Medicago species were tested for their ability to bind and activate the nuclear receptor PPARγ by SPR experiments and transactivation assay, respectively. The SPR analysis proved to be a very powerful and fast technique for screening a large number of compounds for their affinity to PPARγ and selecting the better candidates for further studies. Based on the obtained results, the sapogenin caulophyllogenin was proved to be a partial agonist towards PPARγ and the X-ray structure of its complex with PPARγ was also solved, in order to investigate the binding mode in the ligand binding domain of the nuclear receptor. This is the first known crystal structure of a sapogenin directly interacting with PPARγ. Another compound of the series, the echinocistic acid, showed antagonist activity towards PPARγ, a property that could be useful to inhibit the adipocyte differentiation which is a typical adverse effect of PPARγ agonists. This study confirms the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating the metabolic syndrome.A series of saponins and sapogenins from  PPARγ is a crucial regulator of glucose and lipid homeostasis and an important pharmacological target for treating metabolic diseases1257Some medicinal plants have been traditionally used to treat this kind of metabolic diseases because of their hypoglycemic and antidiabetic properties. Saponins are a class of chemical compounds found in particular abundance in various plant species which have been reported to exhibit hypoglycemic potential in diabetic states10121314Saponins are biologically active plant-derived glycosides consisting of a sugar moiety linked to a hydrophobic aglycone (sapogenin) with a triterpenoid or a steroid structure. They may have one (monodesmosidic) or more (bi- and tridesmosidic) linear or branched sugar chains linked to the aglycone mojety through an ether or ester bond. Due to their variety of chemical structures, naturally occurring saponins display a broad diversity of polarity, hydrophobicity and acidity that determine their various biological and pharmacological featuresPlatycodi radix have been shown to improve homeostasis in type 2 diabetic states, partly by enhancing hepatic and adipocyte insulin sensitivity which is achieved by activating PPARγ19Pulsatilla koreana inhibited TNFα-stimulated NF-κB activation in a dose-dependent manner, with IC50 values ranging from 0.75–8.30 μM, repressing the expression of the iNOS and ICAM-1 genes, which play important roles in the inflammatory response2150 values up to 1 μM. Moreover, protopanaxatriol, a monoglucoside sapogenin present in the root of Panax ginseng, showed antagonist activity towards PPARγSaponins from Medicago species were considered and used to study their effects on PPARγ. In the genus Medicago, saponins are a complex mixture of triterpenic pentacyclic glycosides with medicagenic acid, hederagenin, bayogenin, zanhic acid and soyasapogenol B as the most abundant aglycones. The most abundant monosaccharide units found in the Medicago saponins are: arabinose, rhamnose, xylose, glucose and glucuronic acid. These compounds have been shown to possess a broad spectrum of biological properties such as fungicidal, molluscicidal, insecticidal, nematicidal, allelopathic, antiviral, cytotoxic and hemolytic activitiesBased on the above reported evidence, saponins and sapogenins from Medicago species were tested for their ability to bind and activate PPARγ by SPR experiments and transactivation assay, respectively. Based on the obtained results, one of the most active compounds was also considered for further studies by resolving the X-ray structure of its complex with PPARγ, in order to investigate the binding mode in the ligand binding domain (LBD) of the nuclear receptor. ITC experiments were also performed on this compound in comparison with the fatty acid 13-(S)-HODE, a natural agonist of PPARγ showing a similar binding mode.In the present paper a series of previously purified and characterized saponins and sapogenins from d) and rate constants  for PPARγ receptor/saponin or sapogenin interactions are reported in d obtained by this analytical method (2.34 μM) basically confirmed that previously achieved from ITC experiments (3.66 μM)1–17), and sapogenins (compounds 18–24). For saponins, different values of affinity towards PPARγ were registered: higher affinity (Kd less than 100 μM) were found for saponins 1 (that shows the lowest equilibrium dissociation constant Kd = 18.33 μM), 3, 4, 6–8, 10, and 17; a middle affinity (Kd 125–150 μM) was found for saponins 2, 5 and 9, while a very low or no affinity (Kd from about 500 to >1000 μM) was observed for saponins 11–16  and caulophyllogenin (19) showed considerable affinities for PPARγ, with Kd = 9.70 and 54.82 μM, respectively, whereas the other tested sapogenins had very low or no affinity  showed a binding profile that differed from all the other tested compounds. In fact, the data obtained from kinetic analysis of 18 were more suitable for a 2:1 molecular interaction model  and a lower dissociation rate constant koff (1.4·10−3versus 0.3–64 s−1), therefore a slow kinetics of association and dissociation can be hypothesized. A similar behavior has been observed for other nuclear receptor antagonistsInterestingly, echinocistic acid  and soyasaponin I (17) did not show cytotoxicity at concentrations up to 100 μM. The other tested compounds showed cytotoxic activities at values ranging from 3 to 20 μM . Only 19 behaved as a partial agonist with EC50 = 12.6 ± 2.7 μM and efficacy = 9.4 ± 0.6%. 18 displayed similar activity only at concentrations up to 6.25 μM  of PPARγ is shown in 19 can be easily fitted. The ligand was accommodated between the helix3 and the β-sheet, where its carboxylic oxygens formed a hydrogen bond (2.1 Å) with the NH group of S342, belonging to the β-sheet, and the CO of L340 (3.3 Å), respectively. The 16α-hydroxy group is H-bound to the CO of G284 on helix 3 (2.7 Å). The 3β-hydroxy group made a H-bond with the hydroxyl group of the Y327 side-chain (2.9 Å). Moreover, there were extensive vdW interactions between the sulphur atom of C285, on the helix 3, and the carbon atoms of the rings C and E of the ligand. The ring E was also engaged in vdW interactions with the aromatic ring of F363, on helix 7. The position of 19 in the LBD of PPARγ was very similar to that of the partial agonist R enantiomer of LT175 (pdb 3D6DS)-HODE  of this rigid sapogenin with those of the more flexible fatty acid 13-(S)-HODE. The results . Moreover, the comparison of the thermodynamic parameters between the two compounds showed that although the two compounds have similar Kd (6.5 and 9.4 μM for 19 and 13-(S)-HODE, respectively), the binding of 13-(S)-HODE, whose higher flexibility allows better interactions with the protein, shows a more favorable enthalpic contribution , instead the binding of 19 is associated to a more favorable entropic term , also due to the minor loss of translational and rotational degrees of freedom of this more rigid ligand upon binding. Anyway, both binding interactions seem to be entropy-driven and more hydrophobic in character.ITC experiments were performed to confirm the interaction of  results  indicateA selected series of previously purified saponins and sapogenins was screened by SPR techniques to test their affinity to PPARγ in order to select the most promising compounds for the activity test and for X-ray analysis. All the tested compounds had the same pentacyclic triterpene structure with differences in number and position of hydroxyl and carboxylic groups on the triterpene skeleton and nature and number of the sugars linked at C-3 and C-28 position.β position of the aglycone seemed to lower the affinity. Saponins of 2β-hydroxy oleanolic acid (compound 2) and bayogenin (compounds 9 and 10) showed, in general, higher dissociation constants Kd compared to saponins of oleanolic acid (compound 1) and hederagenin  in which the 2β-hydroxy group was not present.The SPR experiment showed that, among saponins, the compounds with higher affinity were, in general, short sugar chains mono- and bidesmoside saponins characterized by the presence of a methyl or alcoholic group at C-23 position on the triterpenic nucleus. On the contrary, a carboxylic group in the same position, as in the derivatives of medicagenic and zanhic acid, significantly reduced the affinity. In addition, the hydroxyl group at the 24, 6, 7 and 8), bayogenin (compounds 9 and 10) and soyasapogenol B (compound 17) even though their aglycone portion, hederagenin (20), bayogenin (21) and soyasapogenol B (24), didn’t show affinity to the nuclear receptor. This allows to suggest that sugars, in particular when linked at the C-3 position, are of relevance for the interaction mechanisms with the binding site of PPARγ. Sugar chains could either allow a correct positioning of the aglycone moiety in the affinity site of the nuclear receptor or favor its accommodation into an alternative binding site. In fact, the X-ray structure of the complex PPARγ/19 showed that there could be additional space for the accommodation of the glycoside moieties in the region tipically occupied by full agonists, towards the helix 12.The glycosidic portion of the molecule seemed to be an additional important feature for the interaction with PPARγ. As reported in Also the sugar chain length, in particular at C-28 position, seems to be of importance to improve affinity. The most active saponins are both monodesmosidic or monosaccaride C-28 substituted compounds.18) and caulophyllogenin (19) possessed a good affinity