min-1, N2 pressure 10 psi, N2 flow rate 2 L min-1, number of passes 12, flow rate 100 µL min-1 and track spacing 3 mm. In negative-ion mode, 9-AA matrix  was sprayed using the following parameters: nozzle temperature 90 °C, nozzle velocity 1200 mm min-1, N2 pressure 10 psi, N2 flow rate 3 L min-1, number of passes 2, flow rate 120 µl min-1, track spacing 3 mm and drying time 30 s between passes. Each slide was vacuum-dried before analysis.Fresh frozen 10-µm sections of human muscle were mounted on conductive ITO-coated glass slides 75 × 25 mm  and stored at -80 °C until analysis. Matrix solutions were applied with an automatic microsprayer HTX TM-Sprayer  as previously described7H6O4 , C7H15NO3 , C7H6O4 , C5H14NO4P , C9H17NO4 , C14H8O6 , C21H12O9 , C40H80NO8P , and C42H82NO8P . In negative-ion mode the assigned peaks were C13H10N2 , C17H26N6O6 , C10H15N5O10P2 , C10H16N5O13P3 , C23H39N7O7 , C30H46N6O8  and C39H77O9P . Data size was set at 2 million points for a transient length of 0.87 s, and spectra were acquired with a 97% data file reduction. A single MSI measurement has been performed by specimen. Images were captured using FTMS control and FlexImaging  software. Images were processed with SCiLS Lab Pro software . The total ion current method was used for normalization, and m/z intervals were automatically set at ± 1 ppm. Images were viewed using both FlexImaging and SCiLS Lab software .Data were acquired on a FTICR instrument  equipped with a 12-T superconducting magnet and a dynamically harmonized ICR cell. This instrument is also equipped with both a laser desorption ionization source  and an electrospray (ESI) source. Each MALDI spectrum for each position is the result of 1 scan and 500 consecutive laser shots. Spectra were acquired over an 80 µm raster. Before imaging analyses, the instrument was externally calibrated in the required mode by NaTFA infusion via ESI source, then internally calibrated by assigning known metabolites from m/z 150–1000 via MALDI source. The instrument was autocalibrated during image acquisition. In positive-ionization mode, calibration was performed by assigning CIon intensities have been log-transformed. Univariate analyses were performed using t-tests to identify discriminatory ionic species between the assessed groups. Age has been taken into account by adding it as covariate. Spearman correlation analysis was performed using R software. Euclidean distance was used as a similarity measure in the clustering analysis. False discovery rates were corrected using the Benjamini–Hochberg–Yekutieli method, and p < 0.05 was considered statistically significant.50 and HMDB 51, using a threshold of ± 2 ppm. For some metabolites, the precise raw formula led to one hit. Others were identified via “on-tissue” tandem mass spectrometry experiments using MALDI tandem MS/MS. Ions of interest were first isolated using a window of ± 1 Da, then fragmented by collision-induced dissociation with energy levels between 10 and 40 eV. For each MS/MS analysis, 50 scans were accumulated for better sensitivity. Spectra were reprocessed using Data Analysis 4.4 software  and recalibrated with the single-point calibration option.Preliminary assignments based on accurate mass measurements were performed using the mass spectrometry databases, METLIN All muscle samples used in this study belong to a collection declared to the French Ministry of Health  located in the Pathology Department (Prof. Annie Laquerrière) of Rouen University Hospital in accordance with the relevant guidelines and regulations and with permission of the local authorities. Written informed consents were obtained from the parents when the patient is under 18 or from the adult patient in order to perform any investigation related to their pathology. The study protocols have been approved by ethics committee of NORD WEST1 – Rouen University Hospital .Supplementary Tables.Supplementary Figures.


C. elegans lifespan assays, we conducted a worm metabolite screen and identified an eukaryotes conserved metabolite, myo-inositol (MI), to extend lifespan, increase mobility and reduce fat content. Genetic analysis of enzymes in MI metabolic pathway suggest that MI alleviates aging through its derivative PIP2. MI and PIP2 are precursors of PIP3, which is negatively related to longevity. The longevity effect of MI is dependent on the tumor suppressor gene, daf-18 (homologous to mouse Pten), independent of its classical pathway downstream genes, akt or daf-16. Furthermore, we found MI effects on aging and lifespan act through mitophagy regulator PTEN induced kinase-1 (pink-1) and mitophagy. MI’s anti-aging effect is also conserved in mouse, indicating a conserved mechanism in mammals.Aging is characterized by the loss of homeostasis and the general decline of physiological functions, accompanied by various degenerative diseases and increased rates of mortality. Aging targeting small molecule screens have been performed many times, however, few have focused on endogenous metabolic intermediates—metabolites. Here, using  2 to PIP3 and is encoded by the age-1 gene known to regulate lifespan. Here the researchers found that the metabolite myo-inositol, which can be converted to PIP3 extends worm lifespan and alleviates worm as well as mouse health decline during aging.Phosphatidylinositol 3-kinase catalyzes the reaction from PIP The aging process is closely linked to metabolism2. Metabolism is composed of chemical reaction networks, which can be represented with metabolites as nodes and enzymes as edges3. Metabolites, such as NAD+ and related metabolites6, spermidine7, pyruvate8, N-acetylglucosamine (GlcNAc)9, and α-ketoglutarate10, have been reported to alleviate aging in model organisms.Molecules that promote healthy aging have been searched and studied because of their potential to target human aging13. To investigate the influence of available metabolites on C. elegans lifespan, we conducted a preliminary screen on worm 100 metabolites and found that an evolutionarily conserved metabolite, myo-inositol (MI), significantly increased lifespan across a large range of concentrations.The number of metabolites varies in magnitudes across the four kingdoms: tens of thousands in plants, thousands in humans, and one thousand in worm17, whose isomers or derivatives participate in many fundamental physiological processes18. Among its nine possible stereo-isomers, MI is the most abundant18. MI is a common ingredient in food19, and it is also used as a food supplement to alleviate a variety of diseases20, including diabetes21 and polycystic ovary syndrome (PCOS)22. An isomer of MI, D-chiro-Inositol, which does not exist endogenously in either C. elegans or Drosophila melanogaster, has been reported to extend fly lifespan through unknown mechanisms and was speculated to act through dFOXO23. Inositol in the form of MI itself has not been reported to influence aging.MI is an extensively studied metabolite26. Among them, abnormally high levels of PIP3, a key lipid component in the insulin and insulin-like growth factor signaling pathway (IIS), is known to promote cancer and shorten lifespan through activation of AKT in mouse/mammals27. Conversely, another derivative, IP7 is reported to inhibit AKT in mouse28, but has not been reported to influence aging. Both PIP3 and IP7 in aging were indirectly studied by perturbations of their synthases, i.e., loss of function (LOF) of worm Phosphatidylinositol 3-kinase (age-1)29, mouse liver phosphatidylinositol-4,5-bisphosphate 3-kinase (Pi3k)30 and LOF of inositol hexaphosphate kinase (Ip6k1) in mouse28, respectively. It remains unknown whether or how the other derivatives influence aging.There are scores of MI derivatives, classified into three groups, phosphatidylinositol phosphates (PIs), inositol phosphates (IPs), and glycosyl phosphatidylinositols (GPI)2, despite MI and PIP2 being precursors of PIP3. Moreover, its effects are completely dependent on the well-known tumor suppressor gene, daf-18 , which has many functions besides being an enzyme in the MI metabolic pathway, and partially depends on its downstream mitophagy regulator pink-1 rather than its classical downstream daf-16. The anti-aging effects of MI were recapitulated in mice at phenotypic and transcriptome levels, suggesting a conserved mechanism in mammals.Here we found that MI extended worm lifespan and promoted healthspan, in other words, prevented the age-related decline in physiological functions. Genetic dissection of the MI metabolic pathway suggests that MI alleviates aging through its derivative PIP18. The