Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion.Caffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p < 0.01). 1,3-dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively by HPLC in A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively.Seven tea-derived fungi were collected and identified by ITS, β-tubulin and calmodulin gene sequences, A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.For the first time, we confirmed that isolated  Camellia sinensis) and caffeine (Coffea arabica) [2 + NH3 [Camellia plants [Caffeine  is the most abundant methylxanthine alkaloids in tea, and also one of the major tea flavor content causing bitterness . Until n[2 + NH3 , 5. The a plants , 7. Theoa plants , 9.Pseudomonas sp. [Pseudomonas putida [Serratia marcescens, Fusarium solani [Stemphyllium sp., Aspergillus tamarii, and Penicillium commune [N-demethylation and oxidation [N-demethylation of caffeine, which present marked differences from bacteria that theobromine  or paraxanthine  are major metabolites in caffeine catabolism [P. putida and Pseudomonas sp. were established to not only use caffeine, theobromine, paraxanthine  and 7-methylxanthine, but also degrade theophylline and 3-methylxanthine [P. putida and Pseudomonas sp. were established to use caffeine, theobromine, paraxanthine  and 7-methylxanthine, and they also degrade theophylline and 3-methylxanthine). In addition, Aspergillus niger, Talaromyces marneffei and Talaromyces verruculosus isolated from cocoa pod husks were established to degrade theobromine and produce methylxanthine [P. putida CBB5 was confirmed to degrade theophylline via N-demethylation [Although caffeine level remains stable in the processing of general teas  , 11, sevonas sp. , 13, Pses putida , 15, Serm solani , 17, Ste commune . Two posxidation . Theophytabolism . Moreovexanthine , 20  impact on caffeine metabolism and potential value in theophylline production through aerobic fermentation [Pu-erh tea is one of the most representative dark tea and natural microorganisms involved in solid-state fermentation (SSF) play an important role in tea processing , 24. Mic-erh tea –31. Theo-erh tea –34. Caff-erh tea –39. We fn in SSF . After fentation , 42.A. sydowii causing the production of theophylline in SSF, theophylline-degrading fungi also could be found in SSF. In this paper, two theophylline-degrading fungi were isolated from the SSF and identified as A. ustus and A. tamari based on colonial morphology and ITS, β-tubulin and calmodulin gene sequences, respectively. Theophylline degradation metabolites and pathways were analyzed in fungi by high-performance liquid chromatography (HPLC). The application in production of methylxanthines was investigated by using A. ustus and A. tamarii, respectively.In this study, we found that theophylline content had a highly significant (p < 0.01) decrease during the later period of SSF after a highly significant (p < 0.01) increase. Therefore, apart from an isolated caffeine-degrading fungus identified as C. sinensis var. assamica) with moisture content 6.25% by weight were obtained from Yunnan Province, China. Caffeine, theophylline, 3-methylxanthine, 1-methylxanthine, xanthine, 1,3-dimethyluric acid, 1-methyluric acid, 3-methyluric acid and uric acid were purchased from USA Sigma Company. SP fungal DNA kit was purchased from USA Omega Company. DNA marker, polymerase chain reaction (PCR) spread reagent, primers [Sun-dried green tea leaves . Samples collected on days 20 and 25 were stored at 4 °C and selected for further isolation and identification of theophylline-degrading fungi.The SSF of pu-erh tea was based on the natural microbiota existing on tea leaves and fermentation environment. A 2 kg sample of sun-dried green tea leaves was mixed with 885 mL tap water to achieve given moister content of 35% (w/w) . During Fungal strains were isolated using potato dextrose agar (PDA) medium and they were counted by dilution-plating method . Microscg for 5 min after cultivation in 20 mL of Czapek Dox medium at 30 °C for 2 days on an incubator shaker (250 rpm) and freeze-dried at − 80 °C [μM) of each primer, and 36.5 μL of sterile distilled water were used to implement amplifications [Fresh cells were obtained by centrifugation at 1700 − 80 °C , 41. DNA − 80 °C . The finications , 41. Theications . It was The PCR was produced in an ABI3730 automatic