for the PPARγ LBD . Both compounds are characterized by the presence of a 16α-hydroxyl group in the molecule that probably is another important feature in conferring affinity to PPARγ. The same 16α-hydroxy substituent is also present in zanhic acid but this character is probably not sufficient to contrast the strong binding inhibition performed by the carboxylic group at C-23 position.Concerning sapogenins, the SPR experiment showed that only echinocystic acid  indicated that 17 and 19 did not show any cytotoxic activity at concentrations up to 100 μM concentration, but only caulophyllogenin 19 showed a partial agonist activity towards PPARγ. Its low potency and efficacy  should not be underestimated because very similar to those of the well-known selective modulator metaglidasen2819 showed an accommodation of the ligand in the LBD between helix3 and the β-sheet, typical of known partial agonists, where it formed H-bonds with the first strand of the β-sheet and with helix3. Hypothetically, in this orientation of the ligand it could be very easy to accommodate glycoside moieties linked to the carbon atom 3 that protrude towards the helix12, in the region occupied by full agonists.The MTT assay test and the transcriptional activity test performed on the most affine compounds  deserves to be deepened to establish a possible role of this sapogenin as potential anti-cancer in combination with other anti-tumorigenic drugs, as already evidenced in previous papers30Very interestingly, echinocystic acid 18 and caulophyllogenin 19 used in this investigation were extracted from M. polimorpha (Leguminose family), but they are also components of Crysantellum americanum, a plant of the Asteraceae family which contains several triterpen saponins, whose therapeutic properties as hypolipidemic and hepatoprotective agents are knownEchinocystic acid 19 as a promising partial agonist of PPARγ and echinocistic acid 18 as PPARγ antagonist.In conclusion, the present study confirm the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating type 2 diabetes, obesity, metabolic syndrome and inflammationFinally, the SPR analysis has been proved to be a very powerful and fast method for screening a large number of compounds and selecting the better candidates as agonists and also antagonists of the nuclear receptor PPARγ.Medicago species as described by Tava et al.353634353637Saponins were extracted and purified as previously described34353637E. coli BL21 DE3 were grown in LB medium with 30 μg of kanamycin/mL at 310 K to an OD of 0.6. The culture was then induced with 0.1 mM isopropyl-β-D-thio-galactopyranoside and further incubated at 291 K for 20 h. Cells were harvested and resuspended in a 20 mL/liter culture of Buffer A , pH 8) in the presence of protease inhibitors . Cells were sonicated, and the soluble fraction was isolated by centrifugation . The supernatant was loaded onto a Ni2+ -nitrilotriacetic acid column  and eluted with a gradient of imidazole 0–300 mM in Buffer A (batch method). The pure protein was identified by SDS PAGE. The protein was then dialyzed over buffer A to remove imidazole, and it was cleaved with thrombin protease  (10 units/mg) at room temperature for 2 h. The digested mixture was reloaded onto a Ni2+ -nitriloacetic acid column to remove His tag and the undigested protein. The flow-through was loaded onto a Q-Sepharose HP column  and eluted with a gradient of NaCl 0–500 mM in Buffer B  with a BioLogic DuoFlow FPLC system . Finally, the protein was purified by gel-filtration chromatography on a HiLoad Superdex 75 column  and eluted with Buffer C . The protein was then concentrated at 8 mg/mL using Amicon centrifugal concentrators with a 10 kDa cutoff membrane . Part of the protein was used for SPR experiments. Crystals of apo-PPARγ were obtained by vapor diffusion at 18 °C using a sitting drop made by mixing 2 μL of protein solution with 2 μL of reservoir solution . The crystals were soaked for 8 days in a storage solution  containing the sapogenin 19 (0.25 mM). The ligand dissolved in DMSO was diluted in the storage solution so that the final concentration of DMSO was 0.5%. The storage solution with glycerol 20% (v/v) was used as cryoprotectant. Crystals of PPARγ/19 belong to the space group C2 with cell parameters shown in PPARγ LBD was expressed as N-terminal His-tagged proteins using a pET28 vector and then purified as previously described19 belonging to C2 space group. A final step of refinement was performed with the software Phenix19 have been deposited in the Brookhaven Protein Data Bank (PDB) with the code 5F9B.X-ray data set were collected at 100 K under a nitrogen stream using sinchrotron radiation . The collected data were processed using the programs MOSFLM and SCALAN-hydroxysuccinimide (NHS):200 mM ethyl-3(3-dimethylamino) propyl carbodiimide (EDC). Fifty μL of a 0.25 mg/mL PPARγ solution  were injected for 5 min at 10 μL/min on channels 1 and 3 (channel 2 was used as ref. 464714 and 15) over the three channels at 20 °C (association phase of 180 s). Five buffer blanks were injected for double referencing . The regeneration of the surfaces between binding cycles was not necessary because all the analytes dissociate quickly in the 120 s dissociation phase. A DMSO calibration plot was constructed (buffer sample containing 1–3% (vol/vol) DMSO) to correct for bulk refractive index shiftsd) was determined by the ratio koff/kon. Constant reported in Surface plasmon resonance analyses were performed by using Pioneer AE optical biosensor equipped with COOH5 chips (SensiQ). PPARγ surfaces were prepared by using standard amine-coupling proceduresS)-HODE (10 μg with 83 μL of buffer to obtain a 400 μM solution) and the SAP19 stock solution (25 mM in DMSO). DMSO was added to the protein solution at the same percentage of the ligand solution (2%) for the experiment with SAP19. The protein solution (50 μM) was placed in the sample cell, and ligand solution (500 μM) was loaded into the syringe injector (PPARγ 40 μM and the ligand 400 μM for the experiment with 13-HODE) . The titrations involved 19 injections of 2 μL each at 150 s intervals. A reference titration of ligand into buffer was used to correct for heats of dilution. Thermodynamic data was processed with Origin 7.0 software . Fitting the isotherms with one-site binding model yealded the values of the association constant (Ka).ITC experiments were performed at 22 °C by using a ITC200 microcalorimeter . PPARγ was extensively dialyzed against a solution of HEPES  and TCEP (1 mM) with Amicon Ultra filters, and the final exchange buffer was used to dilute 13- containing five repeats of the Gal4 response elements upstream of a minimal thymidine kinase promoter that is adjacent to the luciferase gene were described previously−1 of penicillin G, and 100 μg·mL−1 of streptomycin sulfate at 37 °C in a humidified atmosphere of 5% CO2.Human hepatocellular liver carcinoma cell line HepG2  was cultured in Minimum Essential Medium (MEM) containing 10% heat-inactivated fetal bovine serum, 100 U·mL5 cells per well were seeded in a 96-well plate and transfected after 24 h with K2 Transfection System (Biontex Laboratories GmbH), according to the manufacturer’s protocol using 0.20 μg/well of DNA. Cells were transfected with expression plasmids encoding the fusion protein Gal4-PPARα-LBD or Gal4-PPARγ-LBD, pGal5TKpGL3, and pCMVβgal to normalize the transfection efficacy. 24 h after transfection, medium was replaced with fresh complete growth medium supplemented with test compounds (ranging from 200 nM to 100 μM), reference compounds Wy-14,643 (10 μM) and rosiglitazone (2 μM), or DMSO 0.1%. After further 24 h of incubation cells were lysed and luciferase activity in cell extracts was determined by a luminometer  and normalized for β-galactosidase activity. Fold induction activity was calculated and plotted using GraphPad Prism5 software.For transactivation assays, 1·105 cells/well into 96-well flat bottom culture plates containing test compounds , in a final volume of 100 μL. Test compounds were dissolved in DMSO . Control wells lacked inhibitor. After 24 h of incubation at 37 °C in a 5% CO2 atmosphere, 3--2,5-diphenyltetrazolium bromide  was added to a final concentration of 0.5 mg/mL. To control for background absorbance, six wells of cells were lysed by adding Triton X-100  immediately prior to the addition of MTT reagent. After incubation under the same conditions for further 3–4 h, the culture medium was removed, the insoluble product dissolved by the addition of 100 μL of solvent , and the absorbance of the well was measured at 570 nm using a PERKIN–ELMER Victor V3 plate reader. Cell growth inhibition was then calculated using the following Equation,Cell viability was measured using the MTT assay. HepG2 cells were seeded at a density of 1·10V% is the percentage of cell viability, A is the absorbance of treated cultures, Ab is the absorbance of background control, and Ac is the absorbance of control cultures.where All experiments were performed in triplicate and were repeated at least twice with similar results. The results were expressed as mean ± SEM. The responses to agonists were calculated as a percentage compared to control, which was set to 100%.Accession codes: The coordinates of PPARγ /19 have been deposited in the Brookhaven Protein Data Bank (PDB) with the code 5F9B.How to cite this article: Montanari, R. et al. Screening of saponins and sapogenins from Medicago species as potential PPARγ agonists and X-ray structure of the complex PPARγ /caulophyllogenin. Sci. Rep. 6, 27658; doi: 10.1038/srep27658 (2016).