major reactions in the MI pathway are conserved from worm, mouse to human. Here we define MI metabolic pathway as the reactions forming an unbroken chain with MI upstream or downstream as annotated by the three KEGG pathways: Inositol phosphate metabolism, Phosphatidylinositol signaling system and Glycosylphosphatidylinositol (GPI)-anchor biosynthesis.MI is a cyclohexanehexol Fig.  that is p < 0.01, Fig. 31, we used 50 mM mannitol (M) as an osmotic control, because it cannot be further metabolized in worms.Through a small molecule screen with endogenous metabolites, we found that various concentrations of MI, from 1 up to 700 mM, and optimally at 500 mM, could extend worm lifespan , the rate-limiting enzyme to synthesize MI in most species, including worms and mammals32 as a fusion protein with GFP, and backcrossed to N2 six times. We found that this inos-1::GFP overexpression (OE) strain had a 19% increase in lifespan compared with GFP only OE strain  or Y6B3B.5, to produce MI from the three types of inositol monophosphate (IP). IP is produced by INOS-1 or the degradation product of inositol phosphates. The p < 0.0001, Fig. p = 0.1466, Fig. We further examined MI’s effect on the transcriptome changes. As expected, MI induced a transcriptome change opposite to the changes induced by aging, in particular, aging downregulated genes were significantly upregulated by MI  4 to day 10. Both MI treatment and 33, and we found that MI also decreased worm fat storage after examining lipid oil red O (ORO) staining. In contrast, the osmotic control M increased worm body fat, compared to untreated control  model , PCA of all nine samples  revealed that MI treated muscle samples were closer to CTL samples on PC1 and similar to Young_CTL samples on PC2  by clustering analysis. Cluster 0 and 1, related to cell adhesion and adaptive immunity, respectively, were downregulated genes comparing 12 vs 3-month mice, and were upregulated by MI; cluster 4 and 5 related to growth regulation and ion transport, respectively, were upregulated genes comparing 12 vs 3-month mice, and downregulated by MI. We also found MI induced downregulation of blood vessel development-related cluster 2 and upregulation of transcription related to cluster 3 genes, which were not drastically changed from M3 to M12 Fig. .8 by knocking down its synthases, F46F11.1 (step 27) and F30A10.3 (step 28), decreased worm lifespan P2. They are 1-phosphatidylinositol-5-phosphate 4-kinase , phosphatidylinositol phospholipase C, beta  and four other enzymes in two reversible reactions, i.e., ppk-1  and synaptojanin , age-1  and daf-18 . ppk-2 has been reported not to be a main PIP2 producer36, although our data showed that knocking down ppk-2 by RNAi did not obviously influence worm lifespan P2, ppk-1 , age-1 (step 15) and egl-8 (step 18) all extended lifespan P2, which makes it impermeable to plasma membranes, we could not test whether PIP2 supplementation promotes longevity.Due to the instability when given as a supplement, and negative charge of PIPppk-1 , the main enzyme to produce PIP2, shortened worm lifespan P2 has been reported to bind and activate PTEN to dephosphorylate PIP339, we tested a PTEN ortholog null-allele in the worm, daf-18-NL (ok480). We found that ok480 could fully block MI lifespan extension  mRNA, protein status or activity. Since there was not a good antibody to detect worm DAF-18 protein, we examined PTEN levels with or without MI supplementation, in several tissues of mice sacrificed after health parameter assays. We found that mouse PTEN protein showed a trend towards upregulation in the muscle Pion Fig.  or inos- OE Fig.  induced ing Fig.  and fat-ing Fig. . Next, w3, which activates AKT, which subsequently inhibits DAF-16/FOXO. However, akt-1(ok525) and akt-2(ok393)  P2 to PTEN might be essential in other functions of PTEN.As a lipid phosphatase, DAF-18/PTEN dephosphorylates PIP50) Fig.  could no45, we examined whether the mitophagy regulator, pink-1 mediates the longevity effect of MI46. We found pink-1-NL (ok3538) mostly, though not completely, blocked the longevity phenotype  partially blocked MI induced lifespan extension  as an indicator of autophagic flux48, we found MI decreased SQST-1::GFP puncta in worm muscle, which indicates the enhancement of autophagy  transgenic worms, MI promoted the co-localization GFP:: LGG-1 and mitotracker, indicating enhanced mitophagy were induced by MI treatment P-16 Fig. .49, the osmotic pressure would differ between the two at an equal concentration. However, both did not result in strong osmotic effect at 50 mM, and when we compared their effect in RNA level at 500 mM, the RNA-seq based differential gene expression analysis showed M induced stronger alteration of osmotic stress-related genes, especially the osmotic marker gene gpdh-150 P2, also shortens lifespan. Abnormal PIP3 elevation is a feature of cancers and many other diseases, while aberrant PIP3 decline results in diabetes. Therefore, a balance between PIP2 and PIP3 appears more vital than simply inhibiting PI3, i.e., controlling PI3 level should not be achieved by blocking PIP2 production, otherwise it might undermine beneficial PTEN functions. Our results comprehensively demonstrated that as an endogenous metabolite, MI enhances mitophagy downstream of PTEN, making it one of the very few endogenous mitophagy activators known53, and thus open a door to many potential implications and applications of MI to degenerative diseases where mitophagy is compromised. This is supported by the MI-supplementation mediated decrease of α-SYNUCLEIN puncta in PD models shown here.Although aberrant PIPGiven that MI has been safely used in human as a supplement for a long time, and that our analyses suggest its anti-aging benefits extend from worms to mice through highly a conserved metabolic and signaling pathway present in human, we envision a promising translation potential of MI supplementation to promote healthy human aging Fig. , b, 5.Worm strains are listed in Supplementary Table Psir5::inos-1::GFP::let858.3-3’UTR construct, a fragment from inos-1 start to the last amino acid codon was cloned between NotI and XbaI sites of the vector pPD158.87. The empty vector was used as control. Plasmids were injected to young adult worm gonad line to get transgenic worms, without genome integration. Transgenic worms were maintained by picking under fluorescent microscopy.To create https://www.dkfz.de/signaling/e-rnai3/, the fragments were inserted into L4440 vector, between Spe I and Xho I sites. Other RNAi strains are from another library54.For RNAi constructs, RNAi sequences were designed using an online tool, 55. In brief, worms were bleached to get synchronized eggs, these eggs were cultured until breeding, then the naturally bred eggs were picked for lifespan assay. Adult worms of 12 h-after-breeding were deemed as AD_1 and transferred to lifespan assay plates. OP50 were seeded on NGM plates, about 12 h before killing with a Hoefer UVC 520 cross-linker (100 mJ/cm2 for 4 min). Killed OP50 were used to feed worms at least 6 h after the UV procedure. The plates were refreshed daily. Initial about 100 animals were used for each treatment. Dead worms were counted from AD_8. Worms of no response to nose-touching were picked out of the bacteria lawn, then those motionless worms were scored as dead. Worms with an abdomen explosion were censored. Statistical analysis of lifespan data is shown in Supplementary Table Lifespan assay was conducted according to a standard protocol with killed OP50 (Amp supplemented), without FUdR unless otherwise notedFor RNA-seq sample preparation, worms were collected in 1.5 ml tubes, washed several times with M9 to remove bacteria, RNA was extracted with Trizol reagent according to the manufacture’s instruction. For mouse samples, equal weight tissues were dissected on ice, collected in 1.5 ml tubes, RNA was extracted with Trizol reagent. All RNA samples passed the quality control of RIN > 8 and 18S/28S > 1.0. Sequencing was implemented with Illumina HiSeq 2000 system.56 and Rsubread57. The read counting was implemented by HTseq using annotations from Worm Base and Gencode, for worm and mouse respectively58. The quantification and normalization of reads are conducted by the R package, DESeq259. The term enrichment analyses were conducted with GSEA60.The RNA-seq reads were trimmed and mapped to WS256 genome (from Worm Base) for worm data and mm10 genome (from UCSC genome browser) for mouse data, using STAR (version 2.5)Worms were pretreated as in lifespan assays. On the inspection day, 100 μl