Saccharomyces cerevisiae. However, their functions remain largely unknown in plant fungal pathogens. Here, we report the identification and functional characterization of a plausible ortholog of yeast Hsp104 in Fusarium pseudograminearum, which we termed FpHsp104. Deletion mutant of FpHsp104 displayed severe defects in the resistance of heat shock during F. pseudograminearum mycelia and conidia when exposed to extreme heat. We also found that the protein showed dynamic localization to small particles under high temperature. However, no significant differences were detected in osmotic, oxidative, or cell wall stress responses between the wild-type and Δfphsp104 strains. Quantitative real-time PCR analysis showed that FpHsp104 was upregulated in the conidia, and disruption of FpHsp104 gene resulted in defects in conidia production, morphology, and germination. The transcript levels of conidiation-related genes of FpFluG, FpVosA, FpWetA, and FpAbaA were reduced in the Δfphsp104 mutant vs. the wild-type strain, but heat-shocked mRNA splicing repair was not affected in Δfphsp104. Moreover, Δfphsp104 mutant also showed attenuated virulence, but its DON synthesis was normal. These data from the first study of Hsp104 in F. pseudograminearum strongly suggest that FpHsp104 gene is an important element in the heat tolerance, development, and pathogenicity processes of F. pseudograminearum.Heat shock protein Hsp104, a homolog of the bacterial chaperone ClpB and plant Hsp100, plays an essential part in the response to heat and various chemical agents in  Fusarium pseudograminearum is a soil-borne plant pathogen that causes Fusarium crown rot (FCR) in wheat and barley. FCR has caused serious economic losses worldwide  pre-mRNAs are alternatively spliced in response to heat stress in mammalian cells  medium was used to induce conidia at 25°C and 150 rpm for 4 days.The is study . All strS. cerevisiae and Hsp98 (XP_957228.1) protein from Neurospora crassa (F. pseudograminearum genome (FpHsp104 (FPSE_03525) was blasted and downloaded from National Center for Biotechnology Information (NCBI). FpHsp104 homologs from Chaetomium thermophilum (XP_006692738.1), Magnaporthe oryzae (XP_003717107.1), and Fusarium graminearum (XP_011324022.1) identified in NCBI were downloaded and used to construct neighbor-joining trees for Hsp104 with the MEGA 5.05 software package, using the neighbor-joining method with 1,000 replicates for bootstrap analysis. ClpB from Escherichia coli and Hsp101 from Arabidopsis thaliana were used as the out-group. The domains of these proteins were predicted using the SMART website.1The Hsp104 (NP_013074.1) protein from a crassa  were usem genome  by BlastFpHsp104 expression during infection, WT conidia were collected in CMC liquid. Wheat coleoptiles were inoculated with 1 × 107 conidia/ml conidial suspensions for 18, 30 h, 2, 3, 5, and 7 days. To test FpHsp104 expression under heat shock pressure, the WT conidia were first cultured in YEPD at 25°C for 12 h, heated to 34°C for 1 h, and then returned to 25°C for 0, 3, 6, 12, 24, or 48 h. To determine the expression of conidiation-related genes in the WT, Δfphsp104, and Δfphsp104-C strains, conidia were produced in CMC at 25°C for 3 days. Total RNA from lyophilized mycelia, conidia, and infected plants was extracted using a KKFast plant RNApure Kit  and transcribed into complementary DNA (cDNA) with HiScript III RT SuperMix for qPCR (+ gDNA wiper) . Each cDNA was used as a template for qRT-PCR analysis using ChamQ Universal SYBR qPCR Master Mix . FpTEF1a and FpActin genes were used as internal standards. The relative normalized transcript level of each gene was computed using the 2–ΔΔCt method. The primers used for qRT-PCR are listed in t-tests in Excel.To analyze FpHsp104 gene-replacement constructs (FpHsp104 gene were amplified by PCR with primer pairs F1 + R1 and F2 + R2 (hph) gene (H) was amplified by PCR with primer pair HYG/F + HYG/R. These three fragments were used as templates for overlapping PCR amplification to obtain a 1,378-bp fragment containing the FpHsp104 and hph upstream cassette (A1H1) and a 1,751-bp fragment containing the FpHsp104 and hph downstream cassette (H2A2), respectively. The resulting PCR products were purified and transformed into protoplasts by the polyethylene-glycol-mediated fungal transformation method . The putative transformants were confirmed by PCRs with the primer pairs H2F + H2R, F3 + H1R, H1F + R3, and G1 + G2  and Sout2F + H2R  was usedFpHsp104 gene driven by 1,087-bp native promoter amplified by primer pair G1 + G2  agar plates. Mycelial plugs (5 mm × 5 mm) from 3-day-old PDA/MM plates were transferred onto new plates at 25°C. Colonies were photographed and measured at 3 days. For heat response analysis, mycelial plugs of equal size were inoculated on PDA/MM plates at 25°C for 36 h, at 34°C for 24 h, and then at 25°C again for 2 days. Mycelial morphology was observed using a CARL ZEISS Axio imager M2 microscope. For stress response assays, mycelial plugs of equal size were placed on freshly prepared PDA plates with 1 mM of CuSO4, 10 mM of MnCl2, 0.05% sodium dodecyl sulfate, and 5 mM of hydrogen peroxide or 50 mg/ml of congo red for 3 days at 25°C in the dark. All experiments were repeated three times, with three replicates each time. Data were analyzed using t-tests in Excel.Vegetative growth of the WT, Δfphsp104, and Δfphsp104-C strains were inoculated on PDA plates at 25°C for 2 days; then, 5-mm-diameter mycelial plugs were punched from the edges of plate colonies and transferred into liquid CMC. Mycelia were cultured with shaking (150 rpm) at 25°C for 4 days. Conidiophore morphology was observed using a CARL ZEISS Axio imager M2 microscope. Conidia were filtered through one layer of miracloth and counted with a hemacytometer under a CARL ZEISS Axio imager M2 microscope. Calcofluor White stain  was used for septa staining. Conidia were shocked in 34°C condition for 3 h and assayed using propidium iodide (PI) . Stained cells were imaged and counted using a CARL ZEISS Axio imager M2 microscope. PI-positive cell proportions were determined after examining at least 500 cells in each of three independent experiments. Conidia were shocked in 34°C condition for 2 h and inoculated on MM plates at 25°C for 3 days to measure the mycelia growth.For conidiation, the WT, Δ4 conidia/ml) were, respectively, prepared by distilled water (ddH2O), potato dextrose broth (PDB) media, or MM media and dropped onto the surface of a slide, which was placed in a moistened box at 25°C. The conidial germination rate was determined as percentage of germinated conidia to total conidia. More than 200 germinated conidia were counted for each strain, and slides were photographed at 3–6 h under the microscope; more than 200 germinated conidia were counted for each strain in each of three independent experiments. Data were analyzed using t-tests in Excel.Conidial suspensions of 100 μl  with a DIG Northern Starter kit .To analyze mRNA splicing in the WT, Δt-tests in Excel.Seedlings of the wheat cultivar Aikang 58 were grown at 25°C with a photoperiod of 16 h/8 h (light/dark cycle) for 4 days. Barley was cultured under the same conditions for 7 days. Then, 5-mm-diameter mycelial plugs were punched from the edges of plate colonies and inoculated on wheat coleoptiles and barley leaves. Agar plugs were used for negative control. The mycelial plugs were removed after 24 h post-inoculation, and lesion formation was examined at 4 days post-inoculation (dpi). Inoculated wheat epidermal cells were viewed under a Nikon Ti-s microscope. Post-cultural infection assays were carried out as previously described . All expfphsp104, and Δfphsp104-C strains were inoculated on PDA plates at 25°C for 3 days; millet seed was sterilized at 121°C for 20 min and then inoculated with five 5-mm-diameter F. pseudograminearum mycelial plugs at 25°C for 7 days. Flasks were shaken daily until the mycelia colonized the millet. In controls, sterile millet was used. DON contamination in millet was assayed using an ELISA kit from Huaan Magnech Bio-Tech, Beijing, China, strictly following the manufacturer’s instructions. The experiment was repeated three times for each strain, and data were analyzed using t-tests in Excel.The WT, ΔF. pseudograminearum , transcripts of FpHsp104 showed a 20-fold increase in conidia  were constructed by transforming WT protoplasts with the fusion PCR products  and 3′-UTR of FpHsp104 indicated that FpHsp104 had been replaced by hph gene , containing the full-length sequence except the stop codon of FpHsp104 and GFP under the control of the FpHsp104 native promoter, were also generated . Expression of FpHsp104 was inhibited by exposure to the lethal temperature and then substantially upregulated after the temperature was returned to 25°C for 36–48 h  treatment caused FpHsp104 to appear as green fluorescence punctate in conidiophores, conidia, and conidial germlings  of Δstaining , as is aermlings . The smaF. pseudograminearum  to monitor the effects of FpHsp104 on mRNA splicing in F. pseudograminearum. There was no time-course change of actin gene mRNA splicing when F. pseudograminearum germinated conidia were cultured in optimal temperature, and the actin gene appeared similar to mRNA splicing in WT, Δfphsp104, and Δfphsp104-C strains   has shown that F. pseudograminearum is more common in cooler and drier regions, in contrast to F. graminearum, which is often found in warmer regions with higher rainfall  . Neverthnditions . The disrainfall . We raisF. pseudograminearum and presented insights into its basic biological functions. The protein is indispensable for acquisition of thermotolerance and appears in heat-treated cells as small irregular gathers. The decreased proficiency of Δfphsp104 mutant in conidia production and formation and their attenuated virulence suggest that FpHsp104 may be a subtle regulator of cell fitness independently from its role in maintaining thermotolerance.In conclusion, we have identified