DNA sequencer  . The rec7 CFU/mL for inoculation after eluting by using sterile saline solution. Qualitative screenings were carried out in Petri dishes containing a solid culture medium contained 20 g/L agar, 4.0 g/L NaNO3, 1.3 g/L KH2PO4, 0.19 g/L Na2HPO4·7H2O, 0.26 g/L CaCl2·2H2O, 0.19 g/L MgSO4 and 20 g/L dextrose as carbon source (control culture) or a selection medium with theophylline instead of dextrose in three different concentrations: 600, 1200 and 1800 mg/L per plate, respectively [The isolate strains were transferred into PDA medium and incubated aerobically at 30 °C for 72 h on an incubator shaker (250 rpm), respectively. The spore suspension was adjusted to 1.0 × 10ectively . Fungal 3, 1.3 g/L KH2PO4, 0.19 g/L Na2HPO4·7H2O, 0.26 g/L CaCl2·2H2O, 0.15 g/L MgSO4, 2.0 g/L sucrose and 300 mg/L theophylline in distilled water [7 CFU/mL and all given TLM was adjusted for pH 6.0 by phosphate buffer before inoculation. For each isolate, control and experimental mediums (25 mL each) were inoculated with spore suspension with 4% inoculum size (v/v) that 1 mL spore suspension was inoculated into each medium, and biocidal treatment was defined as the control. Theophylline concentration was determined and mycelium was collected after cultivation at 30 °C for 5 days on an incubator shaker (130 rpm), respectively. The collected mycelium was filtered in a Buchner funnel, and rinsed in 20 mL of water: ethyl acetate (1:1) [Theophylline liquid medium (TLM) was prepared using 4.0 g/L NaNOed water . To invete (1:1) . The mycte (1:1) . Theophyte (1:1) .Through comparison, additional sucrose could promote theophylline degradation in liquid culture. Therefore, TLM-S was selected as the optimal medium to analyze theophylline degradation by selected isolates. A series of TLM-S mediums with different initial theophylline concentrations  were set up each day of incubation and a 7-day period cultivation of each selected isolates were carried out on an incubator shaker . At intervals of up to 24 h for 7 days, an aliquot of each culture was filtered through a 0.45 μm syringe filter, and theophylline concentration and related metabolites were determined by HPLC according to the method from Mensah et al. .Standard calibration curves were prepared from solutions of theophylline, 3-methylxanthine, 1-methylxanthine, xanthine, 1,3-dimethyluric acid, 1-methyluric acid, 3-methyluric acid and uric acid. 2 mL aliquots from each collected culture were filtered and analyzed by HPLC for theophylline, 3-methylxanthine, 1-methylxanthine, xanthine, 1,3-dimethyluric acid, 1-methyluric acid, 3-methyluric acid and uric acid. Internal standard method was used to aid in identification of metabolites. The concentrations of main degradation products (3-methylxanthine and xanthine) were analyzed in inoculated culture medium by selected isolates.N-demethylation was the main theophylline degradation pathway in fungi, and 3-methylxanthine and xanthine were main demethylated products. To explore the effects of selected isolates on 3-methylxanthine and xanthine, 3-methylxanthine and xanthine liquid mediums were prepared as above described with 5 g/L sucrose as carbon source and 100 mg/L 3-methylxanthine or 100 mg/L xanthine, respectively. Each isolate was inoculated with 4% inoculum size (v/v) and biocidal treatment was defined as the control. 3-Methylxanthine and xanthine concentrations were determined by HPLC after cultivation at 30 °C for 5 days on an incubator shaker (130 rpm), respectively.Analysis of theophylline degradation metabolites showed that Three biological replications were carried out to ensure validity and repeatability. All data are presented as mean value ± standard deviation (SD). One-way analysis of variance (one-way ANOVA) was carried out by Duncan’s multiple-range test using SPSS 20.0 for window to determine whether the significant difference at p < 0.05 level or the highly significant difference in p < 0.01 level exist.5 CFU/g. Because of the metabolic activity of fungi, caffeine content  with theophylline. directly , 46. TheA. pallidofulvus, A. sesamicola and P. mangini had no ability to use theophylline. Theophylline utilization of A. niger and A. sydowii was restricted in liquid culture, theophylline removal ratios were about 1.03% and 5.19% in TLM-S, respectively. Only A. ustus and A. tamarii could utilize caffeine significantly