M9 buffer was pipetted onto the surface of an empty 35 mm NGM plate, one worm was picked into the buffer, and allowed 30 s to recover from the transfer. Then the number of worm thrashing during 30 s was counted, four times for each worm, 10 to 15 worms for each treatment. One worm from one treatment was counted, then one worm from another treatment, then the third treatment, and so on, the cycle was repeated to avoid the interference of environment or operation.Worms were pretreated as in lifespan assays. All treatment plates were taken out from the incubator to room temperature (RT). The counting was conducted directly on the treatment plates. The number of pumping within 30 s was counted, repeated four times for each worm, 10 to 15 worms per treatment. One worm from one treatment was counted, then one worm from another treatment, then the third treatment, and so on, as thrashing assay. Counted worms were picked out from the culture plates, picked back when the intraday experiments finished.2EGTA, 1 mM spermidine-HCl, 0.4 mM spermine, 30 mM Na-PIPES pH 7.4, 0.2% β-mercaptoethanol). After that, samples were washed twice with 100 mM Tris-Cl (pH 7.4). After wash, the samples were treated with 10 mM DTT in 100 mM Tris-Cl (pH 7.4). Then samples were resuspended in 70% isopropanol in 1× PBS, and incubated for 15 min to dehydrate. Next, the worms were stained with Oil-Red-O solution overnight with shaking . The next day, the staining solution was discarded and animals were resuspended within pure water. Worms were mounted on 2% agarose pad and imaged under 10× microscope61. The intensity was analyzed with an in house MATLAB script.Worms were washed several times with 1× PBS to remove bacteria, then fixed and permeabilized for 30 min by shaking within MRWB buffer containing 2% paraformaldehyde, ]), the tail region was imaged under 63× CONFOCAL microscopy. To avoid the influence of levamisole on autophagy, all slides were freshly made and imaged within 5 min. The images were analyzed with the in-house MATLAB scripts.Worms were anesthetized with 25 mM levamisole, mounted on a 2% agarose pad, with cover clips and fixed with finger nail oil. For NL5901, worm tail region was imaged under 63× CONFOCAL microscopy, one worm per image, 30 animals for each treatment. And the representative picture of five worms was imaged under ×20 objective. For the strain  by gavage. Drug was given twice weekly, at 580 mg/Kg for an average mouse body weight of 40 g. The food consumption and bodyweight were assessed weekly. After three months, body temperature, serum biochemical indexes, mobility data were acquired. After that, mice were dissected, tissues were instantly frozen in liquid nitrogen, and kept in −80 °C fridge for further use.63. The color of nodes and edges were labeled according to the RNA expression data and lifespan data in this article.MI metabolic pathway from KEGG was imported to Cytoscape with the plug-in application KEGGscape2 quantified according to the kit manual .About 400 worms per sample were homogenized with Bead Rupter 24 Elite (OMNI). The lipid was extracted, and PIPg for 10 min at 4 °C, and the supernatant was used for immunoblotting analysis. Bio-Rad Protein Assay Dye Reagent was used for measuring protein concentrations in lysates. 20–50 μg of protein was used for immunoblotting. The blotting was conducted following standard protocol.For immunoblotting assay, cells or mouse tissues were homogenized and lysed at 4 °C in lysis buffer  Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 μg/ml pepstatin). The cell or muscle lysates were centrifuged at 16,000 × myo-tube-like C2C12 was treated with or without 50 mM MI for 24 h in DMEM. Oxygen consumption assay was conducted according to the kit manual, and measured with XF24_3 Extracellular Flux Analyzer. 10 mM Glucose, 1 mM Pyruvtae, and 1 mM GlutaMAX were supplemented in the assay medium. Final 1 μM Oligomycin, 2 μM FCCP, 0.5 μM Rotenoe/Antimycin A were used to interfere with the mitochondria respiration complex. The data were analyzed with Wave (Version -2.6.0.31).C2C12 cell line was obtained from the Core Facility for Stem Cell Research in SIBS, and cultured in DMEM with 20% FBS. Cells were seeded on Agilent Seahorse XF24 microplate (Part No. 100777-004), grown to 100% confluency, then 2% horse serum was used to induce differentiation. The differentiated 64.For the oxygen consumption assay of the worm, 40 worms of each treatment, were picked to M9 buffer in XF24 microplate, washed several times to remove bacteria. 10 μM FCCP was used to induce max respiration. Both basal and max respiration were measured six timesDA2123 worms were treated with MI as lifespan assay until AD_8, then were stained with 0.5 mg/ml Mitotracker  in M9 buffer for 3 h. After washing, worms were mounted on a 2% agarose pad, with cover clips and fixed with finger nail oil. Images were made under 63× CONFOCAL microscopy. Manders’ coefficient calculated by Coloc2 in Fiji (version 2.0.0) is used as colocalization index in quantification. Both M1 and M2 are calculated.n labels the sample size, which was determined by experience rather than previous statistics, N indicates the biological replicates. Student t-test was used for significance test, log-rank test was used for survival data analysis. The p-values for the GSEA test statistics were calculated by feature permutation for 1000 times.All experiments were repeated at least twice, unless otherwise stated. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Reporting Summary


We prepared low-density polyethylene (LDPE) nanofiber, a few hundred nanometers in diameter, using polyvinyl butyral (PVB) and a laser melt-electrospinning (M-ESP) device. We blended PVB with LDPE via an internal melt mixer, removed the PVB after M-ESP by ethanol treatment, and studied the influence of PVB on fiber diameter. A substantial diameter reduction with improved crystallinity of LDPE fiber was observed with increased PVB content in the blend. PVB inclusion also increased the polarity of the LDPE/PVB blend, resulting in better spinnability. The removal of PVB from LDPE/PVB blend fiber caused a massive drop in the LDPE fiber diameter, due to fiber splitting, particularly in PVB-rich samples. Fourier transform infrared (FTIR) spectroscopy of fibers confirmed that the prepared nanofiber was the same as pure LDPE fiber. Electrospinning is an electrohydrodynamic process that draws super-fine continuous fibers using electrostatic force to collect the liquid jet from a polymer solution or melt. This process is widely used to fabricate nanofibers from 50 or more varieties of polymers, due to its simplicity and suitability ,2. It ha2 laser heating source, we introduce a new electrospinning technique that simplifies the high-temperature setup of M-ESP .,20.19,20Sheng et al. have reported ultra-drawn high-quality PE nanofibers with a diameter of only 50–500 nm, using the two-stage heating of polymer gel . Moreove2 laser beam M-ESP device. In particular, we investigated the effect of PVB content on fiber spinnability, crystallinity, and diameter.Polymer density is the main classifier of PE grades, and the first PE grade developed was low-density polyethylene (LDPE). LDPE is an excellent amorphous polymer with a relatively low melting point and good flow behavior, due to long side-chain branching . As a reρ) of 1.12 g/cm3, melting point (mT) of 140 °C, and a melt flow rate (MFR) of 14 g/10 min, was a gift from the Kuraray Co. Ltd., Tokyo, Japan. LDPE with MFR: 13 g/10 min, mT: 105 °C and ρ: 0.919 g/cm3 was the kind gift of the Tosoh Corp., Japan. Ethanol (99.5%) was also procured from the Nacalai Tesque, Inc., Kyoto, Japan.A powder form of the blend PVB component, grade B60HH, comprised of 80–87% vinyl butyral, 12–16% vinyl alcohol, and 1–4% vinyl acetate, with a density  was employed at 190 °C with a pressure of 25 MPa to fabricate LDPE/PVB sheets of 120 mm × 60 mm × 0.2 mm for preparation of nanofibers by laser M-ESP. Pure PVB and LDPE films were also made for comparison.Various specimen blends were prepared with a kneading extruder , by adding 10, 30, 50, 70, and 90 weight-% (wt.