CO2 concentrations. We grew freshly germinated seedlings of up to 28 C3 species  in climate chambers at 160 ppm, 450 ppm, and 750 ppm CO2. We determined biomass, allocation, SLA (specific leaf area), LAR (leaf area ratio), and RGR (relative growth rate), thereby doubling the available data on these plant responses to low CO2. High CO2 increased RGR by 8%; low CO2 decreased RGR by 23%. Fast growers at ambient CO2 had the greatest reduction in RGR at low CO2 as they lost the benefits of a fast‐growth morphology (decoupling of RGR and LAR [leaf area ratio]). Despite these shifts species ranking on biomass and RGR was unaffected by CO2, winners continued to win, regardless of CO2. Unlike for other plant resources we found no trade‐offs in morphological and growth responses to CO2 variation, changes in morphological traits were unrelated to changes in growth at low or high CO2. Thus, changes in physiology may be more important than morphological changes in response to CO2 variation.Evolutionary adaptation to variation in resource supply has resulted in plant strategies that are based on trade‐offs in functional traits. Here, we investigate, for the first time across multiple species, whether such trade‐offs are also apparent in growth and morphology responses to past low, current ambient, and future high  This is encapsulated in the equation RGR = LAR*ULR (Evans 2 leaf per g plant) and ULR . Differences in carbon capture may be driven by either: the chemical and physiological traits underlying ULR; tissue carbon content and photosynthesis rate; or the allocation and morphology traits underlying LAR, leaf mass fraction , and SLA   with a small decrease of SLA and LAR  move away from the trait values that, at the interspecific levels, are associated with fast growth (higher SLA = higher RGR), possibly due to a disproportionate increase in photosynthesis per unit leaf area. The very limited experimental results so far have shown that at low CO2 representing the Pleistocene Ice ages a whole suite of traits is drastically altered compared to ambient CO2. Morphological traits are strongly adjusted in response to low CO2. Thinner and less dense leaves  Which plant functional types  lose or will gain the most in terms of growth rate at low and high CO2, respectively? (3) How are changes in RGR related to changes in underlying allocation and morphological traits?Thus, our study had the following research questions: (1) Do species‐specific responses in RGR at low and high CO3 plant functional types in walk‐in climate chambers at a wide range of CO2 concentrations, 160 ppm, 450 ppm, and 750 ppm CO2.To that end, we performed an experiment to quantify variation in growth rate and morphological and allocation traits among 28 different species belonging to a wide variety of C2 concentration, we obtained seeds from a wide range of temperate  woody, forb, and grass C3 species. These had been field collected in Sheffield, U.K., and the Chongqing region, SW China, as well as supplied by B&T World Seeds, Sheffield Seed co., USA, and Kruythoeck Seeds, Netherlands. The seeds were set out to germinate by placing them on either wet sand or wet tissue paper. Special pretreatment of seeds  was carried out according to supplier instructions and the authors' experience. This resulted in successful germination of 28 different species (Table S1) which consisted of six woody species , 16 forb species, and 6 grasses. Shortly after germination, individual plants were transferred to experimental CO2 conditions at the Phytotron labs at Utrecht University, The Netherlands. The growth experiment was spread out over the period October 2012 – October 2013, during which batches of different species were sequentially screened in the standardized environmental regimes.To determine the response of a variety of plant growth forms to variation in CO2 levels: low, ambient, and high. These levels broadly (±50 ppm) represented the large range from Pleistocene past to future high CO2. The low CO2 concentration of 160 ppm (peaking to 180 ppm when handling the plants inside the chamber) was achieved by scrubbing CO2 from ambient air ventilating the room using a molecular sieve . The ambient level (450 ppm) was slightly higher than outside air due to elevated levels inside the office building. The high level (750 ppm) was achieved by adding fossil fuel derived CO2 from high‐pressure tanks to ambient air in the climate room. CO2 levels inside the chambers were digitally monitored  and scrubber or valve capacity adjusted accordingly. Low levels of CO2 while handling plants were maintained using a gas mask to capture exhaled breath in a large airtight bag.We used three separate custom‐built walk‐in climate rooms  in which we maintained three COμmol light, 18°C night/21°C day temperature, 10‐h photoperiod, and 70% relative air humidity. Total daily photon flux was comparable to that of an average March day in the Netherlands, which is when several of the species would have naturally germinated and start to grow. Pots were watered thrice daily up to field capacity using an automated watering system supplying water from below. To prevent nutrient limitation during the experiment, nutrients were added three times per week with 50 mL full Hoagland solution , 2 mmol L−1 NH4H2PO4, 1 μm KCl, 25 μm H3BO3, 2 μm MnSO4, 2 μm ZnSO4, 0.1 μm CuSO4, 0.1 μm (NH4)6Mo7O24, 20 μm Fe(Na)EDTA). To prevent damage from excess nutrients to young plants, freshly germinated individuals were supplied with an increasing concentration starting with 25% nutrients after germination to full Hoagland at the onset of the first leaf and subsequent growth period.Growth conditions were ~350 2, seedlings were transferred to the CO2 chambers in 400‐mL plastic pots containing coarse sand. Because of the small size, the seedlings were expected to obtain during the duration of the experiment pot size was assumed to be sufficient to maintain less than 2 g plant per L soil to avoid pot size effects  measurements. Leaf area (m2) was then determined by pixel counting using ImageJ version 1.47. Fresh plant material was oven dried at 70°C for 48 h and weighed. After drying, leaves were removed from stems and stems were weighed to calculate leaf and stem mass fraction. Plant leaf area ratio  was then calculated by multiplying SLA with leaf mass fraction.At final harvest, plants were washed to remove sand from roots, and fresh weight (weighed to the nearest mg) was measured for above and belowground plant parts. Images were taken to illustrate the effects of COize Fig. . Leaf ar could not be determined. Two episodes where the low CO2 and high CO2 chamber were unavailable due to emergency maintenance led to 25 species in the low CO2 treatment, 22 species in the high CO2 treatment and 19 species with all three treatments.Due to difficulties in germinating enough seedlings to do a representative baseline harvest, RGR of three species 2 response  measuring a single species at multiple time intervals; and (2) comparing the results of multiple batches of different species at different times. Data on RGR and SLA for Avena sativa grown in different batches with substantial time intervals showed a consistent response to CO2 and supports the robustness of the treatment in the climate chambers ; only one batch was used in this study. While different species grown during different batches show moderately different responses to CO2, the overall effect of low or high CO2 was comparable between species and batches . We are thus confident that a repeat of the experiment with a different “random draw” of species would lead to similar conclusions.Given the limited number of species analyzed for low to high CO2. Rank changes were then tested pairwise between CO2 treatments in a paired‐Wilcoxon‐signed‐rank test with Bonferroni correction. CO2 effects on traits and species differences in traits were tested by comparing the shift in trait value to the trait value at ambient CO2. To improve normality and minimize skew, trait values were natural log (ln) transformed prior to analyzing CO2 effect on trait shifts. The difference in ln‐transformed trait level from ln‐transformed ambient level was then the relative shift in trait level via eln transformed difference−1. This approach had the added benefit that a halving or a doubling in trait value from ambient had the same ln‐transformed difference. Per species we averaged the trait response of individual replicates per treatment. These shifts in species trait values at low or high CO2 (compared to ambient CO2) were then tested by one‐sample t‐tests. Differences between plant types were determined by two‐sample t‐tests on species trait shift with Bonferroni correction for the three comparisons made . To determine whether the reduction and stimulation in growth and biomass was related to trait values at ambient CO2 or shifts in trait value toward low or high CO2, we performed a stepwise model selection procedure selecting models based on AIC using the MASS package , LMF (leaf mass fraction), and LAR at ambient CO2. The initial model to test the relationship between the difference in RGR and shifts in trait level included the shifts in RMF, LMF, and LAR. The relationship between RGR and LAR at all three CO2 levels was determined using ordinary least squares regression as we viewed LAR as a predictor of RGR.Results were analyzed using R  and RStudio . Changes in the interspecific ranking based on RGR were analyzed nonparametrically by determining species rank on RGR at low, ambient, and high CO2 treatments. The photographs of Figure 2 on plant size. In general, plants at low CO2 were tiny compared to ambient CO2 and as expected plants were stimulated by elevated CO2. While different species showed moderately different responses, species grown in different batches in different periods showed comparable responses to CO2 .Plants responded strongly to the low and high COPicea sitchensis growing the slowest regardless of CO2 concentration and the forb Rumex acetosella (missing at high CO2) and semi‐woody scrambler Solanum dulcamara growing fastest at low, ambient, and high CO2  at low CO2  although grass species did not increase their RGR  and high CO2 increased biomass by 32.2% ± 6.7 (P < 0.001) . Low CO. Low CO22, we did find that, in general, species with a higher RGR at ambient CO2 had a stronger reduction in RGR with low CO2 . These RGR differences led to absolute biomass loss at low CO2 compared to ambient being highest for large species .While the ranking of species for RGR and biomass was not significantly altered by COCO2 Fig. A. As the Figure S. These R2, only at the stress of low CO2 did we find significant differences in trait adjustment between types . Grass species had a markedly different response in SLA and LAR. On average, relative to ambient CO2, SLA increased strongly at low CO2 by 59.4% ± 12.4 (P < 0.001) and decreased modestly by 13.8% ± 2.6 at high CO2 (P < 0.001). However, when viewed separately at low CO2, woody and forb species had a very large increase in SLA  whereas grass species did not significantly increase their SLA , and grass and woody species, however, did not significantly reduce SLA. For LAR, a similar result was found .Plant types showed only limited differences in their responses to COpes Fig. . Biomasspes Fig. F with wo2. None of the morphological traits  was significantly related to differences in growth rate or biomass at low CO2. However, species with the highest growth rate or largest biomass at ambient CO2 did show the strongest absolute reduction at low CO2 . However, at low CO2  and allocation , and thereby to changes in leaf area ratio. Indeed, the well‐established positive relationship between LAR and RGR, as seen at ambient and high CO2, broke down entirely at low CO2.This study is novel in that we investigated 19 plant species belonging to different functional types in their performance across the whole range of Pleistocene low, via ambient to future high CO2. However, with some exceptions, species ranking remained similar . This poses the question whether there are clearer winners and losers in interspecific competition now than in the Pleistocene past due to increased differences in growth rate between species.In general in plant strategy theory, there are trade‐offs in species performance across resource supply gradients. For example, species that perform well at high nitrogen supply are poor performers at N‐limited growth conditions  are not generally found in shady habitats. From a resource economics perspective, the extent of CO2 stimulation should be dependent on the availability of other resources  mechanism is unclear.We found comparable trait responses in the three plant types. Over the whole range of CO2, at elevated CO2 growth and biomass stimulation was greatest for woody species  morphology with faster growth. Differences in growth rate at carbon starvation could therefore be driven more by physiological differences. Understanding how physiological traits are affected by carbon starvation and carbon excess will shed more light on the interaction between morphology, physiology, and growth from past low to future high CO2.Plant growth and development are strongly affected by CONone declared.Figure S1 Response of multiple batches of Avena sativa to CO2.Figure S2 Comparison of RGR and SLA response to CO2 of two batches of different species.Figure S3 Plant species biomass ranking at low, ambient and high CO2,Figure S4 RGR and RGR rank at low, ambient and high CO2.Figure S5 Relative biomass lost at low CO2 as compared to ambient CO2.Figure S6 Difference in growth rate and biomass at high CO2 as compared to ambient CO2.Figure S7 Relationship between leaf mass fraction (LMF) and relative growth rate (RGR) at past low, ambient future high CO2.Click here for additional data file.Table S1 Species grown and their classification.Click here for additional data file.