a conserved Hsp104 of NP_013074.1. (2) Hsp104 sequences from filamentous fungi were downloaded from NCBI under the accession numbers XP_006692738.1 (Chaetomium thermophilum), XP_009254919.1 (Fusarium pseudograminearum), XP_003717107.1 (Magnaporthe oryzae), XP_011324022.1 (Fusarium graminearum), XP_957228.1 (Neurospora crassa). (3) ClpB sequence from Escherichia coli was downloaded from NCBI under the accession numbers WP_020239930.1. (4) Hsp101 sequence from Arabidopsis thaliana was downloaded from NCBI under the accession numbers NP_565083.1.The original contributions presented in the study are publicly available. This data can be found here: (1) Hsp104 sequence from yeast was downloaded from NCBI under the accession numbers All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, the effects of changing carbonate chemistry on biomacromolecules, such as protein and carbohydrate of coccolithophores, are less documented. Here, we disentangled the effects of elevated dissolved inorganic carbon (DIC) concentration  and reduced pH value (8.04 to 7.70) on physiological rates, elemental contents, and macromolecules of the coccolithophore Emiliania huxleyi. Compared to present DIC concentration and pH value, combinations of high DIC concentration and low pH value (ocean acidification) significantly increased pigments content, particulate organic carbon (POC), and carbohydrate content and had less impact on growth rate, maximal relative electron transport rate (rETRmax), particulate organic nitrogen (PON), and protein content. In high pH treatments, elevated DIC concentration significantly increased growth rate, pigments content, rETRmax, POC, particulate inorganic carbon (PIC), protein, and carbohydrate contents. In low pH treatments, the extents of the increase in growth rate, pigments and carbohydrate content were reduced. Compared to high pH value, under low DIC concentration, low pH value significantly increased POC and PON contents and showed less impact on protein and carbohydrate contents; however, under high DIC concentration, low pH value significantly reduced POC, PON, protein, and carbohydrate contents. These results showed that reduced pH counteracted the positive effects of elevated DIC concentration on growth rate, rETRmax, POC, PON, carbohydrate, and protein contents. Elevated DIC concentration and reduced pH acted synergistically to increase the contribution of carbohydrate–carbon to POC, and antagonistically to affect the contribution of protein–nitrogen to PON, which further shifted the carbon/nitrogen ratio of E. huxleyi.Elemental contents change with shifts in macromolecular composition of marine phytoplankton. Recent studies focus on the responses of elemental contents of coccolithophores, a major calcifying phytoplankton group, to changing carbonate chemistry, caused by the dissolution of anthropogenically derived CO Coccolithophores play a central role in the marine carbon cycle and contribute 1–10% to marine organic carbon fixation through photosynthesis  ynthesis  and 40–6fication . The cosells L−1 . The impells L−1 . Howeverells L−1 . TherefoE. huxleyi received considerable attention with a number of studies investigating their responses to ocean acidification , was obtained from the Roscoff algal culture collection. Cells were maintained in semi-continuous cultures in artificial seawater (ASW) media prepared according to 3, with a salinity of 33psu, under 200μmol photons m−2 s−1 of photosynthetically active radiation  and a 12:12h light/dark cycle (light period: 8:00 to 20:00) and 20°C in a thermo-controlling incubator . The DIC-free ASW media was enriched with 64μmolL−1 NO3−, 4μmolL−1 PO43−, f/8 concentrations for trace metals and vitamins  and hydrochloric acid  were added stepwise into the media to achieve four different carbonate chemistry conditions  value were set to 400μatm and 8.04 under low CO2 and high pH treatment , 400μatm and 7.70 under low CO2 and low pH treatment , 1,000μatm and 8.04 under high CO2 and high pH treatment , and 1,000μatm and 7.70 under high CO2 and low pH treatment , respectively. In each condition, the ASW was put into the incubator at 20°C for 24h and then filtered  and carefully pumped into autoclaved 100ml, 1,120ml, and 2,350ml polycarbonate bottles with no headspace to minimize gas exchange. 100ml seawater was used to determine initial total alkalinity (TA) and pHTotal of seawater, 1,120ml bottles were used to acclimate cells to experimental conditions (one replicate), and the main experiment culture was conducted in 2350ml bottles (four replicates). The cells were inoculated to achieve an initial concentration of about 3,000 cell ml−1 and cultured in each experimental condition for 2days and then diluted to the initial cell density again , POC, and nitrogen (PON), protein, carbohydrate, and pigment. It should be mentioned that E. huxleyi can rapidly alter the rates of essential metabolical processes in response to changing seawater carbonate chemistry within 26h. In this study, E. huxleyi cells were incubated under each treatment with 4 biological replicates for 2days, thus the results found here were comparable with other studies , 40ml and 50ml samples were, respectively, filtered  5h after the onset of the light period (at 13:00) and used to measure the pHTotal and TA. The bottles were filled from bottom to top with overflow, and closed immediately without a headspace. The pHTotal was measured immediately at 20°C using a pH meter which was calibrated with buffers  at pH 4.01, 7.00 and 10.00. The pH value was not corrected with a standard buffer of defined pH in seawater  using the CO2SYS program  was calculated for each replicate according to the equation: μ=(ln Nt−ln N0)/d, where Nt and N0 refer to the cell concentrations in the second day and beginning of the main experiment, respectively, and d was the growth period in days.Twenty fiveml samples were taken daily 6h after the onset of the light period (at 14:00) and fresh ASW with the same carbonate chemistry as in the initial treatment conditions were added as top-up. Cell density was measured using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter). The diameter of detected particles was set to 3–7μm in the instrument, which excluded detached coccoliths . Cell co−2 s−1 were applied in nine steps and 45s each in fast light response curve measurements. The instant minimal (F0ʹ) and maximal fluorescence (Fmʹ) were, respectively, determined at the end of each A-PAR and saturating light pulse . The effective photochemical quantum yield (Yield) was calculated as: Yield=(Fmʹ−F0ʹ)/Fmʹ (rETR) was calculated as: rETR=Yield×A-PAR  represents the light-saturating level of rETR, and light use efficiency (a) was derived from the slope of each electron transport rate (ETR) vs. light curve. Saturating light intensity, Ik, is calculated from the expression rETRmax/a and is characteristic for the onset of light saturation.The photosynthetic fluorescence parameters were determined using a pulse amplitude modulated fluorometer . 2ml samples were taken from the incubation bottles and kept in the dark for 15min at 20°C. The assay light levels (A-PAR) between 0 and 1,120μmol photons mF0ʹ)/Fmʹ . The relld×A-PAR . The parg for 10min to remove glass fibers. Absorbance (A) of the supernatant on 750, 664, 647, and 480nm was measured using a spectrophotometer . The chlorophyll a (Chl a) concentration was calculated as: Chl a (μgml−1)=−1.93×(A647–A750)+11.93×(A664–A750). The carotenoid concentration was calculated as: Carotenoid (μgml−1)=4× with low vacuum pressure (<0.02MPa) at 15:00 and soaked in 5ml 90% acetone overnight at 4°C in the dark. The extracts were centrifuged at 4300×μ (d−1), respectively, (Two hundredml samples for determinations of TPC and POC (PON) were, respectively, gently filtered onto GF/F filters (pre-combusted at 450°C for 6h) at the same time (15:30) in each treatment, rinsed three times with DIC–free ASW media, and then stored in the dark at −20°C. For determination of POC (PON), samples were fumed with HCl for 12h to remove inorganic carbon. TPC and POC (PON) samples were dried at 60°C for 12h and analyzed using an Elementar CHNS analyzer . PIC quota was calculated as the difference between TPC and POC quota . PIC, POctively, .−1 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (protease inhibitor), 125.00mmolL−1 ethylene diamine tetraacetic acid, 28.07mmolL−1 Tris base, 21.10mmolL−1 Tris-HCl, 1085.89mmolL−1glycerol and 73.44mmolL−1 lithium dodecyl sulfate). Cells were lysed using a FastPrep–24 machine  at 6.5ms−1 with 4cycles and 1min each, and samples were chilled in an ice bath for 2min between 2cycles. Then, the samples were centrifuged at 10,000×g for 5min , and extracted protein in the supernatant was determined at 562nm using the BCA Assay with Bovine gamma globulin as a standard, using a spectrophotometer  and onto pre-combusted GF/F filters at 15:30 and then stored in the dark at −80°C. Protein samples were put into 2ml MP Biomedical tubes  containing large ceramic beads. After being freeze-dried, protein samples were extracted by a mixture of 0.7ml 1X protein extraction buffer (0.10mmolLotometer .