%) PVB to LDPE. Melt blending was conducted under N2 laser melting device, at a room temperature of 25 ± 2 °C, and 68% ± 2% relative humidity; further details regarding the equipment were provided in our previous paper [From LDPE/PVB blended film, fibers were fabricated by the M-ESP system equipped with a line-like COus paper . The ble−1, respectively. The crystallinity  of produced films and fibers was carried out using DSC-60 . Samples were heated from 30 to 200 °C, held for 5 min, and cooled to 30 °C at a rate of (+) 10 °C and (−) 4 °C minequation : (1)Xc(%88 J g−1 ), and ꞶP−1. Then, frequency sweep tests were carried out between 0.1 and 100 rad s−1 at a strain amplitude of 5%. All tests were performed at 190 °C under a nitrogen atmosphere. Disc polymer samples, 25 mm in diameter and 1-mm thick, were prepared using a hot press for tests.The rheological properties of samples were measured using a modular compact rheometer . A parallel plate, 25 mm in diameter and with a gap of 1 mm was used. To determine the linear viscoelastic region, strain sweep experiments were conducted at a frequency of 1 rad sD and σ), respectively, from the SEM images. Measurements were conducted for 3–5 SEM images of different areas of a sample, and more than 100 fibers were employed. Additionally, histograms were prepared for all individual sets of evaluations. Scanning electron microscopy (SEM) of fibers and film surfaces was carried out using a Keyence microscope . An ion coater  was used to gold-sputter coat the sample surface before SEM observation. Image analysis software (Photo Ruler) was used to measure the average and standard deviation of the PVB particle size in blend films and fiber diameters  equipped with a diamond/ZnSe crystal embedded single reflection ATR accessory . The characteristic band for pure PVB, pure LDPE, blended LDPE/PVB, and PVB-removed LDPE fibers were recorded.The Fourier transform infrared (FTIR) spectrum was measured at room temperature within a range of 400–4000 cmDSC measurements of the LDPE/PVB blend films verified the melting behavior of the compounds. From the DSC results, the crystallinity (%) of LDPE was calculated, as shown in D = 19.75 µm) diameter with a higher standard deviation (σ = 11.46 µm) was found for pure LDPE fiber. The addition of PVB increased the number of Taylor cones per cm , 1463 cWe successfully minimized the fiber diameter of LDPE nanofibers, producing fibers that are a few hundred nanometers in diameter by laser M-ESP system, using PVB as a blend component. Here, the process involved two-step fiber diameter reduction: during M-ESP via the inclusion of PVB and by subsequently removing PVB from LDPE/PVB blend fiber. A massive blend fiber diameter drop was observed with even a low 10% inclusion of PVB, and the trend continued linearly up to 90% PVB content. In addition, PVB improved the polarity and drawing effect of LDPE/PVB blend films during laser melting, which facilitated the production of a more delicate fiber with better spinnability. A further drop of LDPE fiber diameter was achieved after PVB was removed from LDPE/PVB blend fibers, due to the fiber splitting, mostly in PVB-rich samples. Consequently, LDPE nanofibers with a diameter of 235 ± 141 nm were obtained from blended fiber using 90% PVB. DSC results also revealed that the improved crystallinity of LDPE in the film was decreased for M-ESP but regained after ethanol treatment. These superfine polyethylene nanofibers constitute a promising material for battery separators and filtration media for waste-water treatment.


In the frame of graphene-based material (GBM) hazard characterization, particular attention should be given to the cutaneous effects. Hence, this study investigates if HaCaT skin keratinocytes exposed to high concentrations of few-layer graphene (FLG) or partially dehydrated graphene oxide (d-GO) for a short time can recover from the cytotoxic insult, measured by means of cell viability, mitochondrial damage and oxidative stress, after GBM removal from the cell medium. When compared to 24 or 72 h continuous exposure, recovery experiments suggest that the cytotoxicity induced by 24 h exposure to GBM is only partially recovered after 48 h culture in GBM-free medium. This partial recovery, higher for FLG as compared to GO, is not mediated by autophagy and could be the consequence of GBM internalization into cells. The ability of GBMs to be internalized inside keratinocytes together with the partial reversibility of the cellular damage is important in assessing the risk associated with skin exposure to GBM-containing devices. Togetgraphite ,4,5. Thegraphite . On the graphite .However, the use of GBMs in various technological fields requires the assessment of their potential impact on both human health and the environment. For this reason, the risk assessment of GBMs must be an integral part of the innovation process . To dateCompared to toxicity studies related to other exposure routes, the characterization of GBM toxicity after cutaneous exposure is based on rather limited data, derived from in vitro studies on skin fibroblasts , keratinFLG and dehydrated GO (herein named d-GO) were endotoxin-free to avoid false-positive results due to lipopolysaccharide (LPS) contamination. Both materials were the same as used in our previous study, in which full physicochemical characterization after endotoxin removal by heat treatment was carried out .Endotoxin-free FLG. Endotoxin-free FLG was prepared by heat treatment of FLG powder, prepared through a solvent-free ball-milling under air atmosphere, as previously described [2 conditions, as previously described [escribed ,18. Heatescribed .Endotoxin-free GO (d-GO). Endotoxin-free GO was prepared by heat treatment of commercially available GO  at 200 °C for 1 h in a protective Ar atmosphere. As previously demonstrated, since Raman and thermogravimetric analyses showed slight physicochemical alterations of GO samples after the heat treatment, ascribed to water loss, this material was accordingly renamed dehydrated GO (d-GO) [O (d-GO) .−2 M L-glutamine, 1.0 × 10−4 g/mL penicillin and 1.0 × 10−4 g/mL streptomycin at 37 °C in a humidified 95% air/5% CO2 atmosphere. All reagents were purchased from Sigma-Aldrich . Cell passage was performed once a week and 2 days post-confluence. All the experiments were performed between passage 63 and 74. HaCaT cells were routinely checked for the absence of Mycoplasma infection.HaCaT cells are spontaneously immortalized non-tumor keratinocytes, cultured in high glucose Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1.0 × 10Cells were exposed to GBMs (1–100 µg/mL) under different conditions: (i) continuous exposure for 24 or 72 h and (ii) recovery exposure, consisting of 24 h cell exposure to GBMs, followed by three washes in PBS to remove cell-unbound materials and additional cells culture for 48 h in GBM-free fresh medium (24 + 48 h). Representative optical images of cells exposed to FLG or d-GO showing materials sediments above cells are available in v/v). As a positive control, cells were exposed to 5% sodium dodecyl sulphate (SDS). Absorbance was subsequently determined at 450 nm by an Automated Microplate Reader EL 311s . Data are reported as % cell viability as compared to negative control (cells not exposed to GBMs).The effects of GBMs on cell viability were evaluated by the WST-8 reduction assay, based on the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium probe, as previously described . BrieflyMitochondrial damage was evaluated by means of mitochondrial depolarization using the JC-1 Mitochondrial Staining Kit , as previously described . BrieflyROS production was evaluated using the 2′,7′-dichlorofluorescin diacetate (DCFDA) probe , as previously described . Brieflyw/v) sodium dodecyl sulfate (SDS); 10% (v/v) glycerol and 5% (v/v) β-mercaptoethanol. Cell lysates were separated on a 4–20% gradient gel (Biorad), transferred onto a polyvinylidene difluoride (PVDF) membrane and blocked with PBS added with 0.1% (v/v) Tween 20 (PBS-T) and 5% (w/w) non-fat dry milk for 1 h. Membranes were then incubated for 50 min at 4 °C with 1 µg/mL primary antibodies in PBS-T containing 5% non-fat dry milk. The antibodies used were specific for the LC3 autophagic marker  and the β-actin loading control . After washing with PBS-T, membranes were incubated for 30 min at room temperature with goat anti-rabbit IgG antibody  conjugated to horseradish peroxidase (HRP). The signal was detected using enhanced chemiluminescence detection reagents  and visualized on radiographic film in a Kodak processor .To evaluate autophagic activity, HaCaT cells were exposed to 1–100 µg/mL GO or FLG for 24 h, and with or without 5 µM ammonium chloride for the last 3 h. Then, whole HaCaT cell proteins were extracted using Laemmli buffer, constituted by 125 mM Tris-HCl buffer pH 6.8, 2%  with Epon™. After soaking, final inclusion of Epon™ into the cells was done by polymerizing Epon™ at 40 °C for 30 min then incubating at 60 °C for 48 h. Afterwards, the