in all given TLM. Hence, A. ustus, A. tamarii, A. niger and A. sydowii were potential theophylline-degrading fungi for theophylline degradation in liquid culture.For theophylline biodegradation in liquid culture, seven isolates were inoculated into TLM with the presence of theophylline and sucrose or dextrose as carbon source, or ammonium sulphate as nitrogen source, respectively. Theophylline concentration and fungal dry mass were determined after cultivation at 30 °C for 5 days. Results are showed in Fig. A. ustus and A. tamarii through enhancing cell density in liquid culture. Therefore, TLM-S was selected to analyze characterization of theophylline degradation in liquid culture.The presence of additional carbon or nitrogen sources had a significant impact on theophylline degradation and pathway. The optimum liquid medium was chose by comparing theophylline removal ratios in different mediums. In contrast with other mediums , theophylline degradation level had a highly significant (p < 0.01) improvement in TLM-S. In addition, extra sucrose promoted theophylline degradation in TLM-S inoculated by Aspergillus ustus, A. tamarii, A. niger and A. sydowii were inoculated into TLM-S with increasing theophylline concentrations , and Tissue-culture bottles were incubated in an orbital shaker . The inoculation bottles were took every 24 h for the determination of theophylline and related metabolites by HPLC, and results are presented in Fig. A. ustus and A. tamarii, theophylline decreased highly significantly (p < 0.01) in all substrate concentrations. However, theophylline decreased slightly (p > 0.05) in all concentrations inoculated by A. niger and A. sydowii. Therefore, A. ustus and A. tamarii had more advantage in theophylline degradation than A. niger and A. sydowii. Both A. ustus and A. tamarii could degrade theophylline completely in low concentration (100 mg/L theophylline). However, A. ustus only degrade 79.00% theophylline in high concentration (300 mg/L theophylline), while A. tamarii could degrade theophylline almost completely in all given concentrations, which showed that A. tamarii had a higher theophylline degradation capacity.N-demethylation at the position N-1 of theophylline in A. ustus and A. tamarii. Xanthine was a further demethylated metabolite in theophylline degradation found in A. ustus and A. tamarii through N-demethylation at the position N-3 of 3-methylxanthine. In contrast to A. ustus that additional metabolites including 1,3-dimethyluric acid and 3-methyluric acid were identified in the culture through the oxidation of theophylline and 3-methylxanthine, respectively, only uric acid was identified in A. tamarii culture as the oxidation product of xanthine, which showed the differences in degradation metabolites and pathways between A. ustus and A. tamarii.A series of experiments was conducted to find out theophylline degradation pathway through the identification of catabolic intermediates by HPLC using internal standard method Table . 1,3-DimA. ustus and A. tamarii. Microbial utilization of 3-methylxanthine and xanthine were investigated in liquid culture of isolates. 3-Methylxanthine and xanthine concentrations were determined by HPLC after cultivation for 5 days. As shown in Fig. A. ustus and A. tamarii had a significant (p < 0.05) or a highly significant (p < 0.01) impact on 3-methylxanthine degradation with a removal ratio of about 27.05% and 84.29%, respectively. Additionally, A. tamarii had a highly significant (p < 0.01) impact on xanthine degradation with a removal ratio of about 51.77%. Associated with the metabolites detected in theophylline degradation, 3-methyluric acid and xanthine were 3-methylxanthine degradation metabolites through oxidation and N-demethylation, respectively.Several xanthine derivatives including 3-methylxanthine have been synthesized chemically for use in medical industry . Except A. ustus and A. tamarii, respectively. To investigate the application in production of 3-methylxanthine and xanthine by using theophylline-degrading fungi with theophylline as feedstock, quantitative determinations of 3-methylxanthine and xanthine were carried out in all theophylline concentrations inoculated by A. ustus and A. tamarii, respectively. 3-Methylxanthine and xanthine concentrations in A. ustus and A. tamarii cultures are presented in Fig. A. ustus and A. tamarii. 