One of the C=F bonds is formed by transfer of electrophilic fluorine from the hypervalent iodine reagent, while the other one arises from the tetrafluoroborate counterion of silver. Deuterium-isotope-labelling experiments and rearrangement of methyl styrene substrates suggest that the reaction proceeds through a phenonium ion intermediate.An air- and moisture-stable fluoroiodane in the presence of AgBF Of these major areas the introduction of a CF2 group is probably the least developed. is the BF4−, which must not necessarily be added as part of a silver salt. However, the yields of 4 are much higher with stoichiometric amounts of AgBF4. Therefore, we employed one equivalent of AgBF4 as a mediator and secondary fluorine source in additional applications . In the case of using Zn(BF4)2 or Cu(MeCN)4BF4, instead of AgBF4, we could observe the difluorination reaction and isolate 4 a in the corresponding yields of 46 and 32 %. However, particularly with a copper salt, several other byproducts were formed (entries 7 and 8).Deviation from the above mentioned [Eq. ] optimal a Table . Applica4(BF4)2] proved to be a rather efficient catalyst, thus affording 4 a with 50 % yield [Eq. 4− counter ion exchanged to Cl− the outcome of the reaction was completely different. When using [PdCl2(MeCN)2] as the catalyst 4 a did not form at all but the reaction gave the iodofluorinated product 5, which could be isolated with 43 % yield. Further studies indicated that in the geminal difluorination reaction with 1, AgF, ZnF2, or CuF2 were not able to serve as fluoride sources , while using toluene led to formation of only traces of 4 a (entry 11).The reaction with palladium salts was rather interesting. -2 a. When [D2]-2 a was reacted with 1 in the presence of AgBF4 under the usual reaction conditions the compound [D2]-4 a was obtained in a very clean reaction [Eq. 2]-2 a was expected. The double deuterium shift suggested a significant deuterium isotope effect. Therefore, we performed a competitive difluorination reaction between 2 a and [D2]-2 a  catalyst [Eq. (2)] the faith of 7 was probably different and it underwent C(sp2)=I bond fission to result in 5. The only possible source of iodine in 5 is 1. However, in the case of AgBF4  the fluorinated phenonium ion 8 forms 4(BF4)2] [see Eq. (2)] may serve as a fluorine source in the activation of (SiMe3)2 for catalytic silylation of allyl alcohols. Gandon and co-workers[4− from AgBF4 is an excellent fluorinating reagent for organometallic compounds. The present study is probably one of the few examples demonstrating that a C=F bond can also be formed under mild reaction conditions using BF4− as a fluorine source. An additional remarkable feature of the above-presented reaction is that the opening of the cyclopropyl ring of 8 is highly regioselective. We could not observe formation of α-,β-difluorinated regioisomers of 4 a in the 1H and 19F NMR spectra of the crude reaction mixtures. Opening of the cyclopropane ring of the fluorinated carbon in 8 also easily explains the apparent migration of the methyl group in the difluorination of 2 j–l  induce geminal difluorination of styrenes. These air- and moisture-stable reagents react under mild reaction conditions to perform a selective formal F2 addition. Using equimolecular amounts of 1 and styrene derivatives resulted in yields over 50 %, thus indicating that one of the C=F bonds is created by electrophilic fluorinating reagent 1, while the other one results from fluorine transfer from BF4−. The deuterium-labelling experiments indicate that the process probably proceeds via phenonium intermediates. The reaction is suitable for mild synthesis of β-difluoro aromatic compounds, which are bioisosters[1.In summary, we have shown that the hypervalent iodine oisosters of natur1 , AgBF43 , and the styrene 2  were mixed in CDCl3 (0.5 mL) and this mixture was stirred at 40 °C for 18 h. Then product 4 was isolated by chromatography.The iodane


Base glasses with different contents of CaO/SrO, P2O5, and F− were prepared within the glasses of the SiO2–Li2O–K2O–CaO/SrO–Al2O3–P2O5–F system. Preliminary studies of nucleation by means of XRD and scanning electron microscopy (SEM) of the nucleated base glasses revealed X-ray amorphous phase separation phenomena. Qualitative and quantitative crystal phase analyses after crystallization were conducted using XRD in combination with Rietveld refinement. As a main result, a direct proportional relationship between the content of apatite-forming components in the base glasses and the content of apatite in the glass–ceramics was established. The microstructures of the glass–ceramics were investigated using SEM. Microstructural and mechanical properties were found to be dominated by Li2Si2O5 crystals and quite independent of the content of the apatite present in the glass–ceramics. Biaxial strengths of up to 540 MPa were detected. Ca5(PO4)3F and Sr5(PO4)3F influence the translucency of the glass–ceramics and, hence, help to precisely tailor the properties of Li2Si2O5 glass–ceramics. The authors conclude that the twofold crystallization of Li2Si2O5–Ca5(PO4)3F or Li2Si2O5–Sr5(PO4)3F glass–ceramics involves independent solid-state reactions, which can be controlled via the chemical composition of the base glasses. The influence of the minor apatite phase on the optical properties helps to achieve new combinations of features of the glass–ceramics and, hence, displays new potential for dental applications.The combination of specific mechanical, esthetic, and chemical properties is decisive for the application of materials in prosthodontics. Controlled twofold crystallization provides a powerful tool to produce special property combinations for glass–ceramic materials. The present study outlines the potential of precipitating Ca Li2Si2O5 is still subject of a multitude of glass–ceramic research. Nucleation mechanisms have been extensively studied . Micrographs of the glass plates after nucleation, polished with a 0.5-μm diamond grit grinding disk and subsequently etched by soaking into 20% H3PO4 for 2 min, were taken by means of scanning electron microscopy (SEM) . Prior to the analysis, the samples were coated with an approximately 2 nm Au–Pd layer by sputtering.Besides nucleation, the first heat treatment was required to relief stresses in the glass structure and, hence, allows further machining of the glass. The cooled glass blocks were placed in a furnace pre-heated to 500°C. After a dwell time of 30 min, the glass blocks were allowed to cool to room temperature. To study the effect of the dwell time at 500°C, one glass block of glass D Table  was heat® type . Small glass plates of approximately 2 mm thickness, cut from the nucleated glass blocks, were used. A detailed investigation of the temperature-dependent crystal phase formation was conducted considering glass D. The nucleated glass plates were heat treated for 30 min at 520, 540, 560, 580, 600, 700, or 800°C. After the dwell time, the samples were allowed to cool to 500°C in the furnace before opening it. Prior to XRD analysis, conducted as described above, the surface of the glasses and glass–ceramics was removed using a 40-μm diamond grit grinding disk.The second heat treatments took place in an oven of the Programat2O3 in acetone. The solvent was evaporated in an oven pre-heated at 80°C. Al2O3 was used as an internal standard in a concentration of about 50 wt.%. After recording the powder XRD patterns from 10° to 100° 2θ in 0.014° steps, the quantification of the crystal phases was done with the TOPAS software from Bruker.A quantitative study of the crystal phase formation was conducted applying Rietveld refinement. Glass–ceramics nucleated at 500°C for 30 min and subsequently crystallized via a second heat treatment for 30 min at 800°C were used. The glass–ceramic material was crushed and comminuted in a mortar grinder  and subsequently sieved <45 μm. Then the powder was elutriated with approximately the same mass of AlThe microstructures of the glass–ceramics crystallized at 800°C for 30 min were investigated applying SEM. Small plates of the nucleated glass blocks were crystallized at 800°C for 30 min and subsequently polished with a 0.5-μm diamond grit grinding disk. The surface of the samples was etched with vapor of 40% HF acid and coated with a 1–2 nm Au–Pd layer prior to SEM analysis. In order utilize the contrast provided by elements with high specific weight via the detection of backscattered ions during SEM, glass–ceramic G was determined. The sample preparation was conducted as described above; however, the surface was etched by soaking into 3% HF acid for 10 s.® InLab milling machine . Crystallization of the plates took place as described above via a second heat treatment for 30 min at 800°C. The samples were prepared for the testing of the biaxial fracture strength according to ISO 6872 including a surface finishing with a 15 μm diamond grit grinding disk. The measurements were conducted using a universal testing apparatus . The biaxial strengths are given as the means of data sets ±SD. Ten samples of each series were tested. Normality of the data sets was determined applying a Jarque–Bera test. Significant deviations (>95%) between the different data sets were examined using the Student’s t-test.Small circular plates were milled from the blocks of nucleated glass using a CERECSmall glass plates measuring approximately 10 mm × 10 mm × 2 mm were cut from the nucleated glass blocks and crystallized at 800°C for 30 min. Afterwards the glass–ceramic plates were prepared for translucency (CR) measurements and color evaluation according to BS5612, DIN5033, and DIN6174, respectively. A spectrometer of the type CM-3700d  was used for the spectroscopic analysis.O of the glass–ceramics was estimated using an equation derived by Kobashigawa et al. . While the a-value gives a measure for the intensity proportion of red and green, the b-value deals with yellow and blue. The Δ values a and b describe the differences of these parameters detected in either the transmission or reflection mode. Glass–ceramic plates measuring approximately 10 mm × 10 mm × 1 mm were used.The opalescence a et al.  