−1 sulfuric acid (H2SO4) in the dark for 1h and then diluted by milliQ water for a final H2SO4 concentration of 1.20molL−1. Samples were then sonicated for 5min and vortexed for 30s, and boiled at 90.0°C for 3h in a water bath  treatments and were 1,010μatm under high DIC (HC) treatments , carbonate (CO32−) and TA concentrations, and under high pH treatment, high DIC concentration (HCHpH) represents high CO2, HCO3−, CO32−, and TA concentrations , and saturating light intensity  and increased chlorophyll a (Chl a) and carotenoid content by 13.56 and 7.17%, respectively , respectively , ocean acidification  did not significantly affect growth rate, trations , 2. Compt low pH –C, increectively , 3 and d2 concentrations, the effects of reduced pH value on growth rate, Chl a and carotenoid contents, rETRmax, a and Ik were negative, which can be seen by comparing these variables in low pH condition with their paired high pH condition , reduced a by 8.16% at LC and by 14.60% at HC  and reduced Ik by 14.68% at LC and by 8.52% at HC , respectively  and POC/PON ratio , PIC/POC ratio (p=0.01) and POC/PON ratio (p=0.07) did not significantly affect POC content and significantly reduced PON content.Compared to present DIC concentration and pH value (LCHpH), ocean acidification (HCLpH) reduced PIC content by 40.93% and PIC/POC ratio by 44.05%, and increased POC content by 13.75% and did not significantly affect PON content ; in high DIC concentration, it reduced POC content by 21.24% and PON content by 19.78% . In low CO2 concentration, reduced pH value did not significantly affect protein content (p=0.05), and in high CO2 concentration, it significantly reduced protein content (p<0.01).Compared to present DIC concentration and pH value (LCHpH), ocean acidification (HCLpH) did not significantly affect protein content and increased carbohydrate content by 38.20% , gray. Tp<0.01). These results showed that elevated DIC concentration increased protein content in high pH condition but not in low pH condition and mainly increased carbohydrate content under both low and high pH treatments  and significantly reduced this percentage in high DIC concentration.Compared to present DIC concentration and pH value (LCHpH), ocean acidification (HCLpH) did not significantly reduce the percentage of POC allocated to protein p=0.08; , gray, sp=0.40) and significantly increased this percentage by 34.98% under low pH treatment . The percentages of POC allocated to Chl a (Chl a–C: POC) and carotenoid (Carotenoid–C: POC) were 0.45–0.78% and 0.38–0.65%, respectively, under different carbonate chemistry treatments  under high pH treatment . Under low or high DIC concentrations, the effect of reduced pH value on percentage of PON allocated to protein was negative, which can be seen by comparing this percentage in low pH conditions with their paired high pH conditions (p<0.05). These results showed that elevated DIC concentration and reduced pH value acted synergistically to increase the percentage of POC allocated to carbohydrate  was positive, which can be seen by comparing this percentage in high DIC concentrations with their paired low DIC concentrations . Elevatenditions . Reducedohydrate , and antohydrate .rETRmax .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MMP1, MMP8, MMP13), gelatinases , stromelysins , membrane-type MMPs , tissue inhibitors of metalloproteinases , and ADAMTS proteinases . It was found that MMP, TIMP, and ADAMTS are expressed in all parts of the aneurysm with different patterns. A developed aneurysm has such a disturbed expression of the main participants in extracellular matrix remodelling that it is difficult to infer the causes of the disorder development. MMP12 secreted by macrophages at the onset of inflammation may initiate extracellular matrix remodelling, which, if not controlled, initiates a feedback loop leading to aneurysm formation.Abdominal aortic aneurysm refers to abnormal, asymmetric distension of the infrarenal aortic wall due to pathological remodelling of the extracellular matrix. The distribution of enzymes remodelling the extracellular matrix and their expression patterns in the affected tissue are largely unknown. The goal of this work was to investigate the expression profiles of 20 selected genes coding for metalloproteinases and their inhibitors in the proximal to the distal direction of the abdominal aortic aneurysm. RNA samples were purified from four lengthwise fragments of aneurysm and border tissue obtained from 29 patients. The quantities of selected mRNAs were determined by real-time PCR to reveal the expression patterns. The genes of interest encode collagenases (The online version contains supplementary material available at 10.1007/s13353-021-00642-3. Abdominal aortic aneurysm (AAA) is an abnormal, asymmetric distension of the infrarenal aortic wall of 3 cm or greater . The patients who underwent surgery for AAA included in this study were of both sexes, ranging in age from 57 to 82 years (mean 67.5 ± 6.35 years). The AAA patients excluded from the study were those who fulfilled the following criteria: (a) chronic obstructive pulmonary disease (COPD); (b) diabetes; (c) creatinine level > 1.0; (d) reconstruction of coronary vessels and thoracic aorta (CABG); (e) reconstruction of carotid artery (ICA); (f) diagnosed generalized atherosclerosis (AO); (g) family history of AAA or inherited cardiovascular syndromes; and (h) lack of ability to provide informed consent for surgical treatment. The research plan was approved by the Bioethics Committee of the Medical University of Silesia in Katowice, protocol no. KNW/0022/KB1/55/14 and its further extension no. KNW/0022/KB1/55/1/14/17.Fragments of AAA, usually approximately 50 mm long, were collected from the patients upon surgery. Non-aneurysmal aortic samples of the aneurysm neck (unaffected samples) were simultaneously collected for use as controls Fig. .Fig. 1AbAll surgical procedures were performed in the planned mode. Briefly, the material collected for the research was part of the aneurysm excised during an OAR. The samples were collected intraoperatively in the General and Vascular Surgery Clinic  and secured immediately in the operating room at room temperature in sterile 50 mL tubes filled with 25 mL of Dulbecco’s modified Eagle’s medium  supplemented with glucose (4.5 mg/mL) (high-glucose DMEM), penicillin , streptomycin (10 mg/ml), and amphotericin B (25 µg/ml) . The procedures were established to maintain living cells because the specimens were also used for cell isolation and their culture to characterize the cell types in the separated layers of the AAA wall. Immediately upon arrival to the cell culture facility, the aneurysm was divided into 4 fragments, border, control/border (C); neck, upper/proximal (1); aneurysm bag, middle/central (2); and the end segment, bottom/distal region (3) Ziaja , where tTotal RNA was isolated in duplicates using Zymogen Quick RNA Mini Prep  following sample homogenization in TissueLyser II . Quality and quantity evaluation was performed using a NanoDrop 2000 spectrophotometer . Total RNA 1 to 2 µg) was transcribed using a cDNA transcriptor first-strand cDNA synthesis lit  using random hexamers. Expression analyses with Real Time ready Custom Panel 384–96  and LightCycler 480 Probe Master (Roche) were performed using a LightCycler 480 II (Roche). The genes analysed in this report are listed in Table  to 2 µg GAPDH, GusB, PPIA, and RPL13a . Raw data were subjected to normalization to sample amount followed by normalization to the reference genes MMP11 the distribution was normal. Due to the small sample group size, the data for the analysis were based on the calculations of the median and sem  were calculated to determine the correlation between genes  showed the lowest expression in the aneurysm sac in comparison to adjacent tissues.Group I (Online Resource TIMP4 (3.82 ± 0.002), and no differences have been detected between those fragments for TIMP1, MMP12, MMP10, MMP11, ADAMTS13, and MMP8 (Table ADAMTS8 (2.92 ± 0.13). No differences in expression were detected for MMP1, MMP7, MMP9, MMP10, MMP11, MMP12, MT-MMP1, MT-MMP3, ADAMTS13, TIMP2, and TIMP3). Significantly large changes in the expression of the analysed genes were observed between the proximal and distal parts, with the greatest difference for MMP3 (-4.33 ± 0.41). No expression changes were detected for MMP8, MMP10, MMP11, MMP13, MT-MMP3, MT-MMP2, ADAMTS1, ADAMTS8, and ADAMTS13.The highest fold change between proximal fragment and aneurysm sac was observed for TIMP4 3.8 ± 0.002,MMP9  in aneurysm sac and 1.1 in the distal segment) could be a result of dysregulation of their expression in the segments. Moreover, only moderate correlation (rS = 0.41) was found between the relative expression of MMP1 and TIMP1 along the aneurysm tissue. Soluble protease, MMP1, breaks down interstitial collagens, including types I and III, both present in the arterial walls. MMP1 is only inhibited by TIMP1, and therefore, the regulation of the two gene products is critical for proper matrix remodelling.Here, the expression of mRNAs encoding metalloproteinases and their inhibitors was found to be irregular and variable along AAA. For example, the ratio MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 play key roles  or fibroblasts and is activated by MMP3 and the urokinase plasmin activator (uPA)/plasmin system. Other groups also reported lower expression of TIMP1 than MMP1 Supplementary file2 (PDF 137 KB)Supplementary file3 (PDF 79 KB)Supplementary file4 (PDF 88 KB)Supplementary file5 (PDF 195 KB)Supplementary file6 (PDF 138 KB)Supplementary file7 (PDF 139 KB)Supplementary file8 (PDF 138 KB)Below is the link to the electronic supplementary material.


Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with addition of parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D . Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilaginous genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including  As the bone marrow niche contains numerous differentiated and progenitor cell types, studying the properties of MSCs in vivo is challenging. Thus, cultured MSCs are useful to determine the cell-specific responses of MSCs apart from other cell types; however, there are several challenges with current cell models.The ability of mesenchymal stem cells (MSCs) to differentiate into multiple lineages makes them a valuable tool for the investigation of tissue development and responses to various stimuli. While MSCs are present in numerous tissues, bone marrow derived MSCs are essential for cartilage formation, bone remodeling and repair, and can also form into adipose tissue2 and bone marrow3 in both human and animal models. Although culture conditions can be optimized to promote growth of MSCs over other cell types, these cultures typically remain highly heterogenous, containing hematopoietic cells and other cell types. In addition to the contamination of other cell types, there is wide variation among donors  and even between isolations within the same donor. Primary MSC cultures often display differing growth kinetics and variation in the proportion of cell populations therein. Such variabilities create challenges in obtaining consistent phenotypic and functional results and may even lead to incorrect interpretation of data3.Primary MSCs can be extracted from various tissues, but are most commonly isolated from adipose4. While the use of human MSCs provides a powerful tool that may be more easily translated to human studies, human cells often vary greatly in their responses compared to cells derived from animals. Furthermore, most of the available MSC lines require complicated protocols to induce differentiation, often including the use of proprietary cell culture medium, which is both costly and introduces undefined components (likely various growth factors) that may influence downstream experimental outcomes. Additionally, the majority of these cell lines require upwards of 4 weeks in culture to achieve osteogenic and adipogenic differentiation7.In contrast to primary cells, immortalized cell lines provide greater homogeneity and are thus capable of producing more consistent experimental outcomes. Numerous human MSC cell lines are available through commercial vendors, some of which are immortalized, enabling greater expansion in the laboratory setting. Human MSC cells derive from various tissues, most commonly adipose aspirates, but also from bone marrow9. This is especially useful to generate cells from mice with transgenic modifications; however, the limitations with heterogeneity and contamination with other cell types remain.Mice are an extremely useful biological model, and the ability to induce transgenic modifications in mice makes them a powerful tool for discovery. As such, mouse cell lines are useful for in vitro confirmation of in vivo results, often providing essential data to translate findings from animal to human studies. Unfortunately, there are relatively few established immortalized mouse MSC cell lines, and the lines that are available have similar challenges to those presented by human cell lines. In the absence of readily available MSC cell lines many groups isolate primary cells for each experiment, requiring the use of numerous animals. A few groups have developed improved methods for the isolation of MSCs from mice that allow for multiple passages of the isolated cellsC-terminal forms of fibroblast growth factor 23 protein (cFGF23). While several other cell lines produce Fgf23 mRNA, there are very few available that secrete FGF23 protein. As such, these cells provide a useful tool not only to study responses of mesenchymal progenitors, but also to examine development of multiple tissue types.To overcome some of the common issues with primary MSC cultures, we developed two novel multi-potent cell lines capable of differentiating into the osteogenic, adipogenic, and chondrogenic lineages. These cells were isolated from the bone marrow of C57BL/6 mice and are referred to as Murine Progenitor Cells 1 and 2 (MPC1 & MPC2). MPC1 and MPC2 cells were expanded from single cell clones and harbor the temperature sensitive large T-antigen, providing a unique ability to regulate proliferation and differentiation based on the incubation temperature. In addition to the tri-lineage capacity, MPC cells produce very high expression of several commonly studied molecules. In particular, both MPC1 and MPC2 cells express and secrete the intact (iFGF23) and Cell culture media, trypsin–EDTA, and antibiotics were purchased from Invitrogen . Fetal bovine serum (FBS) was purchased from Atlanta Biologicals . Ascorbic acid, β-glycerophosphate, alizarin red, and the alkaline phosphatase staining kit were obtained from Sigma Aldrich .Antibodies (Abs) recognizing PPARγ (#2443), GAPDH (#5174), and perilipin (#9349) were purchased from Cell Signaling . Antibody against fatty acid binding protein 4 (FABP4) was purchased from ProSci, Inc.  (XG6174). The anti-adiponectin Ab (PA1-054) was obtained from Affinity BioReagents . Aggrecan Ab was from Abcam (ab3778). Antibody against collagen I was purchased from Millipore (AB765P), and the collagen X Ab was purchased from Calbiochem . For flow cytometry analysis, anti-mouse CD44 (#553131), anti-mouse Sca-1 (#557403), anti-mouse CD105 (#550546), anti-mouse CD106 (#553330), anti-mouse CD90 (#553016), Rat IgG2a control (#559073), were purchased from BD Biosciences . Anti-mouse CD146 (#134702), anti-mouse CD73 (#127202), rat IgG2c control (#400702), and rat IgG2b control (#400602) were purchased from Biolegend . The mouse monoclonal Ab recognizing the SV40 T antigen  was purchased from Santa Cruz Biotechnology .9. Briefly, mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Femurs and tibias were dissected and kept on ice in Roswell Park Memorial Institute (RPMI) media with FBS , horse serum , and penicillin/streptomycin (100 μg/ml). Marrow was flushed from long bones and passed through a nylon mesh cell strainer (70 μm). Cells were spun down (7000 rpm), resuspended in RPMI media, and plated in a T175 flask. Cells were cultured for two passages in RPMI media, then passaged twice more in Iscove’s Modified Dulbecco’s Medium (IMDM) containing FBS , horse serum , and penicillin/streptomycin (100 μg/ml). Cells then were expanded and cryogenically preserved. All mouse handling and cell isolation was performed in accordance with protocols approved by the Indiana University Institutional Animal Care and Use Committee and ARRIVE guidelines. Additionally, all methods were carried out in accordance with relevant guidelines and regulations.Mesenchymal progenitor cells were isolated from 8-week-old male C57BL/6 mice as previously described11. The sequence was ligated into the pLVX Puro lentiviral plasmid  using the same restriction sites and sequenced to confirm correct orientation. Plasmid DNA was amplified in One Shot Stbl3 E. coli (Thermo-Fisher Scientific) and purified using a Plasmid Maxiprep kit (Qiagen).The temperature-sensitive large T antigen SV40 sequence, SVU19tsa58, was digested from pZipSVU19tsa58 plasmid  using the BamH1 restriction site as previously describedHuman embryonic kidney (HEK) 293 T cells were transiently transfected with the immortalizing plasmid and plasmids encoding for Tat, Rev, Gag/Pol and VSV-G using Fugene-6 transfection reagent as per the manufacturer’s instructions (Roche) for 8 h. Media was replaced with DMEM containing FBS  and cells were incubated for 48 h. The media containing virus particles was collected, centrifuged (2000 rpm) to pellet cell debris, the supernatant was passed through a filter (0.45 µm), and stored at − 80 °C.4 cells/cm2 in T25 flasks the day prior to transduction. The following day, media was removed and replaced with a mixture of culture media (50%) and crude virus (50%) containing Polybrene . Transduction media was removed and replaced with fresh culture media 24 h later. After an additional 48 h, fresh media containing puromycin  was added to select for infected cells. Puromycin selection was maintained for 10 days.MSCs were seeded at a density of 2 × 10MPC cells were plated in 6-well plates . After 24 h cells were transfected with an eGFP vector  using Fugene-6 HD reagent according to the manufacturer's protocol. Cells were incubated at 33 °C for the for at least 24 h before being imaged with a fluorescence on a microscope (Leica).Stably transfected cells were cloned by limiting dilution in 96-well culture plates. Over 20 clones were created, of which 10 were differentiated in osteogenic media containing alpha minimal essential media (α-MEM), FBS , β-glyerophosphate (5 mM) and ascorbic acid (50 µg/ml) for 21 days. The two mesenchymal progenitor cell clones showing the greatest mineralization, as determined by alizarin red staining, were selected for further characterization and designated as “MPC1” and “MPC2”.5 cells were aliquoted into polypropylene tubes and pelleted as above. Cells were resuspended in 100 µl of primary antibody at a final concentration of 10–20 µg/ml. Cells in replicate tubes were incubated with the relevant isotype-control supernatant antibody. Following incubation on ice for 45 min, cells were washed twice as above then resuspended in 100 µl blocking buffer containing secondary anti-rat IgG-PE conjugated antibody at 10 µg/ml and incubated on ice for 30 min. After washing twice with cold wash buffer (2 ml), cells were resuspended in fixative (0.5 ml) containing formalin , D-glucose (0.1 M), sodium azide , in phosphate buffered saline for flow cytometric analysis.Cells were removed from flasks with a brief trypsin/EDTA digestion and the suspension filtered through a cell strainer (70 µm). After washing in Hank’s Buffered Salt Solution (HBSS) containing FBS  by centrifugation at 1400 rpm for 8 min at 4 °C, cells were resuspended in blocking buffer  and incubated on ice for 30 min. 2 × 102 in six-well dishes  as previously described12. Cells were incubated overnight (ON) in IMDM media to allow them to adhere. The following day cells were moved to 37 °C and IMDM media was replaced with osteogenic media consisting of α-MEM ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM) or adipogenic media containing dexamethasone (0.1 μM), insulin (5 μg/ml) and indomethacin (50 μM). For osteogenic cultures, media was changed every 48 h. To determine osteocytic responses of endocrine factors MPC cells were differentiated in osteogenic media for 28 days followed by addition of PTH (50 mM) or 1,25D (10 nM) for 24 h, after which time RNA or media were collected for analysis.Cells were cultured at 33 °C to allow for proliferation. For osteogenic and adipogenic differentiation MPC1 and MPC2 cells were seeded at 6000–10,000 cells/cm−8 M), and TGF-β1 (10 ng/mL) in DMEM. Cells were centrifuged for 5 min (250xg), fitted with vented caps and incubated for 48 h in normoxia. After 48 h chondrogenic cultures were moved to hypoxic conditions (5% O2) and cultured for 28 days, as previously described14.Induction of chondrogenic differentiation was accomplished by adding 250,000–500,000 MPC1 or MPC2 cells to polypropylene tubes containing growth media containing sodium L-ascorbate (50 nM), insulin (6.25 μg/mL), sodium selenite (6.25 ng/mL) and penicillin/streptomycin  in DMEM or with chondrogenic media composed of insulin (6.25 μg/mL), transferrin (6.25 μg/mL), sodium selenite (6.25 ng/mL), sodium L-ascorbate (50 nM), dexamethasone (103VO4 (1 mM), aprotinin (1 μg/ml), leupeptin (1 μg/ml), pepstatin (1 μg/ml), and phenylmethylsulfonylfluoride  were added fresh, just prior to lysis. Total protein lysates (20 μg) were separated on SDS polyacrylamide gradient gels (4–12%) and transferred to polyvinylidene difluoride (PVDF) membranes, as described previously15. Membranes were blocked with milk  diluted in tris-buffered saline containing tween-20 . Blots then were incubated ON at 4 °C with the appropriate primary antibody. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution)  at RT for 1 h with chemiluminescent detection using ECL Plus substrate . Images were developed and acquired with an iBright CL1000 machine (Applied Biosystems).Whole cell lysates were prepared using radio immunoprecipitation assay (RIPA) lysis buffer  sodium deoxycholate,1% (w/v) Igepal, pH 7.5) with protease and phosphatase inhibitors. Inhibitors including NaF (1 mM) and Na16. mRNA was reverse transcribed, and genes were amplified with a BioRad CFX Connect™ qPCR machine, using gene-specific primers as previously described17. PCR products were normalized to Gapdh mRNA expression and quantified using the ΔΔCT method.Total RNA was isolated by using the RNeasy kit  as described previously2O prior to addition of 1 mL of Alizarin Red  per well. The plates were incubated in the dark at RT for 10 min. After removal of unincorporated dye, wells were washed three times with dH2O, re-aspirated, and stored at room temperature (RT). In identical cultures for the mineralization assay, monolayers of MPC1 and MPC2 cells were washed twice with 1X PBS and fixed for 30 min in 4% PFA. PFA was removed and cells were washed again in 1X PBS, then stained using the Leukocyte Alkaline Phosphatase Kit (Sigma) as previously described18. Plates were stored at RT. Images were taken at 10X magnification on an inverted Leica microscope. Alizarin red staining was quantified using cetylpyridinium chloride (CPC) extraction for 3 h as previously described19 and absorbance was measured at 544 nm. Intensity of alkaline phosphatase staining was quantified using ImageJ, as previously described20.Mineralization was induced on confluent monolayers in 12-well plates by addition of osteogenic media. Monolayers were washed with 1X PBS and fixed for 1 h with cold ethanol , then washed 3 times with excess dH2 vitamin D (10−8 M) (Sigma) or vehicle (DMSO) for 24 h. Media was aspirated and centrifuged to remove unattached cells and debris. Media was concentrated in Amicon Ultra Centrifugal Filters (Milipore) and stored at − 80 °C. Adherent cells were lysed with 300 µL of 1X Lysis buffer  with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) protease inhibitor (1 µg/mL) according to the manufacturer’s directions . Total cell lysate protein concentrations were determined with the Better Bradford Kit (Thermo-Fisher Scientific) according to the manufacturer’s instructions. Secreted FGF23 protein was assessed using both the rodent-specific ‘intact’ FGF23 (‘iFGF23’) and ‘C-terminal’  ‘cFGF23’ ELISAs  and normalized to total protein concentration.To test FGF23 protein production, MPC1 or MPC2 cells were seeded into 6-well plates and grown to confluence. Cells were differentiated in osteogenic media for 14 or 21 days then treated with 1,25(OH)21.Chondrogenic pellets were fixed in neutral buffered formalin  for 48 h and embedded in paraffin. Paraffin embedded pellets were sectioned (10 μm), deparaffinized, and rehydrated prior to staining with either Safranin-O/Fast Green or Alcian Blue, as previously described2O) then inactivated by exposure to H2O2 for 20 min. at RT and then blocked in PBS containing BSA  and goat serum  for 30 min at RT. Sections were incubated with primary Ab (1:100) in a humidified chamber at 4 °C ON. After rinsing with PBS, horse radish peroxidase-conjugated secondary Ab was added for 1 h at RT and rinsed again with PBS. Sections then were exposed to 3,3’ Diaminobenzidine (DAB) chromogenic substrate, rinsed with ddH2O, counterstained with Gill No. 1 hematoxylin, dehydrated, cleared with Xylene, and mounted on slides prior to imaging.Immunohistochemical staining of chondrogenic pellets was performed by first deparaffinizing sections and rehydration of sections. Epitope retrieval was achieved by incubation with chondroitinase ABC (2 mg/ml) for 30 min at RT (aggrecan and collagen X) or by incubation in citrate buffer  for 20 min at 90 °C, followed by a 30 min incubation at RT. Sections were rinsed with distilled/deionized water . All assays were replicated at least three times, using biological replicates, to assure reproducibility.As both MPC cell lines were immortalized using the SV40 large T-antigen, we examined expression of SV40 at both 33 °C and 37 °C by Western blot following 1–6 days of culture in growth media. In MPC1 cells, expression of SV40 was detectable when cells were cultured at 33 °C at each time point examined (1–6 days) Fig. A. In conTo ensure that changes in expression of SV40 correlated with restriction of proliferation, MTT assays were performed. For MPC1 cells, there was no significant differences in cell viability during the first 4 days of culture when comparing cells grown at 33 °C to those at 37 °C Fig. A. HoweveExpression of SV40 was also examined in MPC2 cells grown at 33 °C and 37 °C. As expected, SV40 was present in cells cultured at 33° but essentially absent in cells cultured at 37 °C Fig. B. Cell v23.MPC1 and MPC2 cells were examined for the expression of cell surface markers of mesenchymal stem cells  for 7–28 days, followed by alizarin red staining for calcium deposition. Compared to the maintenance growth media, when cultured in osteogenic media both MPC1 and MPC2 cells displayed increased alizarin red staining Fig. . QuantifMPC cells exposed to osteogenic differentiation conditions were also stained for the osteoblast marker alkaline phosphatase (Alkphos). The pattern of Alkphos staining was similar to that of alizarin red. Following 7 days of osteogenic differentiation MPC1 and MPC2 cells showed minimal Alkphos staining Fig. A, B, whiSost) mRNA expression was significantly higher in MPC2 cells at day 0 (1.4-fold) and day 7 (14.6-fold)  and 28 days (7260-fold) in OM. MPC2 cells had significantly increased Sost expression at 7 (35-fold), 14 (31-fold), and 21 days (63-fold) of culture in OM, compared to day 0  Fig. A. No sig 28 Fig. A. Comparion at 7 5-fold, 1Dmp1) was significantly higher in MPC2 cells compared to MPC1 following 7 (55-fold), 14 (4.8-fold), and 21 days (1.4-fold) of osteogenic differentiation , 14 (469-fold), 21 (2237-fold), and 28 days (5193-fold) of culture in OM. MPC2 production of Dmp1 was significantly increased at 7 (3412-fold), 14 (3739-fold), 21 (5297-fold), and 28 days (6436-fold) compared to 0 days of OM culture  mRNA, an endocrine phosphaturic hormone produced by osteocytes, was significantly higher in MPC2 cells compared to MPC1 at 7 (fourfold), 21 (6.7-fold), and 28 days (fourfold) of culture , 21 (73-fold), and 28 days (511-fold). MPC2 cells had significantly increased Fgf23 expression compared to day 0 when cultured in OM for 7 (54-fold), 14 (238-fold), 21 (1750-fold), and 28 days (7429-fold)  and 7 (13.6-fold). After 28 days of culture in OM media Mepe expression in MPC1 cells was 12-fold higher than MPC2 cells  and 28 days  of osteogenic differentiation. MPC2 cells expressed significantly increased Mepe, compared to day 0, at 7 (32-fold), 14 (37-fold), 21 (73-fold), and 28 days (1334-fold) of osteogenic differentiation  and 7 1.6-fold. ) and 7 1.6-fold. E11) is highly expressed in early stages of osteocyte formation24. Expression of E11 was significantly higher in MPC2 cells compared to MPC1 at day 0 (153-fold), 7 (114-fold) and 14 (12-fold). MPC1 cells produced significantly higher E11 levels at 21 (0.55-fold) and 28 days (2.7-fold) of osteogenic culture , 14 (23-fold), 21 (1849-fold), and 28 days (6644-fold) of culture. Osteogenic differentiation of MPC2 cells produced significantly increased E11 at days 21 (6.7-fold) and 28 (15.7-fold) compared to day 0  mRNA expression was significantly increased in MPC2 cells compared to MPC1 at day 0 (2.6-fold), day 7 (11.6-fold), day 14 (17.4-fold), and day 21 (5.8-fold). Compared to day 0, MPC1 cells had significantly higher levels of Bglap at 7 (10.7-fold), 14 (17.4-fold), 21 (120-fold), and 28 days (905-fold) of osteogenic differentiation , 14 (114-fold), 21 (264-fold), and 28 days (463-fold) of exposure to OM  is a master regulator of osteogenic differentiation25. Compared to MPC1, MPC2 cells produced significantly greater levels of Runx2 at every timepoint surveyed, with the greatest difference being twofold  or 1,25D (10 nM). PTH significantly suppressed Sost mRNA in both MPC1 (99.9%) and MPC2 cells (98.8%)  and MPC2 (99.4%) cells, whereas exposure to 1,25D resulted in significantly increased production of Dmp1 mRNA by 2.7-fold in MPC1 and 3.7-fold in MPC2 cells  and MPC2 (3.9-fold) cells. As previous studies have demonstrated that expression of Sost and Dmp1 is suppressed by PTH, and 1,25D induces both Dmp1 and Fgf23 expression, our results demonstrate that osteogenic differentiation of MPC cells recapitulates the hormonal responses of bone.Bone is acutely sensitive to hormonal signals, many of which directly influence lineage commitment of mesenchymal progenitors. Parathyroid hormone suppresses 8%) Fig. A. Treatm MPC2 99.