polymerized blocks were removed and sliced into ultrathin sections using a diamond knife attached to a ultramicrotome cutter . The ultrathin sections were then collected on copper grids coated with Butvar® B-98 and stained with 1% uranyl acetate for 30 min followed with lead citrate staining for 2 min. Grids were then examined by TEM . Control cells without FLG underwent the same procedure.For TEM  imaging, HaCaT cells were cultured in 12-well plates at a density of 1 × 10p values < 0.05.Results of cytotoxicity assays are the mean ± standard error (SE) of at least 3 independent experiments performed in triplicate. Statistical analysis was performed by a one-way or two-way ANOVA followed by Bonferroni’s post-test  and statistical significance was considered for To investigate the reversibility of GBM-induced cytotoxicity toward HaCaT keratinocytes, cells were exposed to FLG or d-GO (1–100 µg/mL) for 24 or 72 h (continuative exposure), or for 24 h followed by 48 h culture without GBMs . Then, cell viability was evaluated by the WST-8 assay.p < 0.01) and 59% (p < 0.001), respectively. The cytotoxic effect of 24 h exposure to FLG or d-GO was slightly, but not significantly (p > 0.05), reverted culturing the cells for further 48 h in GBM-free conditions . In addition, cell viability recorded after 24 h exposure to FLG (10 and 100 µg/mL) or d-GO (100 µg/mL) followed by 48 h recovery in GBM-free medium  was significantly higher than that recorded after continuous exposure to GBMs for 72 h. In particular, as compared to negative controls, viable cells after exposure to 10 and 100 µg/mL FLG under the recovery condition (24 + 48 h) were 99% (p < 0.01) and 84% (p < 0.01), respectively, significantly higher than those recorded after 72 h continuous exposure to the same FLG concentrations . As compared to negative controls, the viability of cells exposed to 100 µg/mL d-GO under the recovery condition  was significantly higher than that recorded after 72 h continuous exposure .As depicted in These data suggest that both FLG and d-GO induce a cytotoxic effect toward HaCaT keratinocytes already after 24 h exposure, which can be partially reverted upon the removal of GBMs, in particular FLG.To investigate the reversibility of GBM-induced mitochondrial damage, HaCaT cells were exposed to FLG or d-GO (1–100 µg/mL) for 24 or 72 h (continuous exposure), or for 24 h followed by 48 h culture in GBM-free medium . Then, mitochondrial damage was evaluated by means of mitochondrial depolarization using the potentiometric JC-1 probe.p < 0.05) and 9% (p < 0.05), respectively, if cells were cultured for a further 48 h under the recovery condition (24 + 48 h). Similarly, 24 h exposure to 10 and 100 µg/mL d-GO increased mitochondrial depolarization at 26 and 47%, respectively, which was significantly reduced to 8% (p < 0.05) and 15% (p < 0.05), respectively, under the recovery condition (24 + 48 h).As shown in p < 0.01), 10% (p < 0.01) and 9% (p < 0.01), respectively. Cells exposed to 1, 10 and 100 µg/mL d-GO for 72 h showed increased mitochondrial depolarization at 28, 35 and 60%, respectively, significantly higher than that recorded under the recovery condition (24 + 48 h): 6% (p < 0.01), 8% (p < 0.01) and 15% (p < 0.001), respectively.Mitochondrial depolarization induced by 24 h exposure to GBMs followed by 48 h recovery in GBM-free medium  was significantly lower than that induced by continuous exposure to the materials for 72 h. In particular, as compared to negative controls, 72 h exposure to 1, 10 and 100 µg/mL FLG increased mitochondrial depolarization at 22, 41 and 41%, respectively, significantly higher than that induced under the recovery condition (24 + 48 h), corresponding to 2%  for 24 or 72 h (continuative exposure), or for 24 h followed by 48 h recovery in GBM-free medium . Then, ROS production was evaluated by the DCFDA fluorescence dye.p < 0.01), 12% (p < 0.05) and 25% (p < 0.05), respectively.As depicted in p < 0.01), 12% (p < 0.01) and 25% (p < 0.05), respectively.In addition, d-GO-induced ROS production induced by each concentration after the recovery condition (24 + 48 h) was significantly lower than that induced by 72 h continuous exposure to this material, whereas no significant difference was recorded for FLG. In particular, as compared to negative controls, 72 h exposure to 1, 10 and 100 µg/mL d-GO increased ROS production at 30, 40 and 128%, respectively, levels significantly higher than those detected under the recovery condition (24 + 48 h): 1%  and the corresponding untreated sample.To better understand the impact of GBMs on autophagy, HaCaT cells were continuously exposed for either 24 or 72 h with 1, 10 and 100 µg/mL FLG or d-GO, before harvesting and blotting with LC3 antibodies. A,B The rAll results were non-significant, with no change in the autophagic flux for d-GO and a non-significant trend to decrease the autophagic flux for FLG E,F.Given that the major reversibility of cytotoxicity was observed in case of FLG, cell internalization analysis was performed considering this material. In control untreated cells , top rawFor this experiment, we focused our attention on FLG. In the FLG 72 h-treated cells  and evaluating the recovery of the damage after their removal. To this aim, we used a research grade FLG and a d-GO, as representative of GBMs. Both materials were endotoxin-free, avoiding result alteration due to false positive data given by LPS contamination, a common feature of GBMs  h and ev. IntriguOn the basis of these considerations, herein, we investigated if the cytotoxic effects of high FLG and d-GO concentrations in HaCaT cells can be reverted by the material’s removal, as an in vitro approach to investigate the reversibility of GBM damage. To this aim, we compared the effects of GBMs under a recovery condition (24 h cells exposure to GBMs followed by 48 h culture in GBM-free media) to those induced by continuous cell exposure to the materials for 24 or 72 h. FLG and d-GO were tested at concentrations previously shown to induce a significant cytotoxic effect (1–100 µg/mL) in HaCaT skin keratinocytes , and a p2-bearing groups in comparison to FLG [Considering ROS production, a different behavior was found between FLG and d-GO. In particular, only ROS production by HaCaT cells exposed to d-GO was significantly reduced upon the material removal from culture medium, whereas no significant effect was observed in case of FLG removal. This observation suggests that only d-GO induced oxidative stress is a partially reversible cytotoxicity parameter. This conclusion could rely on the different oxidative capabilities of the two materials, d-GO being more potent in inducing ROS production, probably due to the higher density of On to FLG ,26. AddiIn general, the results of this study demonstrate that GBMs effects on skin keratinocytes are only partially reversible. This conclusion is particularly evident for FLG, probably because of its slightly lower cytotoxicity in comparison to d-GO on these cells ,13. The Under recovery conditions, such as those employed in our study, a key phenomenon able to recover cells from damages is represented by autophagy. Indeed, autophagy plays an important role in mediating cell survival and maintenance, through the degradation and recycling of cytoplasmic organelles, proteins, and macromolecules. The role of autophagy in the mechanism of nanomaterial toxicity has been well-reviewed by many groups ,28. In tLike what other researchers have reported with FLG in cellular TEM , there iIn conclusion, the results of this study demonstrate a partial reversibility of GBM-induced cytotoxicity in skin HaCaT keratinocytes. In particular, recovery experiments suggest that the cytotoxicity induced by 24 h exposure to GBMs is only partially reduced by the material’s removal followed by culturing of cells for additional 48 h. This partial recovery could be ascribed to the material internalization by cells, which could allow them to continue their cytotoxic effect, even if with a slightly lower potency. In addition, this partial reversibility seems to be not mediated by autophagy activation, as a key phenomenon in mediating cellular damage recovery. Altogether, these results represent a step forward to characterize the hazard potential of GBMs at the skin level. In particular, the ability of GBMs to be internalized inside keratinocytes together with the partial reversibility of the cellular damage acquires a significant importance in assessing the risk associated with skin exposure to GBM-enriched devices, especially in the case of possible GBMs release.