3-Methylxanthine was detected in the culture medium after 24 h for the first time, and increased significantly with cultivation. Over a 7-day period cultivation of A. ustus  or highly significantly (p < 0.01); moreover, the theophylline utilization of A. niger and A. sydowii were restricted. However, at the presence of sucrose as carbon source in liquid culture, A. pallidofulvus, A. sesamicola and P. mangini had no ability to use theophylline. Though the effect of C/N ratio on growth is strain-dependent [The assessment in agar mediums with theophylline as carbon source showed that six es Table  could utependent , increasCamellia sinensis. As mentioned above, the pathway of caffeine degradation metabolism is comparatively clear in microorganisms that both N-demethylation and oxidation were found in caffeine degradation, and N-demethylation was main pathway in fungi [P. putida, A. niger, Talaromyces marneffei, and Talaromyces verruculosus [A. ustus and A. tamarii isolated from pu-erh tea could degrade theophylline in liquid culture. Based on the downstream metabolites detected in liquid culture, hypothetical theophylline degradation pathways were established and shown in Fig. N-demethylation and oxidation were theophylline degradation pathways found in A. ustus and A. tamarii culture. Theophylline and related demethylated metabolites can be oxidized to 1,3-dimethyluric acid, 3-methyluric acid and uric acid, respectively. The degradation metabolites suggested the differences in degradation pathways of A. ustus and A. tamari, except the common pathway that theophylline → 3-methylxanthine → xanthine. Therefore, N-demethylation was main theophylline degradation pathway, which was similar to caffeine and theobromine catabolism in fungi.Except for caffeine, theophylline and theobromine are main purine alkaloid in tea, which both have close connection with caffeine metabolism that theophylline is catabolite of caffeine and theobromine is precursor of caffeine biosynthesis in in fungi , 18, 56.uculosus , 18, 20.A. ustus and A. tamarii cultures. 3-Methylxanthine and other xanthine derivatives have been shown various biomedical effects as adenosine antagonist and inhibitors of Primary Amine Oxidase [A. ustus and A. tamarii would be applied in the production of 3-methylxanthine and xanthine with theophylline as feedstock through microbial conversion. The differences in accumulated concentrations of 3-methylxanthine and xanthine suggested that A. ustus benefits the production of 3-methylxanthine, while A. tamarii benefits the production of xanthine. After a 7-day period cultivation in 300 mg/L of TLM, A. ustus could produce 129.48 ± 5.81 mg/L of 3-methylxanthine, and A. tamarii could produce 159.11 ± 10.8 mg/L of xanthine, respectively.3-Methylxanthine and xanthine were common downstream metabolites detected in  Oxidase , 58. Bes Oxidase , 48. DueA. ustus and A. tamarii isolated from SSF of pu-erh tea and identified based on ITS, β-tubulin and calmodulin gene sequences, were confirmed to degrade theophylline significantly (p < 0.01) in liquid culture through the sequential selections. Extensive experiments were carried out to detect related degradation metabolites by using HPLC, finding N-demethylation and oxidation in theophylline catabolism. Through the absolute quantitative detection, it is showed that 3-methylxanthine and xanthine were main degraded metabolites in A. ustus and A. tamarii respectively, which suggests that A. ustus benefits the production of 3-methylxanthine, while A. tamarii benefits the production of xanthine with theophylline as feedstock. This paper also suggests theophylline degradation pathway in Aspergillus fungi and represents a new microbial synthesis platform for production of methylxanthines using theophylline through the inoculation of A. ustus and A. tamarii, respectively.This paper describes theophylline degradation pathways in tea-derived fungi and explores the application in production of methylxanthines. Additional file 1: Figure S1. Colony characteristics  and conidial structure  of strain PT-6. (a): Front on PDA medium at 25 °C for 7 days. (b): Back on PDA medium at 25 °C for 7 days. (c): Mature conidia heads, conidia stems and antipodal cells (200 ×). (d): Hyohae, conidia stems and conidiums (400 ×). Figure S2. Colony characteristics  and conidial structure  of strain PT-7. (a): Front on PDA