% cells, Fgf23 mRNA, few have demonstrated the ability to secrete functional FGF23 protein. As such, we sought to determine not only if MPC cells produce FGF23 protein, but to quantify the extent to which both intact, bioactive FGF23 (iFGF23) and C-terminal forms of secreted FGF23  were regulated by 1,25D using two distinct ELISAs. After 14 days of osteogenic differentiation, at baseline (no treatment), MPC1 cells released 4.24 × 10−5 ng/μg of intact FGF23, as measured by ELISA of the cell media, which was normalized to total cell protein content. MPC2 cells produced 5.27 × 10−5 ng/μg, which was significantly increased compared to MPC1 cells, consistent with higher Fgf23 mRNA content. No significant differences in intact FGF23 were seen between 14 and 21 days of osteogenic differentiation . Adipogenic differentiation of both MPC1 , collagen 2a1 (Col2a1) (15.3-fold), collagen 10 (Col10), aggrecan (Acan), cartilage oligomeric matrix protein (Comp) (200-fold), and SRY-box transcription factor 9 (Sox9) (89-fold) were all significantly increased when cultured in chondrogenic media , Col10, Comp, and Sox9 (65.4-fold) were significantly increased when cultured in chondrogenic media, compared to maintenance growth media. Levels of Col10, Acan, and Comp mRNAs were undetectable in samples grown in growth media. No changes in expression of Col2a1 or Acan were observed between growth and chondrogenic media  Fig. A. No appIn sum, we have developed two novel mesenchymal progenitor cell lines that mimic the in vivo ability to differentiate into osteoblasts/osteocytes, adipocytes, and chondrocytes. With the capability of responding to exogenous stimuli, known to affect the responses of differentiated cell types, these cells provide a powerful tool to examine responses of progenitors and the differentiated cells. Further, these cells are readily transfected in the undifferentiated state, as we have shown by transfecting GFP Fig. , enablinMesenchymal stem cells serve as the progenitors for multiple tissue types, including adipose, bone, and cartilage. A variety of hormonal, chemical, and physical cues influence lineage allocation of MSCs; however, studying these individual effects in vivo present numerous challenges. Despite the availability of several methods for the isolation and characterization of MSCs, obtaining homogenous cultures capable of faithfully replicating bone, cartilage, and fat cell phenotypes remains challenging. Herein, we describe the development of two novel progenitor cell lines, MPC1 and MPC2, which serve as a useful tool to study the properties of mesenchymal progenitors, but also recapitulate the morphological, genetic, and signaling properties of bone, cartilage, and adipose. These cells can be routinely used to study the factors regulating the transition between these lineages, as well as the terminally differentiated cell types produced from differentiation.29. Thus, osteocyte cell lines that maintain proliferative capacity may not fully replicate the osteocyte phenotype. Incorporation of the temperature sensitive large T-antigen into these cells enables the ability to restrict proliferation at the time point of choosing30, which may be equally useful for the study of hypertrophic chondrocytes.Immortalization of MPC1 and MPC2 cells was accomplished by the expression of the temperature sensitive large T-antigen, enabling proliferation at 33 °C and differentiation at 37 °C. The ability to restrict proliferation is a useful feature for the study of some cell types. In particular, osteocytes, which are the terminally differentiated form of osteoblasts, are not proliferative in vivo15. These stem cell-derived osteocyte (SCD-O) cultures formed bone-like nodules containing osteocytes which replicated the morphology and structure of osteocytes in cortical bone. As osteocyte responses rely heavily on connections to the matrix, this three-dimensional environment is essential for the replication of the in vivo condition. When primary osteocytes are isolated, they are removed from the native matrix environment which defines their morphology and regulates contextual behavior. Thus, primary osteocytes grown in culture dishes quickly lose their osteocyte-like features.In previous work, our group showed that MSCs isolated from mouse bone marrow were capable of differentiating into mineralized nodules31 and MLO-A532 are cultured on a monolayer and thus are also devoid of the matrix environment that is essential for osteocyte responses. However, there have been several recent cell lines produced that form mineralized bone-like structures. These include the IDG-SW330, OCY-45433, and OmGFP6634. In particular, after 28 days of culture in osteogenic media OmGFP66 cells formed highly organized 3D bone-like structures that resembled trabeculae. These cultures had well-defined osteocyte spacing with the characteristic osteocyte morphology34. Importantly, MPC1 and MPC2 cells form mineralized nodules rapidly in culture, providing the matrix environment necessary to support osteocyte differentiation and production/secretion of osteocyte products. In contrast to other commonly used osteocytic cell lines, MPC cells did not require collagen coating of culture dishes to induce or maintain differentiation.Alizarin red and alkaline phosphatase staining of MPC cells demonstrated formation of mineralized bone-like nodules, which were evident as early as 7–14 days. Thus, when grown in osteogenic conditions MPC cultures mimic the in vivo matrix environment. Some of the earlier produced osteocytic cell lines, such as MLO-Y4Sost, Dmp1, Fgf23, and Mepe were also extremely abundant. Notably, osteogenic differentiation resulted in production of these osteocyte-specific genes on the order of 2000–22,000-fold higher than in the undifferentiated stage. Production of these genes at such high levels not only highlights the ability of MPC cultures to serve as a unique source of osteocytic cells, but also serves as an excellent model to study the transcriptional regulation of each of these genes. Sclerostin in particular has received great attention in the last decade as it is an inhibitor of Wnt/Lrp signaling, and the target of the neutralizing monoclonal Ab romosozumab35, a drug recently approved to treat osteoporosis. Our data show that MPC cells treated with PTH have a dramatic reduction of Sost expression as well as Dmp1. These results align well with previously published in vivo and in vitro studies demonstrating hormonal control of these genes and that MPC cells replicate key functional responses of osteocytic genes.In addition to the formation of abundant matrix, MPC cells also produced all of the characteristic markers of osteocytes including the early osteocyte marker E11. Expression of genes that are produced in mature osteocytes, including 37. The UMR-106 line also increased Fgf23 mRNA when challenged with hypoxia-mimetics38 and iron chelation39. Further, response elements in Fgf23 promoter fragments were activated in response to 1,25D in human K562 leukemia cells40. The human osteoblastic cell line U2OS also properly expressed Fgf23 cDNA, and gene targeting was used to identify key glycosylation/phosphorylation events necessary for FGF23 production41. The disadvantage of these lines is that they do not appear to express endogenous FGF23 protein at readily quantifiable levels.The osteocyte-produced hormone FGF23 has been studied in a number of cell models. The MPC cell lines provide a novel system for testing FGF23 biology in regard to this hormone’s expression and actions. MPC cells express FGF23 in response to 1,25D, similar to osteosarcoma lines such as the rat osteoblast/osteocyte cell line UMR-10642, as well as its promoter elements43. Similar to UMR-106 and U2OS cells; however, the detection of measurable FGF23 protein has been elusive in these cells. Whether the lack of mature protein has to do with portions of the protein folding/secretory pathways down regulated over time in the cells or the fact that FGF23 can be proteolytically inactivated prior to secretion44 remains unclear. When differentiated, the MPC cell lines expressed both FGF23 mRNA, and secreted protein as detected by both ‘Intact’ and ‘C-terminal’ FGF23 ELISAs. This capability allows the future study of FGF23 protein processing using inhibitors/activators, as well as the mechanisms underlying secondary modification of the mature FGF23 protein. Primary cultures of isolated osteoblasts from rodents also express Fgf23 mRNA45; however, these cells are somewhat limited in that, as for primary differentiated cells, they are best transfected using specialized viral expression vectors. Groups have also used differentiated primary bone marrow stromal cells (BMSC) to examine FGF23 production under hypoxic conditions and in response to pro-inflammatory stimuli43. Like primary osteoblasts/osteocytes, these cells are typically transduced with viral vectors, and can therefore not be easily targeted by standard genomic modifying reagents such as CRISPR.The mouse osteoblast-like cell line MC3T3-E1 has also been used to study FGF23 expression and its possible roles in osteoblast function34. This line increased FGF23 expression upon differentiation, similar to the MPC lines. FGF23 was also studied in a model osteocyte-like cell line, IDG-SW3, which demonstrated that35 S-labeled FGF23 was cleaved to smaller fragments which were constitutively secreted46. In contrast, intact bioactive FGF23 was more efficiently stored in differentiated than in undifferentiated IDG-SW3 cells. Following osteogenic differentiation of IDG-SW3 cultures, basal Fgf23 mRNA was dose-dependently up-regulated by pro-inflammatory cytokines TNF, IL-1β and TWEAK, and bacterial LPS47. cFGF23 and iFGF23 protein levels also increased, but intact protein only in the presence of furin inhibitors, supporting that FGF23 cleavage controls this hormone’s bioavailability. Further, some osteocyte cell lines, such as MLO-Y4 express negligible levels of FGF23 in the basal