Eq. 1)  O of thea et al. , with a Texo or endothermic Tendo peak temperatures are given in Table Tg is in the range of 450–455°C for samples A–E. A slightly increased Tg can be observed for the F− free reference sample F at about 462°C, while the substitution of CaO by SrO in glass G yields a reduction of Tg to about 442°C. All samples show at least one exothermic peak in the DSC curve indicating crystallization. Increasing the content of the components CaO, P2O5, and F− (samples A–E) clearly decreases the temperature for the first exothermic processes detectable by DSC from about 720 to 565°C. A second exothermic signal indicating crystallization is evident at about 830°C for glass A and at approximately 800°C for glass B. However, the latter is rather weak. While glasses C and D as well as F and G do not show any further exothermic signals during thermal analysis, three more exothermic peaks are evident for glass E at about 610, 680, and 786°C.The results of the thermal analysis of the base glasses after quenching in water are shown in Figure 2O5, and F– contents, with the exception of glass E. All of them show two endothermic peaks between 900 and 965°C. The thermal analysis of the F– free reference sample F reveals only one endothermic signal at about 949°C.A comparison of the endothermic signals, which indicates the dissolution of the crystal phases, reveals that no significant difference can be observed between the glasses having different CaO/SrO, P3PO4. The investigation of the microstructures revealed cloudy separation areas in the range of 1 μm in diameter as well as sharp non-spherical phase separations with dimensions <100 nm  and the highest fraction of Li2SiO3 (2.9 wt.%) of all the glass–ceramics analyzed. The F– free reference sample F shows the largest content of Li3PO4 crystals. Compared to the corresponding F– containing glass C, less Li2Si2O5 crystal content is formed. The substitution of CaO by SrO yields the crystallization of Sr5(PO4)3F in glass G.The qualitative and quantitative composition of the glass–ceramics after crystallization at 800°C for 30 min is shown in Table 2Si2O5 crystals. While the microstructures of glass–ceramics F and G are rather similar, quite different microstructures were found in the glass ceramics A, C, E, and F as shown in Figure 2Si2O5 crystals in the range of >>10 μm are present in the material A. The size of the Li2Si2O5 crystals clearly decreases with an increasing fraction of CaO, P2O5, and F−, as shown in a comparison of A, C, and E. Furthermore, the presence of F− refines the microstructure as can be seen by comparing glass ceramics C and F.After etching of the polished surface of the glass–ceramics crystallized at 800°C for 30 min with HF vapor, only one type of crystal morphology is evident in each of the micrographs presented in Figure 2+, compared to the crystalline structures which obviously comprise rather light ions, such as Li+. Regularly distributed black spots with a diameter of approximately 200 nm are present in both micrographs in Figure The micrographs in Figure The biaxial strength of the glass ceramics is presented in Table 2O5, and F− in the compositions A–C decreases the CR value and, hence, reduces the translucency of the glass–ceramics. The relatively small change of the base glass composition from C to D yields a slight increase of the translucency. Composition E shows the highest CR value of all the series. The F– free reference sample F shows a lower translucency compared to that of the fluoroapatite-containing glass–ceramics C and D, while the SrO-containing glass–ceramic G has a slightly decreased translucency compared to the corresponding CaO-containing sample C.The translucency and opalescence properties of the glass–ceramics are presented in Table 2O5, and F− in the base glass. There is no significant difference in terms of the apatite-free reference sample F. Only a slightly increased opalescence can be observed compared to that of the corresponding apatite-forming composition C.The opalescence of the glass–ceramics increases consistently with an increasing ratio of CaO, P2Si2O5 as main crystal phase and Ca5(PO4)3F or Sr5(PO4)3F as minor crystal phase, have been developed. In a systematic series of experiments, the components CaO, P2O5, and F− were simultaneously increased from base glass A to base glass E in the stoichiometric ratio of Ca5(PO4)3F. The increase of the mentioned components was conducted at the expense of SiO2 and Li2O, ensuring a constant molar ratio of SiO2/Li2O. Besides the components, necessary for the formation of the desired crystal phases, the glass system was extended by Al2O3 and K2O in order to enhance the processability and chemical stability of the glasses.Different base glasses, which allow the controlled volume crystallization of Li2O5 content plays an important role in the studied glass system. It is essential for the nucleation of lithium silicate as well as mandatory for the crystallization of Ca5(PO4)3F. The preliminary study for nucleation by means of SEM 3F in glass D, based on the XRD patterns shown in Figure 2Si2O5 and Li2SiO3 are the first two crystalline phases that formed at temperatures above 540°C, prior to the precipitation of Li3PO4 at temperatures <600°C. The formation of crystalline Li3PO4 occurred surprisingly about 180°C below the temperatures reported so far in the literature for P2O5 containing Li2Si2O5 glass systems 3F crystal phase formation happening at temperatures definitely above the crystallization of lithium silicates as well as Li3PO4 seems to be independent and subordinated, though. However, comparing the crystal phase compositions of the apatite-forming glass–ceramic C with the corresponding F− free reference sample F 3F detected and estimated by means of powder XRD in combination with Rietveld refinement as a function of the theoretically possible fraction based on the chemical composition of the glasses. The content of fluoroapatite increases linearly with the increasing ratio of apatite-forming components. However, according to the slope of the linear regression curve in Figure − on the crystallization of Li3PO4, discussed above, the latter could be explained by the formation of amorphous CaO-P2O5-F rich phase separation sites within the glass microstructure. This phenomenon was already reported in the literature –P2O5 3F-comprising glass–ceramic C and the corresponding apatite-free material F revealed a certain effect of the apatite crystals on this optical property. Although the Li2Si2O5 crystals visible in the micrograph of glass–ceramic F 5(PO4)3F provides a further means of precisely controlling the optical properties of lithium disilicate glass–ceramics, which is a prerequisite for the successful application of this material in prosthodontics. Of major importance is the fact that the precipitation of the minor phase can be specifically controlled without affecting the good mechanical and chemical properties of Li2Si2O5 glass–ceramics, which are relevant to dental applications. Investigations that have been running in parallel to the present study have proved this to be true for the fracture toughness as well as the chemical durability.In summary and with respect to crystallization phenomena, the twofold precipitation of Li5(PO4)3F as a minor phase in Li2Si2O5-based glass–ceramics from bulk glasses via a solid-state reaction can be achieved by adding CaO/SrO, P2O5, and F− in multi-component base glasses.The controlled precipitation of (Ca/Sr)2O5 sites.The investigation of two different phase separation phenomena in the nucleated base glasses indicates the existence of chemically different P2O5 on the crystallization of lithium silicate could be approved, while there is no evidence for the need of any crystalline heterogeneous phase for the nucleation of fluoroapatite. Lithium silicate crystal phase formation starts ca. 160°C prior to the crystallization of Ca5(PO4)3F. The authors conclude on the existence of two different solid-state reactions without competitive character.A strong nucleating activity of amorphous or disordered nanocrystalline P2O5, and F−.The quantity of apatite precipitated in the glass–ceramics is almost directly proportional to the content of apatite-forming components provided in the base glasses. The efficiency of apatite crystallization increases with the increasing content of CaO/SrO, P2Si2O5 crystals. However, the precipitation of Ca5(PO4)3F or Sr5(PO4)3F facilitates the precise control of the translucency and opalescence which is a prerequisite for the successful application of the material in restorative dentistry.The mechanical and optical properties of the high-strength and chemically durable glass–ceramics are for the most part influenced by the morphology and size of the LiThe authors are employees of the company Ivoclar Vivadent AG. A patent was filed on the present subject.