medium at 25 °C for 7 days. (b): Back on PDA medium at 25 °C for 7 days. (c): Conidia heads(200 ×). (d): Conidia stems, sterigmas and conidiums (400 ×). Figure S3. The received sequences of strain PT-6 . Figure S4. The received sequences of strain PT-7 . Figure S5. Neighbor-Joining consensus trees of (a) Aspergillus ustus PT-6 and (b) Aspergillus tamarii PT-7. Identification was based on ITS and calmodulin genes for A. ustus PT-6, and ITS, β-tubulin and calmodulin genes for A. tamarii PT-7. The numbers over branches represent bootstrap confidence values (%) based on 1000 replicates. The scale bar denotes the nucleotide substitution per sequence.Additional file 2: Table S1. Comparisons of fungal dry mass (mg) of each isolate in different theophylline liquid mediums after cultivation at 30 °C for 5 days. TLM-S = theophylline liquid medium with sucrose as carbon source; TLM-D = theophylline liquid medium with dextrose with sucrose as carbon source; TLM-N = theophylline liquid medium with ammonium sulphate as nitrogen source; TLM-SN = theophylline liquid medium with sucrose and ammonium sulphate as carbon and nitrogen sources, respectively. All data were present by mean value ± SD of three replications. The lowercase letters indicated a significant difference at p < 0.05 levels and the uppercase letters indicated a highly significant difference at p < 0.01 levels by using one-way ANOVA of SPSS 20.0. The different letters show significant differences of each isolate between different theophylline liquid mediums.


Aspergillus niger is one of the main contaminants of the International Space Station (ISS). It forms highly pigmented, airborne spores that have thick cell walls and low metabolic activity, enabling them to withstand harsh conditions and colonize spacecraft surfaces. Whether A. niger spores are resistant to space radiation, and to what extent, is not yet known. In this study, spore suspensions of a wild-type and three mutant strains  were exposed to X-rays, cosmic radiation (helium- and iron-ions) and UV-C (254 nm). To assess the level of resistance and survival limits of fungal spores in a long-term interplanetary mission scenario, we tested radiation doses up to 1000 Gy and 4000 J/m2. For comparison, a 360-day round-trip to Mars yields a dose of 0.66 ± 0.12 Gy. Overall, wild-type spores of A. niger were able to withstand high doses of X-ray (LD90 = 360 Gy) and cosmic radiation . Drying the spores before irradiation made them more susceptible toward X-ray radiation. Notably, A. niger spores are highly resistant to UV-C radiation (LD90 = 1038 J/m2), which is significantly higher than that of other radiation-resistant microorganisms . In all strains, UV-C treated spores (1000 J/m2) were shown to have decreased biofilm formation (81% reduction in wild-type spores). This study suggests that A. niger spores might not be easily inactivated by exposure to space radiation alone and that current planetary protection guidelines should be revisited, considering the high resistance of fungal spores.The filamentous fungus  Radiation is the most challenging factor for life in the space environment . On the Bacillus subtilis are known to be highly resistant to extreme space conditions and therefore are currently being used as indicators for decontamination protocols and planetary protection policies  are produced as a natural part of its life cycle , and is covered by an outer layer of rodlets (hydrophobins) and pigments . These outer layers of the cell wall make spores highly hydrophobic and highly pigmented (A. niger spores as an adaptive trait conferring resistance toward UV-A (315–400)  resulted in decreased survival, when exposed to 100 J/m2. This was not the case for an A. fumigatus strain isolated from the ISS, where loss of pksP did neither reduce nor increase viability , and one brown component (∼425 nm) – and were both shown to be FwnA dependent (fwnA gene (ΔfwnA) results in fawn-colored (not white as for A. fumigatus) spores. Knowing if and how pigmentation is involved in spore resistance will be crucial to understand the limits of spore survival, which will, in turn, help develop adequate decontamination approaches. Another important component in fungal spore resistance is DNA repair. When damage occurs, several pathways can be activated: nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), or non-homologous