state; however, modest induction can be seen for Fgf23 mRNA and other osteocyte genes with culture under 3D conditions. Cell lines of other lineages, including HK2  cells have been tested, and Fgf23 and osteopontin mRNAs were expressed in these cells when incubated with TGFβ1; however, these levels were not altered in HK2 cells when treated with 1,25D and high phosphate levels48. Although FGF23 is not normally expressed to a significant degree in kidney in vivo, this cell model may be useful for testing FGF23 expression under specific pathologic conditions. Thus, the novel MPC cell lines faithfully recapitulate many of the critical features of osteocytes, and will allow further understanding of FGF23 transcription, protein modification, and secretion.The MPC lines have the distinct advantage that genes can be targeted by standard transfection techniques during the undifferentiated growth phase, then tested for cell function in the differentiated osteocyte-like state or as we show, during active mineralization. More recently, a clonal osteogenic cell line, OmGFP66, was developed by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter7. MPC2 cells responded to adipogenic conditions with greater increases in adipogenic proteins compared to MPC1 cells. MPC2 cells consistently produced high levels of the transcriptional regulator of adipogenesis, PPARγ as well as other proteins characteristic of adipocytes including adiponectin, perilipin, and fatty acid binding protein 4. Recent studies have shown that accumulation of adipose tissue within the bone marrow is detrimental to skeletal health and is associated with aging and disuse50. Additionally, use of certain drugs, such as rosiglitazone increase bone marrow adiposity, but these effects can be attenuated by exercise51. Several studies have demonstrated that the mechanical contribution of exercise restrict entry of mesenchymal progenitors into the adipogenic lineage53, effects that are influence by actin cytoskeletal organization55, and even shuttling of actin monomers to into the nucleus to regulate YAP/TAZ dynamics56. As the balance between osteogenic and adipogenic fate has important implications for health and disease, MPC cells are a good model to study the genetic and phenotypic changes governing the balance of these two mesenchymal tissues.While there are several cell lines capable of recreating osteoblast and osteocyte phenotypes, MPC cells provide a distinct advantage with the capability to also differentiate into the adipogenic and chondrogenic lineages. MPC1 and MPC2 cells readily formed adipocytes after only 5 days of exposure to adipogenic media, much faster than the 2–3 weeks of adipogenic differentiation required of other MSC lines1. When grown on a monolayer, devoid of the surrounding matrix, primary chondrocytes readily undergo de-differentiation with progressive loss of collagen type II and aggrecan58. Culturing chondrocytes within conditions enabling a rounded morphology promotes maintenance of the chondrocyte phenotype60, as such we allowed MPC cultures to grow within pellets. MPC cells grew into cartilaginous pellets which stained for both alcian blue and safranin-O. MPC1 cells demonstrated increased growth capacity compared to MPC2 cells, and more consistently expressed chondrocyte markers. MPC1 cells exposed to chondrogenic media produced collagens 1, 2, and 10, as well as the chondrocyte transcription factor Sox9 and the matrix molecules aggrecan and Comp. MPC1 cells also stained strongly for aggrecan protein. As such, MPC1 cells reproduced the cartilage phenotype more readily than MPC2 cells. Other cell lines including CFK2, which was established from fetal rat calvariae61, and ATDC5, an embryonal carcinoma cell line isolated from a differentiating culture of AT805 teratocarcinoma62, reproduce the cartilage phenotype in vitro. With the ability to produce cartilage-specific genes and grow within a 3D environment, MPC cells represent a novel cell line to study chondrocyte development, particularly the progression from the progenitor stage.Several different culture models have been developed to study chondrocytes in vitro including explant models, three-dimensional culture systems, and cells grown in monolayers15 which are not clonal nor immortalized. Additionally, there are notable phenotypic differences between MPC1 and MPC2 clones. With respect to osteogenic differentiation, MPC1 cells displayed greater alizarin red staining, but no differences in alkaline phosphatase. Sost and Mepe mRNA expression was greater in MPC1 cells while MPC2 cells had increased Fgf23 and Runx2 compared to MPC1. While MPC1 cells were capable of adipogenic differentiation, MPC2 cells displayed increases in a greater number of adipogenic markers when exposed to adipogenic media. Additional differences were noted during chondrogenic differentiation where MPC1 cells grew into larger pellets than MPC2 under chondrogenic conditions; however, differentiational expression of various chondrogenic genes were noted for each cell type. While the MPC1 and MPC2 are similar in several ways, each has distinct gene expression profiles and differences in lineage commitment, providing investigators options from which to choose when deciding upon the appropriate cell model.While both MPC1 and MPC2 cell lines have several beneficial features, there remain limitations in the use of these cells. As MPC cells are derived from single cell clones, they are clonally similar in the undifferentiated state. This characteristic is beneficial to achieve reproducible results; however, studies seeking to replicate the heterogenous population of the bone marrow niche may opt for a culture system such as the SCD-OSost and Fgf23. Few cell lines secrete FGF23 protein, in particular intact FGF23, a feature that is asset of these cells. In addition to producing these factors, the ability to MPC cells to differentiate into multiple lineages allows investigators the opportunity to study the transition between mesenchymal cell types. An additional strength of this study was that differentiation of MPC cells was achieved across five different laboratories. The consistent growth and differentiation of these cells among several investigators enables greater confidence in achieving reproducible results, providing increased rigor in future studies.The immortalized nature of these cells may also be a limitation for some studies, as the process of introducing the immortalization vector may alter responses. We’ve attempted to circumvent this by using the temperature sensitive large T-antigen, thus the proliferative capacity of the cells can be modulated as needed. Despite some of these limitations, MPC cells produce very high levels of osteogenic genes, especially In summary, we generated and characterized two novel multi-potent cell lines useful for the study of the undifferentiated mesenchymal progenitor state as well as the differentiated osteoblast/osteocyte, chondrocyte, and adipocyte lineages. When cultured in osteogenic conditions, these cells produce abundant mineralized matrix and are capable of expressing the full profile of genes from the MSC precursor, to osteoblast, to osteocytes. MPC cells also quickly differentiate into fat cells, providing a much faster culture model than many currently available cell lines. These cells provide a novel tool to study factors that regulate MSC differentiation as well as the differentiated state of these mesenchymal cell types.Supplementary Figures.


Glycine max L.) containing the phytoene synthase-2A-carotene desaturase gene and wild-type soybean . To investigate metabolic changes between variants, we performed metabolic profiling of leaves (three growth stages) and seeds. Multivariate analyses revealed significant metabolic differences between genotypes in seeds and leaves, with seeds showing accumulation of phytosterols, tocopherols, and carotenoids (BCE only), indicating co-induction of the methylerythritol 4-phosphate and mevalonic acid pathways. Additionally, Hybrid produced intermediate levels of carotenoids and high levels of amino acids. Principal component analysis revealed metabolic discrimination between growth stages of soybean leaves and identified differences in leaf groups according to different genotypes at 8, 12, and 16 weeks, with Wild showing higher levels of environmental stress-related compounds relative to BCE and Hybrid leaves. The metabolic profiling approach could be a useful tool to identify metabolic links in various soybean cultivars.We obtained a new hybrid soybean (Hybrid) by hybridizing β-carotene-enhanced soybean (BCE;  Soybean is consumed worldwide and represents an important crop grown for animal and human consumption as a high-quality protein source ,2. SoybeGlycine max and Glycine soja species according to their habitat [Glycine max is a cultivated soybean that is particularly rich in amino acids and used extensively in breeding and genetic modification to improve soybean production and nutritional value [Glycine soja is a wild species with greater genetic diversity and allele abundance than Glycine max. Additionally, previous studies found that Glycine soja exhibits better tolerance and adaptability to environmental stress, such as low temperature, dehydration, and salt stress, and high levels of phenylpropanoids and isoflavones [Glycine soja is considered a gene source for improving the tolerance of cultivated species to biotic and abiotic stress [Glycine max and Glycine soja could promote the development of new cultivated varieties with improved stress tolerance [Soybean is classified into  habitat . Glycineal value ,12,13. Iflavones ,15,16,17olerance .phytoene synthase-2A-carotene desaturase (PAC) gene into Glycine max L. “Kwangan”, followed by nutritional-composition analysis. Additionally, previous studies have shown that hybridization of carotenoid-biofortified transgenic soybeans with improved nutritional value and wild soybeans promoted the production of soybeans with new