Bone mass loss in aging is linked with imbalanced lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs). Recent studies have proved that histone deacetylases (HDACs) are regarded as key regulators of bone remodeling. However, HDACs involve in regulating BMMSC bio-behaviors remain elusive. Here, we investigated the ability of HDAC9 on modulation of autophagy and its significance in lineage differentiation of BMMSCs.The effects of HDAC9 on lineage differentiation of BMMSCs and autophagic signaling were assessed by various biochemical (western blot and ChIP assay), morphological , and micro-CT assays.HDAC9 elevated in bone and BMMSCs. Importantly, HDAC9 inhibitors recovered the lineage differentiation abnormality of 16-month BMMSCs and reduced p53 expression. Mechanistically, we revealed that HDAC9 regulated the autophagy of BMMSCs by controlling H3K9 acetylation in the promoters of the autophagic genes, ATG7, BECN1, and LC3a/b, which subsequently affected their lineage differentiation. Finally, HDAC9 inhibition improved endogenous BMMSC properties and promoted the bone mass recovery of 16-month mice.Sixteen-month mice manifested obvious bone mass loss and marrow fat increase, accompanied with decreased osteogenic differentiation and increased adipogenic differentiation of BMMSCs. Further, the expression of Our data demonstrate that HDAC9 is a key regulator in a variety of bone mass by regulating autophagic activity in BMMSCs and thus a potential target of age-related bone loss treatment. Osteoporosis is a common aged-related disease and is characterized by decrease bone mass and bone mineral density, leading to bone fragility and a higher risk of fractures , 2. The Bone marrow mesenchymal stem cells (BMMSCs) are a group of cell residual in the bone marrow. They have self-renewal capacity and multilineage differentiation potential. There is considerable data showing that BMMSCs play crucial roles in maintaining bone remodeling, reparation, and regeneration , 6. ImpoOct4 and Nanog [p16 and p53 [During senescence, mesenchymal stem cells (MSCs) undergo epigenetic and transcriptional changes, including decreased expression of stemness genes, nd Nanog , and inc and p53 . Some ad and p53 , 12. HowHDAC1–3 and HDAC8, while class II HDACs include HDAC4–7, HDAC9, and HDAC10. Class III HDACs, also known as named sirtuins (Sirt1–7), differ from the other HDACs, as they are dependent on nicotinamide adenine dinucleotide. Class IV HDAC has only one member, HDAC11. HDAC family members have been shown to be involved in a wide range of aging-associated diseases, including muscle atrophy, loss of physical activity, and neurodegenerative diseases [HDAC3 and HDAC8 supported bone formation [HDAC4, HDAC7, and HDAC9 promoted bone resorption [HDACs are important epigenetic regulators that control gene transcription by removing acetyl groups from lysine side chains in histones and other proteins , 14. Mamdiseases , 15, 16.sorption –19. HoweIn this study, we report that HDAC9 plays an important role in maintaining the balance between osteogenesis and adipogenesis of BMMSCs during aged-related bone mass loss. Furthermore, we found that the downregulation of HDAC9 could partially reverse the differentiation of aging BMMSCs and bone loss by regulating autophagy both in vitro and in vivo. These results suggest that aged-related bone mass loss may be partially controlled by the HDAC9-meditated autophagy of BMMSCs.All animal procedure, operations, and experiments were approved and performed, and experimental protocols were approved by the guidelines of the Animal Care Committee of the Fourth Military Medical University, Xi’an, Shaanxi, China. Two-month-old female C57BL/6J mice were purchased from the Animal Experimental Center of the Fourth Military Medical University. Sixteen-month-old female C57BL/6J mice were purchased from Changzhou Kaiwensi Laboratory Animal Center, Jiangsu, China. All mice were housed under specific pathogen-free conditions  on a 12-h light/12-h dark cycle with food and water easily accessible.The mouse femora at mid-diaphysis were scanned with the GE micro-CT system . X-ray source was set at 80 kV and 80 μA microfocus. Three-dimensional images were reconstructed, and data analysis was performed with GEHC MicroView analysis software. The relevant bone morphometric parameters, including trabecular bone mineral density (BMD), trabecular volume relative to total volume (BV/TV), and cortical bone thickness (Ct.Th), were measured.l-glutamine , 100 U/mL penicillin, and 100 U/mL streptomycin ; and incubated at 5% CO2 at 37 °C. The medium was changed every 3 days. Cells were digested with 0.25% trypsin when confluence reached 90%. BMMSCs used for the majority of experiments were at passage 1 in this study.Bone marrow-derived mesenchymal stem cells were harvested from the femora and tibiae of 2-month-old young C57BL/6J and 16-month-old aged C57BL/6J mice. Mice were sacrificed with cervical dislocation and sterilized with 75% alcohol. The femora and tibiae were separated, and attached muscle was stripped. After epiphyses were amputated and the bone marrow was exposed, primary BMMSCs were drawn out; cultured with basal medium containing α-MEM medium , 20% FBS , 2 mM g, and the pellets were suspended in myoblast growth medium (Ham’s F-10 medium with 10% FBS) and incubated at 5% CO2 at 37 °C. Briefly, the cell suspension was seeded for 15–30 min to allow quick adherence of fibroblasts, thus leaving a purer population of myoblasts in the supernatant, which was then transferred to another dish for subculturing. Myoblasts were digested with 0.25% trypsin when confluence reached 80%.Muscles were harvested from the hind limbs of 2-month-old young C57BL/6J and 16-month-old aged C57BL/6J mice. Firstly, the excessive connective tissues and fat were separated from muscle in cold sterile PBS. Then, muscles were cut into small pieces and enzymatically digested with 400 IU/mL collagenase II (Worthington) at 37 °C for 1 h. The digested slurry was sequentially passed through a 70-μm and then 30-μm cell strainer . The filtrate was centrifuged at 1000The femora were fixed in 4% paraformaldehyde, decalcified with 17% EDTA (pH 7.0), dehydrated with 30% sucrose, and embedded in OCT. Fifteen-micrometer-thick longitudinal sections were prepared and collected on slide for SAbetaGal staining. The β-galactosidase activity was assessed with a SAbetaGal staining kit . Briefly, the slices were washed twice with PBS and fixed with Fixative Solution of the SAbetaGal staining kit at room temperature for 10–15 min. Then, the samples were washed twice with PBS, covered with SAbetaGal staining solution, and incubated at 37 °C overnight. On the next day, the slides were washed with PBS three times and 80% glycerin was mounted on the samples. Then, coverslips were inverted onto slide and excess glycerin was removed. The SAbetaGal positive cells were observed under a microscope.4 cells/cm2 on 6-well or 12-well plastic plates and cultured with basal medium. When cell confluence reached 80%, cells were induced with osteogenic