PRP) injections are an effective way to retard intervertebral disc degeneration, but the mechanism of action is unclear. Activated platelets release some growth factors, such as transforming growth factor‐β1 (TGF‐β1), which positively modulate the extracellular matrix of nucleus pulposus cells. The purpose of this study was to explore the mechanism underlying the PRP‐mediated inhibition of intervertebral disc degeneration. In an in vitro study, we found that the proliferation of nucleus pulposus cells was greatly enhanced with 2.5% PRP treatment. The TGF‐β1 concentration was much higher after PRP treatment. PRP administration effectively increased the collagen II, aggrecan and sox‐9 mRNA levels and decreased collagen X levels. However, Western blotting demonstrated that specifically inhibiting TGF‐β1 signalling could significantly prevent nucleus pulpous cellular expression of Smad2/3 and matrix protein. In a rabbit study, magnetic resonance imaging revealed significant recovery signal intensity in the intervertebral discs of the PRP injection group compared with the very low signal intensity in the control groups. Histologically, the PRP plus inhibitor injection group had significantly lower expression levels of Smad2/3 and collagen II than the PRP group. These results demonstrated that a high TGF‐β1 content in the platelets retarded disc degeneration in vitro and in vivo. Inhibiting the TGF‐β1/Smad2/3 pathway could prevent this recovery by inactivating Smad2/3 and down‐regulating the extracellular matrix. Therefore, the TGF‐β1/Smad2/3 pathway might play a critical role in the ability of PRP to retard intervertebral disc degeneration.Recent studies have suggested that platelet‐rich plasma ( Intervertebral disc degeneration is considered a chief cause of low back pain et al. and Marx et al. Platelet‐rich plasma (PRP), which can be prepared by centrifugation, is an autologous blood product that contains a supraphysiological platelet concentration. Since PRP was first used for maxillofacial reconstruction by Whitman in vitro and animal studies Platelet‐rich plasma has been shown to positively modulate the extracellular matrix of the intervertebral disc in both 5 cells/ml was seeded into the DMEM/F12 medium and incubated at 37°C under 5% CO2 and saturated humidity. When the cell density reached 90%, cells were digested with 0.25% trypsin (Thermo Fisher Scientific) and passaged at a dilution of 1:2.All experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Soochow University and were performed in accordance with the approved guidelines for the care and use of laboratory animals. Intervertebral discs from three rabbits (2–3 months old) were extracted immediately after killing them. All surgical procedures were performed under sterile conditions. The entire thoracolumbar spine was removed, and the front disc attached to the muscles was stripped and rinsed twice with PBS solution . The annulus fibrosus was then incised, and the gelatinous nucleus pulposus was removed. The gelatinous nucleus pulposus was then rinsed twice with DMEM supplemented with F12  and digested in type II collagenase solution  at 37°C for 15–20 min. The digested tissues were pipetted gently, passed through a mesh filter and suspended by centrifugation at 200 × g for 8 min. A total of 1 × 102 solution containing 100 U of bovine thrombin (Sigma‐Aldrich) at room temperature for 60 min. and then centrifuged at 500 × g for 10 min. The activated PRP‐containing supernatant was collected and stored at −80°C.Before isolating nucleus pulposus cells, 10 ml of whole rabbit blood from the same rabbit's central ear artery was extracted and centrifuged at 125 × g for 10 min. Plasma and platelets in the superior layer were collected and centrifuged for another 10 min. at 500 × g The remaining supernatant (approximately 1 ml), including the precipitates, constituted the PRP. The PRP was mixed well and incubated with 110 μl of 10% CaClversus TGF‐β1 concentration. All samples and standards were measured in duplicate.Transforming growth factor‐β1 concentrations were measured using a double antibody sandwich avidin–biotin complex ELISA. To measure the concentrations, ELISA kits for TGF‐β1  were used according to the manufacturer's instructions. The absorbance values of the samples were measured at 450 nm using an ELISA plate reader . The TGF‐β1 concentration was positively proportionate to the absorbance values; therefore, the TGF‐β1 concentrations in the samples were calculated using a standard curve that was plotted as absorbance 4/ml. Each well contained 100 μl of cell suspension. Cell viability in the experimental groups was determined at 1, 2, 3, 4, 5, 6 and 7 days after the induction with 10%, 5%, 2.5% and 1% volume fractions of PRP. The controls were cultured in the regular medium without PRP. The measurement procedure was as follows: 10 μl of Cell Counting Kit‐8 (CCK‐8) solution  was added to each well and the plates were incubated at 37°C and 5% CO2 for 1 hr. After the incubation, the supernatants were carefully aspirated. To lyse the cells and completely dissolve the precipitates, dimethyl sulfoxide (DMSO) was added to each well and the plates were shaken for 15 min. The absorbance was then measured at 450 nm. The background absorbance of the medium in the absence of cells was subtracted. The experiments were independently repeated at least three times.Passage one nucleus pulposus cells were trypsinized, dispersed into single‐cell suspensions and seeded into 96‐well plates 5 × 102O. The PCR conditions were as follows: 95°C for 10 min., 95°C for 15 sec., 60°C for 30 sec. and 72°C for 30 sec. for 40 cycles. Relative gene expression was normalized against GAPDH expression, and the data were presented as the fold change using the formula 2−ΔΔCT, as recommended by the manufacturer. The primer sequences are listed in Table Five days after induction with 2.5% PRP, 2.5% PRP + 10 μmol/l TGF‐βl inhibitor SB431542 , the nucleus pulposus cells were collected and the total RNA was extracted using TRIzol reagent . The cells not subjected to PRP induction were also collected as controls. The RNA samples were quantified and reverse transcribed into first‐strand cDNA. Quantitative polymerase chain reactions (PCR) were performed on a real‐time TP800 system  to detect the genetic expression of the chondrogenic markers collagen II, aggrecan, and sox 9 and hypertrophic marker collagen X. A reference gene  was amplified in parallel with the target genes. The total volume of the reaction mixture used for each PCR was 20 μl, which included 10 μl of SYBR Green mixture, 1 μl of each primer (10 μmol/l), 1 μl of reverse primer (10 μmol/l), 1 μl of cDNA template and 7 μl of ddHNucleus pulposus cells were harvested at 5 days after induction with 2.5% PRP, 2.5% PRP + inhibitor and inhibitor only. Non‐induced cells served as the controls. The cells were washed three times with PBS and suspended in ice‐cold lysis buffer (Bio‐Rad). The lysates were separated by 12% SDS‐PAGE, and proteins were transferred to nitrocellulose membranes. Membranes were blocked with 1× Tris‐buffered saline with Tween‐20 buffer (Thermo Fisher Scientific) containing 5% milk and incubated overnight at 4°C with collagen II  and Smad 2/3  primary antibodies. The next day, the membranes were washed and incubated with the corresponding peroxidase‐conjugated secondary antibodies at room temperature for 1 hr. The membranes were then developed using an enhanced chemiluminescence detection system. The β‐actin expression level was used as an internal control.n = 24) were randomly divided into three groups. Each of the rabbits weighed 2.5 ± 0.3 kg at the beginning of the study, and all were anaesthetized with 30 mg/kg pentobarbital. After the rabbits were shaved, their skin was sterilized with povidone iodine. The rabbits were then placed in the lateral prone position, and the anterior surfaces of two consecutive lumbar intervertebral discs (L4‐5 and L5‐6) were exposed using a posterolateral retroperitoneal approach. The nucleus pulposus tissues of the intervertebral discs were punctured using an 18G needle on a 5‐ml syringe to induce disc degeneration. Four weeks after the initial puncture, either 15 μl of autologous PRP  or 15 μl of PRP + 10 μl of TGF‐βl inhibitor was injected into the centre of the rabbit nucleus pulposus from the contralateral side using a 26G needle with a microsyringe . Other eight rabbits' L4‐5 and L5‐6 discs were approached but not treated and referred to as the ‘control group’. After surgery, all of the rabbits were allowed to move freely in a cage.New Zealand rabbits (Magnetic resonance imaging (MRI) was performed on each rabbit at 0, 4, 8 and 12 weeks after the operation to assess the degree of disc degeneration. Sagittal T2‐weighted images were obtained using a 0.2 T E‐Scan XQ Extremity MRI . In the T2‐weighted images, 16 discs  per group were classified according to the modified Thompson classification from grade I to IV  2).The intervertebral discs were harvested, fixed in 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid and embedded in paraffin using standard procedures. The sections were stained with haematoxylin and eosin to evaluate disc degeneration. For immunohistochemistry detection, sections were incubated with primary antibodies specific to collagen II  and Smad2/3 (Biosynthesis Biotech). The obtained images were captured on an image analysis system and analysed by Image Pro Plus 6.0 software . Integrated optical density was calibrated, and the area of interest was established. The mean optical density was defined as the integrated optical density divided by area .To examine whether the effect of PRP on