nutritional value [Carotenoids are a dietary precursor of vitamin A, a compound nutritionally beneficial to humans ,20. Becaal value ,23. ContGlycine max × Glycine soja) has not been performed.Metabolomics is applied in conjunction with genomics and proteomics to gain a comprehensive understanding of complex networks of biological processes ,25. BecaIn this study, we performed metabolic profiling to understand the metabolic properties of β-carotene-enhanced soybeans (BCE), wild soybeans (Wild), and hybrid soybeans (Hybrid). We applied gas chromatography–time-of-flight mass spectrometry (GC-TOF-MS), GC-quadrupole MS (GC-qMS), and high-performance liquid chromatography (HPLC) to identify primary and secondary metabolites in the seeds and leaves of BCE, Wild and Hybrid.Agrobacterium-mediated transformation using the recombinant PAC gene in cultivated soybean (Glycine max L. ‘Kwangan’) [N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and methoxyamine hydrochloride (MOX) were obtained from Sigma-Aldrich . All chemicals and reagents used in the study were HPLC grade.BCE were developed through wangan’) ,30,31. Pv/v/v) solution, followed by the addition of 60 μL of ribitol solution (0.2 mg/mL in methanol) as an internal standard (IS) to the tube. The mixture was then incubated at 37 °C and shaken at 1200 rpm for 30 min in a thermomixer , followed by centrifugation at 16,000× g and 4 °C for 5 min. The supernatant (0.8 mL) was transferred to a new tube, and 0.4 mL of deionized water was added before centrifugation at 16,000× g and 4 °C for 5 min. The supernatant (0.9 mL) was then transferred to a new tube and dried in a centrifugal concentrator for at least 3 h, followed by freeze-drying for 16 h. For methoxime derivatization, 80 μL MOX (20 mg/mL) in pyridine was added and shaken at 30 °C for 90 min, after which 80 μL MSTFA was added and shaken for 30 min at 37 °C to perform trimethylsilylated etherification. These derivatized samples were analyzed using GC-TOF-MS. Hydrophilic compounds were analyzed on an Agilent 7890A GC  instrument equipped with a Pegasus TOF-MS . The Rtx-5MS column  was added to the GC, and helium gas was passed at a rate of 1 mL/min. Thereafter, 1 μL of extracted sample was injected at a 1:25 ratio in split mode, and the inlet temperature was set at 230 °C. The oven temperature was programmed as follows: starting at 80 °C for 2 min, followed by ramping to 320 °C at 15 °C/min and holding at this temperature for 50 min. The ion-source and transfer-line temperatures were respectively set at 250 °C and 280 °C. Spectral data were scanned over an m/z mass range of 85 to 600. For quantitative and qualitative analysis, we identified metabolites as compounds as Level 1 in the four-step reporting system recommended by the Chemical Analysis Working Group (CAWG) of the Metabolomics Standards Initiative (MSI) [https://www.nist.gov/, accessed on 1 October 2021), and Wiley 9 . For quantification, the relative peak-area ratio of compounds to that of the IS was acquired based on the selected ions  . Data weted ions . Represew/v) were added to a 15 mL tube and vortexed. The samples were then placed in a water bath at 85 °C for 5 min. The mixture was then saponified with 120 μL of 80% potassium hydroxide in water (w/v) in the 85 °C water bath for 10 min. After saponification, the samples were placed immediately on ice for 5 min to stop the reaction, after which 100 μL of 25 ppm trans-β-Apo-8′-carotenal (as an IS), water (1.5 mL), and hexane (1.5 mL) were added to the tube and vortexed. After centrifugation for 5 min at 4 °C and 1200× g, the hexane layer (supernatant) was transferred to a new tube, and extraction was repeated with hexane (1.5 mL). The hexane layer (~3 mL) was then concentrated using nitrogen gas and a vacuum concentrator . After dissolving the concentrate in 250 μL of methanol:dichloromethane  solution, the sample extract was filtered through a 0.5 μm syringe filter into 2 mL autosampler vial and analyzed by HPLC. Carotenoids were separated on a YMC carotenoid HPLC column  using the HPLC system  equipped with a diode-array detector with the wavelength set at 450 nm. The column temperature was set to 40 °C. Solvent A comprised methanol:water  with 10 mM ammonium acetate, and solvent B comprised 100% methyl tert-butyl ether. The binary gradient elution system of solvent A–solvent B was as follows: 0 min, 90% A/10% B; 20 min, 83% A/17% B; 29 min, 75% A/25% B; 35 min, 30% A/70% B; 40 min, 30% A/70% B; 42 min, 25% A/75% B; 45 min, 90% A/10% B; and 55 min, 90% A/10% B. For quantification, a calibration curve was obtained using standard compounds and calculated as the ratio of the peak area of the standard compound to the peak area of the IS , followed by the addition of 50 μL of 5α-cholestane (10 μg/mL) as an IS and incubation in a water bath at 85 °C for 10 min. Saponification was then performed with 120 μL of 80% potassium hydroxide (w/v), and the mixture was placed in a water bath at 85 °C for 10 min, followed by placement on ice for 5 min. The mixtures were then supplemented with hexane (1.5 mL) and water (1.5 mL) and centrifuged at 4 °C and 1200× g for 5 min, after which the supernatants were transferred to a new tube, and extraction was repeated with 1.5 mL hexane. The hexane (~3 mL) was dried using nitrogen gas and a vacuum concentrator. For derivatization, 30 μL MSTFA and 30 μL pyridine were added and incubated at 60 °C and 1200 rpm for 30 min, followed by analysis by GC-qMS using a GCMS-QP2010 Ultra system . For separation, 1 µL aliquots were injected onto an Rtx-5MS column  at a split ratio of 1:10. Helium was flowed at a constant rate of 1 mL/min at an inlet temperature of 290 °C. The initial oven temperature (150 °C) was maintained for 2 min, ramped to 320 °C at 15 °C/min, and held for 10 min. The ion-source and interface temperatures were 230 °C and 280 °C, respectively. Spectra were acquired for m/z 85 to 600, and ions were detected in the selected ion-monitoring mode for peak analysis . PathVisio version 3.3.0 was downloaded from the PathVisio website  and used for visualization of changes in metabolite. A p value < 0.05 was considered statistically significant.All analyses were performed using at least three replicates. The relative quantification data and the absolute quantification data acquired from GC-TOF-MS, GC-qMS, and HPLC were scaled with unit variance scaling before all variables were subjected to the multivariate analysis. Principal component analysis (PCA) was performed using Soft Independent Modeling of Class Analogy software . Pearson’s correlation analysis was performed using the SAS software package  to identify correlations between metabolites. Data analyzed in SAS were imported into MultiExperiment Viewer software  for visualization and generation of hierarchical cluster analysis (HCA) results. The biological pathway was drawn based on the AtMetExpress overview pathway of Glycine soja has high levels of flavonoids and phenylpropanoids [We analyzed the primary metabolites  and secondary metabolites  in seeds of BCE, Wild, and Hybrid using GC-TOF-MS, GC-qMS, and HPLC and obtained a total of 59 metabolites . Analytiopanoids . We then performed Pearson’s correlation analysis and HCA to investigate relationships between the 59 metabolites identified in the three soybean seeds . HCA resphytoene synthase and carotene desaturase genes, with total carotenoid content at 255.8 μg/g, 88.7 μg/g, and 8.2 μg/g in BCE, Hybrid, and Wild, respectively  negatively correlated with 9Z-β-carotene (r = −0.95698), E-β-carotene (r = −0.95513), and 13Z-β-carotene (r = −0.94891), suggesting that pyruvic acid was used as a precursor for the production of carotenoids and resulting in lower levels of pyruvic acid in BCE seeds with higher levels of carotenoids than Hybrid and Wild seeds.PCA and HCA results identified differences in metabolite patterns in BCE, Wild, and Hybrid seeds, with these differences visualized as metabolic pathways  accordinectively . Therefooxidants ,17,38. Aoxidants . MoreovePhenylpropanoid is a defense compound induced by a variety of biotic and abiotic stresses, such as pathogen attack, wounding, and ozone and ultraviolet light exposure, and serves as a precursor to polyphenol-barrier synthesis . TryptopFurthermore, levels of most amino acids were significantly higher in Hybrid seed than in BCE and Wild seeds. Oh et al.  reportedGlycine soja) showed higher levels of shikimic acid, γ-aminobutyric acid, policosanols, and phenolic acids, such as sinapinic acid, p-coumaric acid, and ferulic acid. Phenolic compounds are mainly present in the outer layers of plants to protect against environmental stress, such as dryness and salt [Soybean leaves store energy through photosynthesis and transfer it to storage organs, such as seeds. Given that leaves are closely related to seeds, we performed metabolite profiling of leaves together with the seeds. We analyzed a total of 61 hydrophilic and lipophilic components in the leaves of the soybeans . The twoand salt ,48. The and salt , and Linand salt  reportedGlycine max have higher levels of most amino acids and sucrose than leaves of Glycine soja [In addition, we performed PCA to compare leaves with different genotypes in the same growth stage. A biplot is a standard way to show sample scores and loadings in a single plot. All PCA results at the same growth stage  showed that BCE, Wild, and Hybrid leaves were divided according to genotype . The preine soja ,29. Thisine soja . TherefoThis study represents the first analysis of primary metabolites, including organic acids, amino acids, sugars, and sugar alcohols, and secondary metabolites, including carotenoids, tocopherols, phytosterols, policosanols, and phenolic acids, using GC-TOF-MS, HPLC, and GC-qMS to investigate the metabolic patterns of BCE, Wild, and Hybrid induced by their hybridization. Metabolic profiling has been employed to direct breeding strategies to enhance specific desired balances of food components in fresh food . Multiva