differentiation medium  up to 1 week for western blot assay or 2 weeks for Alizarin Red staining assay, with the medium changed every 3 days. To assess osteogenic differentiation, cells were fixed with 4% paraformaldehyde and stained with 1% Alizarin Red. The expressions of Runx2 and ALP were detected by western blot.Young and aged BMMSCs were seeded at the density of 4.2 × 10Young and aged BMMSCs were cultured using the same method described above. When cell confluence reached 85%, cells were induced with adipogenic differentiation medium  up to 5 days for western blot or 7 days for Oil Red O staining, with the medium changed every 3 days. Lipid droplet formation in cells was detected by staining with Oil Red O solution. The expression of PPAR-γ was detected by western blot.For cell Oil Red O staining, the BMMSCs were fixed with 4% paraformaldehyde for 15 min at room temperature and then stained with Oil Red O staining for 15 min at room temperature. The stained cells were washed twice with PBS and then observed under microscope. The images were analyzed with Image Pro Plus software.2O. Then, the cells were stained with 1% Alizarin Red (Sigma Aldrich) for 3–5 min and washed twice with ddH2O. Quantitative parameters of the mineralized area were analyzed with Image J software.Alizarin Red staining was performed to determine mineralization after 14 days of osteogenic induction. BMMSCs were fixed with 60% isopropanol for 90 s and washed once with ddH2O and the positive areas were observed under microscope.Alcian blue staining was carried out to detect the chondrocytes in growth plates of the femora. Firstly, the samples of decalcified femora were cut into 10 μm thick and mounted on slides. Then, samples were stained with 0.1% Alcian blue staining solution containing acetic acid for 20 min at room temperature. Then, slides were washed twice with ddH4. Cells were treated with or without the autophagy-flux inhibitor chloroquine (CQ) (50 μM) 3–5 h before staining. In sequence, cells were fixed with 4% paraformaldehyde at 4 °C for 10–15 min, washed with PBS, incubated with 0.5% Triton-100 at room temperature for 10 min, and blocked with PBS containing 1% BSA at room temperature for 40 min. Next, the samples were incubated with primary antibodies to LC3 , aggrecan , collagen II , OCN , PPAR-γ , and TRAP  overnight at 4 °C and subsequently incubated with fluorescent secondary antibodies, respectively. The positive cells were examined under a laser scanning confocal microscope . Quantitative histomorphometric analysis was conducted with Image Pro Plus software.For cell immunofluorescence staining, cells were seeded on 3.5 cm confocal dish at the density of 3 × 10Cells were harvested and fixed in 4% glutaraldehyde in 0.1 M PB (pH 7.4) for 24 h, followed by 1% osmic acid for 2 h. After fixation, cells underwent osmosis by acetone and 812 resins. Then, samples were embedded with Epon 812 and kept in a thermostatic drying oven for 4 h at 36 °C, 6 h at 45 °C, and 12 h at 60 °C. Afterwards, embedding blocks were successively cut into semithin sections (1–2 μm) and ultrathin sections (50–100 nm). Then, samples were stained with uranyl acetate and lead citrate. Finally, images were captured with a transmission electron microscope  at an accelerating voltage of 80–120 kV.Total RNA was extracted from BMMSCs, myoblasts, bone marrow, and muscle by RNAiso plus  according to the manufacturer’s instruction. Then, the mRNA was reversely transcribed into cDNA by PrimescriptTM RT master mix . Real-time PCR was performed with SYBR Premix Ex TaqTMII (TaKaRa) and detected by CFX96 Trademark Real-time PCR detection system . The primers used in real-time PCR were listed in the Supplementary Table Total proteins were harvested from BMMSCs, bone marrow, and other organs with RIPA lysis buffer  and quantified by BCA assay. Next, the proteins were separated on sodium dodecyl sulfonate-polyacrylamide gels (SDS-PAGE), transferred to PVDF membranes , blocked in TBST containing 5% BSA, and incubated in first antibodies with Beclin1 , ATG7 , LC3 , p62 , HDAC9 , H3K9ac , H3K18ac , H4K16ac , H3 , p53 , phospho-p53 Antibody , Runx2 , ALP , PPAR-γ , and GAPDH , respectively. Then, the membranes incubated in secondary antibodies which coupled to peroxidase . Finally, the signals were detected by an enhanced chemiluminescence kit .BMMSCs were cultured in the medium with Trichostatin A  at 50, 100, and 200 (nmol/L) or sodium butyrate  at 50, 100, 200, and 400 (μmol/L). To assess the effects of TSA or NaB on inhibition of HDACs in cells, HDAC1–11 were detected by qRT-PCR. The expressions of HDAC9 and acetylated H3k9 were detected by western blotting. To examine the effects of HDACs on BMMSC differentiation, the cells were treated with or without NaB or TSA at their effective concentrations selected above.Small interfering RNA (siRNA) targeting mice BECN1  or HDAC9  were transfected into cells at a final concentration of 50 nM using riboFECT™ CP . siNC  was used as negative controls. All steps were performed according to the instruction in the riboFECT™ CP kit. The following experiments were performed according to the experiment designed. In detail, siRNA silencing HDAC9 was transfected into BMMSCs to investigate the effects of HDAC9 on osteogenic and adipogenic differentiation of BMMSCs and binding to the promoter of autophagy-related genes. In addition, aged BMMSCs were co-transfected with siHDAC9 and siBECN1 to investigate the hypothetical HDAC9-autophagy axis which may regulate BMMSC lineage differentiation.To confirm the interaction between HDAC9 and targeting genes, chromatin immunoprecipitation assays were performed according to the manufacturer’s protocol . Antibodies against HDAC9  and polyclonal anti-histone H3 (acetyl K9)  were incubated with randomly interrupted genome DNA samples. Normal rabbit IgG (Merck Millipore) was used as a negative control. Then, antibody-DNA complexes were captured by protein A/G magnetic beads. Finally, the precipitated DNA samples were detected by qRT-PCR, and the results were normalized to the input value. The primers designed according to the promoters of Atg7, BECN1, LC3a, and LC3b  are listed in Supplementary Table g and the precipitate was resuspended with PBS whose volume is 1/100 of collected medium. The final virus titer was more than 1 × 108/mL. To evaluate the effect of HDAC9 on bone mass/skeletal metabolism in senescence-induced bone loss, aged mice (n = 21) were randomly divided into three groups (7/each group), namely the control group, the shScr group, and the shHDAC9 group, respectively. Aged mice were anesthesized through intraperitoneal injection with 1% pentobarbital sodium and were administrated with 20 μL of lentivirus containing shHDAC9 or empty vector every 2 weeks for 1 month through intra-bone marrow injection in the distal femora. After a 1- or 2-month treatment, the mice from above three groups were sacrificed by cervical dislocation and the femora from each group were collected for micro-CT scanning, Oil Red O staining, and SAbetaGal staining. BMMSCs were