cellular activity was time and dose dependent, we co‐cultured rabbit nucleus pulposus cells with PRP at 10%, 5%, 2.5% and 1% volume fractions and determined cell viability at days 1, 2, 3, 4, 5, 6 and 7 . In contrast, the nucleus pulposus cells treated with PRP and TGF‐β1 inhibitor exhibited low expression levels of collagen II, aggrecan and sox‐9 mRNA and high expression levels of collagen X mRNA. Significant differences were also observed compared with the PRP group (P < 0.05) , respectively, dissolved in DMSO. Real‐time PCR (RT‐PCR) results showed that the administration of 2.5% PRP significantly increased the collagen II, aggrecan and sox‐9 mRNA levels and decreased the collagen X level compared with control    Figs  and 7.et al. Platelet‐rich plasma, defined as a sample of autologous blood with platelet concentrations above the baseline values, is prepared by centrifugation. Different centrifugation steps, forces and times could result in different platelet concentrations, activities and yields. In the United States, there are more than 10 different commercialized PRP preparation systems approved by the Food and Drug Administration. In this study, the PRP was prepared after two centrifugations as described by Aghaloo in vitro. Several studies showed that the positive effect of PRP on cell proliferation is not dependent on PRP concentration. In some cases, high PRP concentrations could suppress cellular proliferation, whereas low PRP concentrations could have a long‐term positive effect on cellular proliferation. Choi et al. reported that the viability and proliferation of alveolar bone cells were suppressed by high PRP concentrations but were stimulated by low PRP concentrations (1–5%) et al. In this study, we investigated the volume fraction of PRP required to best enhance nucleus pulposus cells proliferation in vivo and in vitro results, PRP could retard intervertebral disc degeneration, and the TGF‐β1/Smad2/3 pathway plays a key role in the process.Previous researchers have preliminarily shown that PRP promotes nucleus pulposus cells regeneration by the TGF‐β1/Smad signalling pathway, in which Smad2/3 is phosphorylated by TGF‐β1 and released into the cytoplasm, which induces the nucleus pulposus cells re‐differentiation process in vivo. Compared with the control group, the PRP group showed a significant increase in Smad2/3 according to immunohistochemical staining. This suggested that PRP could activate the biological activity of Smad2/3, which effectively promotes the synthesis and secretion of collagen II and other matrix components that contribute to the structural and functional recovery of intervertebral disc degeneration.Intervertebral disc degeneration is characterized by decreased disc height, progressive fraying and dehydration of the nucleus pulposus, resulting in low T2 signal intensity on MRI. In this study, all rabbits were killed 8 weeks after injection, and their intervertebral discs were subjected to sagittal T2‐weighted MRI analysis. We noted an obvious decrease in the signal intensities of the T2‐weighted images of the discs accompanied by a narrowed disc space in the control and TGF‐β1‐inhibitor injection groups at 8 weeks after injection, but there was no significant difference between the two groups. However, the T2‐weighted images of the discs in the PRP group exhibited higher signal intensity and wider disc space than those in the other two groups, which was similar to the histological and immunohistochemical analysis findings. Haematoxylin and eosin staining showed disordered discs and no nucleus pulposus tissue regeneration in the control or TGF‐β1 inhibitor injection groups. However, the structure of the outer layers of the peripheral annulus tissue was normal in the PRP group and regeneration was observed in the nucleus pulposus, which was further validated by an increase in collagen II found using immunohistochemical analysis. We further confirmed the role of PRP via the TGF‐β1/Smad2/3 pathway. Platelet‐rich plasma injections could also retard intervertebral disc degeneration in a rabbit model. Elucidating the mechanism of PRP therapy would provide valuable information for the clinical treatment of intervertebral disc degeneration.In summary, PRP is rich in growth factors, especially TGF‐β1, which can enhance the nucleus pulposus cells proliferation and anabolic processes The authors declare no conflict of interest.


In ribosome biogenesis, a large fraction of ribosomes is used for producing ribosomal proteins themselves. Here, we applied simulation and experimentation to determine what fraction of ribosomes should be allocated for the synthesis of ribosomal proteins to optimize cellular economy for growth. We define the “r-fraction” as the fraction of mRNA of the ribosomal protein genes out of the total mRNA, and we simulated the effect of the r-fraction on the number of ribosomes. We then empirically measured the amount of protein and RNA in fission yeast cells cultured with high and low nitrogen sources. In the cells cultured with a low nitrogen source, the r-fraction decreased from 0.46 to 0.42 with a 40% reduction of rRNA, but the reduction of the total protein was smaller at 30%. These results indicate that the r-fraction is internally controlled to optimize the efficiency of protein synthesis at a limited cellular cost. Proliferating cells have a high cost in terms of energy and materials for the biogenesis of ribosomesA ribosome consists of four species of rRNA  and about 80 ribosomal proteins (RPs). Ribosome biogenesis involves three classes of RNA polymerase . Pol I specifically transcribes RNA components of the ribosome ; Pol II transcribes mRNA for the protein components of the ribosome (about 80 RPs); and Pol III transcribes another RNA component of the ribosome (5S rRNA). These three classes of RNA polymerase are cooperatively controlled to produce the appropriate number of ribosomes in growing cellsSchizosaccharomyces pombe to understand the mechanism by which the cell allocates the fraction of ribosomes for ribosome synthesis during cell proliferation.In actively growing yeast cells, mRNA of RP genes accounts for about 50% of the total mRNA . The results of the continuous culture of the S. pombe heterothallic haploid L972 strain in the synthetic medium called EMM2 (including NH4Cl 5 g·L−1) are shown in The fission yeast S. pombe was reproducibly detected. The histogram in 34S. pombe has 142 RP genes  and the critical protein number M are given at the start of each simulation.At the start of the simulation, all the RPs have been assembled into a ribosome. The initial number of ribosomes . 8 and R0 = 4 × 105 were given and the number of mRNA for each gene was the same as in The results of the simulation are shown in S. pombe heterothallic haploid strains stop proliferation when nitrogen in the culture medium is exhausted, and the cell cycle is blocked at the G1 phaseS. pombe cells could proliferate in a series of different NH3 concentrations by using the EMM2 medium, and found that the cells could proliferate at 100 times lower NH3 than in the standard EMM2 medium (we refer to it as 1/100 × N-EMM2 and the standard EMM2 as 1 × N-EMM2 in the following). The NH3 concentration at the 1/100 × N-EMM2 was about 0.93 μmol·mL−1; the amount of NH3 for each cell was about 190 fmol at the cell density of 5 × 106 cell·mL−1. On the other hand, the amount of nitrogen included in the cell was estimated at about 170 fmol for a cell volume of 80 fL (see Methods). Thus, 190 fmol of nitrogen is almost equal to the amount of nitrogen included in one cell, and is expected to be consumed in one cell cycle. Based on this estimation, we used 1/100 × N-EMM2 as the minimal concentration of nitrogen that allows S. pombe cells to continue proliferation.Cells of The results of continuous cultures in 1/100 × N-EMM2 are shown in S. pombeIn our measurements, the amount of total RNA per cell was 2.47 pg (SD = 0.24) and 1.46 pg (SD = 0.21) in the 1 × N-EMM2 and 1/100 × N-EMM2 cultures, respectively. We also measured the ratio of 18S and 28S rRNA included in the total RNA by using a BIO-analyzer (Agilent). In addition to the total amount of RNA, the amounts of both rRNA species in the 1/100 × N-EMM2-cultured cells decreased by about 40% compared with those in the 1 × N-EMM2-cultured cells . Thus, iste11 gene, one of the central regulators that induce meiosis upon nitrogen starvationAmong the 4722 genes detected, 59 were reproducibly found in four independent experiments that increased their relative mRNA levels by more than twice in the 1/100 × N-EMM2 culture compared with the 1 × N-EMM2 culture . In thesThe relative number of mRNA in most of the genes in the 1/100 × N-EMM2 culture changed by less than twice compared with the 1 × N-EMM2 culture, as mentioned above  compared with the 1 × N-EMM2-cultured cells  . A reduc8 to 1.8 × 108 combined with two values of mRNA number in the 1 × N-EMM2-cultured cells (r = 0.46) and in the 1/100 × N-EMM2-cultured cells (r = 0.42). The results of the simulation are shown in Thus, we conducted a simulation by adopting the following conditions: values for the critical protein number M ranged from 0.8 × 10S. pombe cells were cultured with limited nitrogen sources. Such experiments have not been possible with a batch culture, but they have become possible by continuous culture.Since ribosomes are necessary for the synthesis of essential cell proteins, including the proteins of the ribosomes themselves, there is a trade-off between RPs and non-RPs in the use of ribosomes for synthesis. This trade-off is more critical for cell survival when resources are limited. In this report, we attempted to