end-joining (NHEJ) recombination (A. niger strains deficient in NHEJ (ΔkusA) are widely used to generate mutant strains, but it was also shown that these strains are more sensitive to UV and X-ray irradiation  . Moreoveiability . Howeverderstood . Pigmentependent . FwnA isbination . A. nigeadiation .A. niger, the stabilization of polarity axes during germination is dependent on the Rho GTPase RacA. A racA deletion displays a hyperbranching phenotype which results in compact colonies . Three mutant strains were included to elucidate the role of pigmentation (ΔfwnA) and DNA repair (ΔkusA) on spore resistance to radiation. In addition, high radiation doses were tested to assess the limits of resistance of fungal spores and their survival potential during long-term space travel. In addition, a fourth strain deficient in polar growth (ΔracA) was tested to assess the impact of UV-C treatment in spore revival and consequent ability for surface colonization.For these reasons, understanding whether 2PO4, 7 mM KCl, 178 nM H3BO3, 2 mM MgSO4, 76 nM ZnSO4, 70 mM NaNO3, 6.2 nM Na2MoO4, 18 nM FeSO4, 7.1 nM CoCl2, 6.4 nM CuSO4, 25 nM MnCl2, 174 nM EDTA, 0.5% (w/v) yeast extract and 0.1% (w/v) casamino acids] by flooding the agar plates with saline solution (0.9% NaCl) and harvesting the spores using a cotton stick. Spore suspensions were filtered using Miracloth (Millipore) to remove hyphal fragments and were kept at 4°C. Counting was done using a Neubauer chamber. All experiments were performed with spore suspensions not older than 2 weeks. Viability assays were done using minimal medium (MM) . For MM or CM agar plates, 15 g agar was added per liter (adapted from Aspergillus niger wild-type (N402) and three mutant strains with defects in pigmentation (ΔfnwA), DNA repair (ΔkusA), and polar growth control (ΔracA), were used in this study and are listed in A. niger strains were exposed to X-ray radiation in PCR tubes (Brand), each filled with 100 μl of saline solution (0.9% NaCl) at a concentration of 107 spores/ml. This concentration was chosen after testing the effect of initial spore concentration (inoculum) on survivability toward X-rays (Spores of d X-rays . Radiatit = time (in minutes); R = desired radiation dose (in Gy), d . Samples were exposed to 50, 100, 250, 500, and 1000 Gy. Given that the average dose rate was ∼20 Gy/min, the maximum time a sample was exposed to X-ray radiation was 50 min (corresponding to 1000 Gy). To test irradiation of dried spores, 25 μl of a spore suspension  either in water (H2O) or in saline solution (0.9% NaCl) was placed in PCR tubes, which were left to air-dry overnight on the bench (22°C) before irradiation. After irradiation, the spores were suspended in 100 μl of water (H2O) or saline solution (0.9% NaCl). Radiation exposure included at least three biological replicates per strain and was performed two independent times (n = 6). Viability was determined by colony forming units (CFUs) (see section “Viability Assay”).where 7 spores/ml. PCR tubes were placed inside Petri dishes stacked together inside plastic bags and placed directly facing the ion beam. Samples were exposed to 10, 100, 250, and 500 Gy. Non-irradiated controls were left at room temperature. Viability was calculated by CFU (see section “Viability Assay”). Radiation exposure included three biological replicates per strain (n = 3). Cosmic radiation exposure was performed at the Heavy Ion Medical Accelerator (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. The helium-ion beam had an energy of 150 MeV/n and linear energy transfer (LET) of 2.2 keV/μm. The iron-ion beam had an energy of 500 MeV/n and LET of 200 keV/μm. Each tested dose was adjusted by exposure time, since beam energy and LET was kept constant.Spores were exposed to helium- and iron-ions (two components of cosmic radiation) in PCR tubes (Brand) each filled with 100 μl of saline solution (0.9% NaCl) at a concentration of 106 spores/ml in 15 ml of saline solution (0.9% NaCl). The concentration of 106 spores/ml ensures a spore monolayer and prevents survival due to shielding by the spores themselves. The UV lamp  was used a UV-C monochromatic wavelength of 254 nm. During irradiation, magnetic stirrers continuously mixed the spore suspension in order to avoid mutual shielding of the spores. The radiation dose [J/m2] was adjusted through exposure time, since the