harvested and cultured from the femora for lineage differentiation analysis and autophagy analysis as above methods.The shRNA sequences for targeting HDAC9 were as follows: forward sequence—5′-CCGGCTGGGCACAAAATTCTGAACACTCGAGTGTTCAGAATTTTGTGCCCAGTTTTTG-3′; reverse sequence—5′-AATTCAAAAACTGGGCACAAAATTCTGAACACTCGAGTGTTCAGAATTTTGTGCCCAG-3′. The shHDAC9 lentivirus was packaged by co-transfecting shHDAC9 lentiviral vector with two packaging systems (pMD2.G and psPAX2) in 293T cells which were purchased from China Center for Type Culture Collection , and the medium containing virus was collected and concentrated 48 h later. Detailedly, the medium was centrifuged at 800 rpm and 4 °C for 10 mins, and the supernatant was filtered by a 0.45-μm filter. Then, the filtrate was mixed with Lenti-X  and kept at 4 °C overnight. The next day, the mixture was centrifuged for 40 min at 1500t test and one-way ANOVA for experiments with more than two groups. All of the experiments were repeated more than three times, with representative experiments shown. The P values of less than 0.05 were considered significant.The data were presented as the mean ± SD. Comparisons were made using HDAC9 expression remarkedly increased, while HDAC5 and HDAC7 expression levels reduced in aged mice. Meanwhile, there was no significant difference in the expression levels of HDAC1–4, HDAC6, HDAC8, HDAC10, or HDAC11 in the bone marrow from young and aged mice  and aged mice (16-month-old) were compared. The micro-CT analysis showed that the aged mice presented typical aged-related bone mass loss, including decreased bone mineral density (BMD) and trabecular bone volume (BV/TV), whereas no cortical bone thickness difference was observed  during osteogenic and adipogenic differentiation. Firstly, BMMSCs were treated with different doses of TSA  and NaB  to examine changes in H3K9 acetylation. Western blotting results showed that 100 nmol/L and 200 nmol/L of TSA . We found that HDAC9 inhibition promoted osteogenic differentiation and repressed adipogenic differentiation in aged BMMSCs , sodium butyrate (NaB), and Our previous study showed that autophagy is a key regulator of BMMSC differentiation in OVX-induced osteoporosis and aged-induced bone mass loss , 21. To HDAC9 expression using HDAC9 siRNA. The results showed HDAC9 expression significantly decreased and H3K9 acetylation levels increased in aged BMMSCs transfected with HDAC9 siRNA  assays were performed in BMMSCs. ChIP analysis was performed using antibodies that individually recognize either acetylated H3K9 (H3K9ac) or HDAC9, and the primers were used to amplify the promoter regions of HDAC9 siRNA effectively improved aged BMMSC differentiation and that HDAC9 regulates autophagy, we next examined whether HDAC9 affect the differentiation of aged BMMSCs by regulating autophagy. We silenced HDAC9 and BECN1, a key gene in the regulation of autophagosome formation [HDAC9 siRNA on improving the osteogenic differentiation of aged BMMSCs was neutralized by silencing BECN1 expression  of aged mice was observed 4 weeks or 8 weeks after shHDAC9 treatment  under inflammatory microenvironment . Kofman  of MSCs , 34. CheAutophagy is a very important degradation system, playing a crucial role in maintaining cell homeostasis in response to cellular stress , 36. AutLC3 transcription by histone methyl-transferase (HMT) G9a [BECN1 promoter [ATG7, BECN1, and LC3a/b, by ChIP analysis. Inhibition of HDAC9 expression in aged BMMSCs by siRNA transfection reduced HDAC9 binding to the promoters of Atg7, BECN1, LC3a, and LC3b, but increased the levels of acetylated H3H9 interaction in the promoter of autophagy-related genes. Thus, we speculated the increased expression of HDAC9 in aged BMMSCs is an important factor leading to autophagic degeneration and this finding may uncover a new mechanism for the regulation of aged-related bone mass loss by HDAC9. However, there are still some unanswered questions. For example, the animal model we used was aged female mice with postmenopausal osteoporosis, which may not have an identical pathogenesis to aged male mice. Therefore, further investigation is required to determine whether the mechanism identified in this study is also applicable to aged male mice. Furthermore, HDAC9 was found to affect bone resorption [Epigenetic modifications associated with the aging process have received our attention over the past few decades. Epigenetic regulations have been widely studied in somatic stem cells to help us understand its roles in governing self-renewal or lineage differentiation. Meanwhile, some studies have reported epigenetic modifications, including acetylation of H4K16 associated with ATG genes , 40, regHMT) G9a , 41, andpromoter , which isorption , which iHDAC9 improved endogenous BMMSC properties and promoted the bone mass recovery in aged mice. Our finding provided a potentially promising target for the prevention of osteoporosis and treatment of aged-related bone mass loss.Taken together, our study suggested a potential role and mechanism of HDAC9 in BMMSC lineage commitment by regulating autophagy activities. Knockdown Additional file 1: sFig. 1 The expressions of HDACs family members in bone marrow and BMMSCs HDACs gene were examined in bone marrow (a) and BMMSCs (b) from young mice and aged mice by qRT-PCR. sFig. 2 The expressions of senescence associated proteins and HDAC9 in skeletal muscle and myoblasts. The expression of p53, HDAC9 and H3K9 acetylation, and HDAC9 in skeletal muscle (a-c) and myoblasts (d-f) were examined by western blotting and qRT-PCR from young mice and aged mice. sFig. 3 HDACs inhibitors promoted osteogenic differentiation and inhibited adipogenic differentiation in aged BMMSCs The expression of HDAC9 and H3K9 acetylation were detected in aged BMMSCs treatment with different dose of TSA (a) or NaB (b) by western blotting. Osteogenic differentiation (c) and adipogenic differentiation (d) were observed in aged BMMSCs treated with TSA or NaB. Scale bars = 100 μm. sFig. 4 The silence efficiency of HDAC9 siRNA HDAC9 and H3K9 acetylation were examined in aged BMMSCs after transfected with HDAC9 siRNA for 48 hours, 3 days and 7days by qRT-PCR  and western blotting . sFig. 5HDAC9 siRNA restored the number of autophagosomes in aged BMMSCs Autophagosomes in young, aged BMMSCs (a) and aged BMMSCs transfected with HDAC9 siRNA (b) were detected by TEM. Scale bars = 1 μm. sFig. 6HDAC9 siRNA decreased H3K9 acetylation in aged BMMSCs. The expression of HDAC9 and H3K9 acetylation were examined by western blotting in aged BMMSCs transfected with HDAC9 siRNA and those cells treated with CQ. sFig. 7 The silence efficiency of BECN1 siRNA Beclin1 was examined by western blotting in aged BMMSCs transfected with BECN1 siRNA 48 hours later. sFig. 8 No changes in growth plate thickness in aged mice were treated with HDAC9 shRNA lentivirus Alcian blue staining were performed to assess growth plate thickness.Additional file 2: Supplementary Table 1. Primer sequences for Real-Time PCR assay. Supplementary Table 2. Primer sequences for ChIP assay.