determine how many of the ribosomes are allocated to produce ribosomes when To evaluate the fraction of ribosomes used to produce ribosomes, we defined the r-fraction as the fraction of the RP mRNA in the total mRNA. Our experimental measurements suggest that RP genes are controlled by a separate mechanism distinguished from non-RP genes, forming the characteristic secondary peak in the mRNA distribution; a change in the r-fraction causes a shift of the secondary peak .Simulations indicate that when the r-fraction is given at a fixed value of M, the number of ribosomes per cell and the number of iterations for cell division reaches a constant value . SimulatThis is consistent with the experimental data; in the cells cultured in low nitrogen, the amount of total RNA and the amount of total rRNA both decreased by 40%, but the total amount of protein decreased by only 30% and the r-fraction decreased to 0.42 compared with 0.46 in the high-nitrogen culture. If the r-fraction did not change, the total protein decreased to the same extent as the decrease in rRNA. However, by reducing the r-fraction from 0.46 to 0.42, the cells were able to keep the total protein at a lower level of reduction (30%) while the rRNA decreased by 40%. Thus, a small reduction of the r-fraction can allocate limited resources to produce essential proteins more efficiently.It is generally thought that the cell maintains the adequate number of ribosomes by controlling transcription of rRNA using Pol I in response to the environmentS. pombe 972 h− strain was used in all experiments. The EMM2 medium4Cl was changed to 0.05 g·L–1, was used for the 1/100 × N-EMM2 culture. Continuous culture was carried out with a chemostat and feed controller BMJ-01NC (ABLE-Biott) at 30 °C and 1.5 vvm (air volume per culture volume per minute) aeration, and stirred with a blade turbine at 600 rpm. The pH of the medium was 5.8. For experiments with the 1 × N-EMM2 medium, the S. pombe cells in the batch culture in EMM2 were transferred at mid log phase to the vessel after washing once with the EMM2 medium to produce 2 × 10−1 in 600 mL EMM2. After culturing for some hours to grow at 5 × 10−1 without feeding, the continuous culture started at a specific DR. During the continuous culture, the cell concentration was kept constant by removing the cell culture at a certain DR and continuously providing a fresh culture medium at the same rate as cells continued proliferating. When the DR was 0.25 per hour, 2.5 mL culture was continuously replaced for each minute [2.5 mL = 600 mL (culture volume) × 0.25 (DR)/60 (minutes)]. We set the DR at the maximum possible rate that did not wash out the cells. The DR was occasionally adjusted to keep the cell density at around 5 × 10−1. The DRs during 72 h culture were averaged. Doubling time (DT) can be calculated from the DR with DT = ln(2)/DR, as described4Cl and washed with the EMM2 with 1/100 × N. Density and viability of cells during culture were checked with a small volume  taken from the culture: when the DR was 0.25 per hour, the volume of culture taken was 2.5 mL corresponding to a one-minute flow. After these samplings, the feed-out pump was stopped for one minute to recover the culture volume. Cell density and cell volume were measured with the particle analyzer CDA-1000 (Sysmex). Cell viability was checked as follows: 300–500 cells for each sample taken at 48 h and 72 h were dissected on YES plates with the dissection microscope MSM (Singer Instruments). After 3 days of incubation at 30 °C, the colonies were counted.The −1.The amount of nitrogen in a budding yeast cell is reported to account for 8% of its dry weightFor calculating the average amount of total protein and RNA for a cell, the total protein and RNA should be isolated from a known number of cells. After 72 h of continuous culture, cells were harvested by filtration. Then, the wet pellet of cells was divided into three parts and the weight of each part was measured. One of the three parts was suspended in a fixed volume of water and the cell number of the cell suspension was counted with the particle analyzer CDA-1000 (Sysmex) to give the number of cells in the wet pellet per weight. Total protein and RNA were isolated from the remaining two parts of pellet.7 cells was suspended in 100 μL of the lysis buffer (2% sodium dodecyl sulfate (SDS); 10% glycerol; 60 mM Tris-HCl, pH 6.6; 8 M urea) and boiled at 100 °C for 5 min, then cooled on ice. Cells were disrupted with 0.5 mm glass beads using the Multi-Beads Shocker (Yasui Kikai) at 2,700 rpm for 1 min (repeated four times). The resulting cell extracts were centrifuged at 100,000 g for 10 min. After recording the volume of the supernatant, its protein concentration was measured using a Bio-Rad DC Protein Assay Kit (Bio-Rad).Total protein was isolated and measured as follows: the cell pellet containing approximately 3 × 10g at 4 °C. The water phase of the sample (500 μl) was mixed with 500 μl of acidic phenol-chloroform in a new phase lock tube. The sample was centrifuged for 5 min at 15100 g at 4 °C. The water phase of the sample (500 μl) was mixed with 500 μl of chloroform in a new phase lock tube. The sample was centrifuged for 5 min at 15100 g at 4 °C. Total RNA was purified from the water phase by ethanol precipitation. After resolving the RNA pellet in a fixed volume of water, the concentration of the isolated RNA was measured with a ND-1000 spectrophotometer (NanoDrop Technology). A small portion of the isolated RNA was also analyzed with a BIO-analyzer 2100 (Agilent) to check its purity and RNA composition.Total RNA was isolated and measured as follows. The cell pellet was frozen with liquid nitrogen. The frozen cell pellet was mixed with 500 μl of TES  pH 8, 0.5% SDS) and 500 μl of acidic phenol-chloroform on ice. The sample was incubated at 65 °C for 1 hour, and then placed on ice for 1 min. The sample was transferred to a phase lock tube (MaXtractTM HighDensity 2 ml Qiagen) and was centrifuged for 5 min at 15100 Cyanine-3 (Cy3)-labeled cRNA was prepared from 200 ng RNA using the LowInput QuickAmp Labeling Kit, one color including Cy3-CTP (Agilent Technologies) according to the manufacturer’s instructions, followed by RNeasy mini column purification . Hybridization was carried out using the Gene Expression Hybridization Kit (Agilent Technologies) according to the manufacturer’s instructions except for the amount of Cy3-labeled cRNA (37.5 ng was used for each hybridization). After hybridization, microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and for 1 minute with GE Wash buffer 2 (Agilent) at 37 °C, then immediately dried. Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8 × 60 K array slides . The scanned images were analyzed using Feature Extraction Software  using default parameters (protocol GE1-107_Sep09 and Grid: 038274_D_F_20120123) to obtain background-subtracted and spatially detrended processed signal intensities.S. pombe sequences and 56 control probes) were spotted multiply to fill 62,976 spots in each array. The median of the gProcessedsignals and an average of the gIsWellAboveBG  of the spots for each probe were calculated to make a data set for 6,767 probes. When the average of gIsWellAboveBG of the spots for each probe was higher than 0.55, the probe signal was considered to be above the detection limit. The median was normalized by the 75 percentile method  to obtain values for the 6,767 probes. The measured values for these spots were assigned to the 6,657 S. pombe genes currently annotated in a public database. The values of probes that represent duplicated genes were divided by the number of duplicated genes to give corrected values to the genes. The values of probes that were cross-hybridized with paralogs were corrected using our custom correction probes (details are described in GPL18374 of Gene Expression Omnibus (GEO). The data sets of the microarray experiments were deposited at GEO (GSE68173).The 6,767 probes , the scaled values of the paralogs of the same ribosomal subunit were summed for each of the 79 subunits.Comparison between the 1 × N-EMM2 and 1/100 × N-EMM2 cultures: we selected 4,722 genes that were reproducibly detected in the four independent experiments either in the 1 × N-EMM2 culture or in the 1/100 × N-EMM2 culture. The geometric mean was scaled so that the total number of mRNA of the 4,722 genes totaled one million.Geometrical means of mRNA numbers for 5,157 protein-coding genes of Theoretical distributions were fitted to the empirical mRNA data by first removing RPs from the dataset to only account for the values contributing to the primary peak. An empirical cumulative distribution function (CDF) was generated from the normalized histogram and used as the basis for comparison with the following theoretical CDFs.log-normal (LN) distribution has two parameters The erf is the Gaussian error function. The CDF of the Gamma distribution is defined as:where with the shape and rate parameters α, β > 0, respectively, and the gamma function Γ. For both, LN and Gamma, maximum likelihood estimators were used for deriving the distribution parameters.0. Its CDF is defined as:We also compared the empirical data with two less well-known distributions that characterize the left and right tails with heavy tail parameters. The double Pareto (dP) distribution0 = 1. Finally, we also considered the double Pareto log-normal (dPLN) distribution152.which we extended from its original definition0 = 63.88), and dPLN .How to cite this article: Chikashige, Y. et al. Cellular economy in fission yeast cells continuously cultured with limited nitrogen resources. Sci. Rep.5, 15617; doi: 10.1038/srep15617 (2015).