height of the UV lamp was kept constant. For that, UV fluence was determined using the dosimeter (UVP UVX radiometer), and exposure time was adjusted for each sample to reach each desired UV dose . UV-C exposure time was calculated as follows:Spores of A. niger were exposed to UV radiation in Petri dishes with an initial concentration of 10t = time (in seconds); R = desired radiation dose (in J/m2), d . After each time point, corresponding to a certain dose, 100 μl of sample were taken in triplicate from the spore suspension and transferred into PCR tubes. Viability was calculated by CFU (see section “Viability Assay”). Radiation exposure included at least three biological replicates per strain and was performed two independent times (n = 6).where 2O2) was measured using a protocol adapted from A. niger were diluted to a final concentration of 108 spores/ml in saline solution (0.9% NaCl), 833 μl of which were placed in a 5 ml tube (Eppendorf). Afterward 167 μl of 30% H2O2 were added (Sigma-Aldrich). The spore-H2O2 suspension was gently mixed at RT (∼22°C) and incubated for up to 15 min in a final concentration of 5% H2O2. 30 μl of the suspension were taken at different time points and diluted 1:10 in saline solution (0.9% NaCl) with bovine catalase (100 μg/ml) to stop the oxidation reaction. Samples were serially diluted and used to determine viability (see section “Viability Assay”). This assay included three biological replicates per tested strain, and was performed two independent times (n = 6).Oxidative stress resistance toward hydrogen peroxide  to facilitate counting. The plates were incubated for 2 days at 30°C before the colonies were counted. This allowed calculation of the survival fraction ratio . To analyze the microscopic morphology, previously irradiated samples were diluted 100-fold before plating 10 μl on a MM agar plate. These were incubated for 1 day at 30°C, after which a Zeiss microscope (Axio Imager.M2) was used to take images of representative areas on the agar plate for further analysis (n = 6).Viability of analysis . Viabili5 spores/ml per well), 100 μl MM, and 100 μl of distilled H2O (dH2O)  and were incubated for 48 h at 30°C. After incubation, the wells were washed three times with dH2O, and 300 μl of crystal violet (0.5%) were added to each well to stain the surface-associated biomass . Excess staining was removed by washing with dH2O. De-staining was carried out by adding 300 μl of 95% ethanol. The absorbance of the ethanol-crystal violet solution was measured at 570 nm. The higher the absorbance value the greater the quantity of biological material. The absorbance was evaluated using the VICTOR Nivo Multimode Microplate Reader . The assay included eight biological replicates per tested strain and was performed twice (n = 16).To assess fungal biofilm formation, i.e., surface-associated growth with production of extracellular matrix, a crystal violet assay was performed, adapted from t-test was performed to analyze the significance between individual data points where a two-tailed p-value ≤ 0.05, was considered significant. Error bars as standard error. Linear regression on survival fraction data was used to calculate the lethal dose for 90% of the population  – which is the same as D10 values (10% survivability).Student’s fwnA) and NHEJ mutant (ΔkusA) strains were exposed to different X-ray doses (up to 1000 Gy) in saline solution. The lethal dose required to inactivate 90% of the spores was similar for both wild-type (LD90 = 366 Gy) and ΔfwnA strains (LD90 = 353 Gy). After the maximum tested radiation dose of 1000 Gy, the ΔfwnA strain demonstrated no significant differences in survival when compared with the wild-type . In contrast, at 500 Gy, ΔkusA was shown to be significantly more sensitive to X-ray radiation (1.25 × 104 ± 1.1 × 103 CFU/ml with a LD90 of 57 Gy), when compared to the wild-type (5 × 106 ± 8.7 × 105 CFU/ml) (p = 0.000). No CFU were detected above 500 Gy for ΔkusA. Survivability and LD90 values of the strains toward X-ray radiation, in all tested conditions, are shown summarized in Spores from the wild-type, color mutant . The highest X-ray dose at which there were detectable colonies was 500 Gy for both wild-type and ΔfwnA strains, and 250 Gy for ΔkusA. Results show that survivability of dried spores was decreased in comparison to wet spores  in all tested strains (6 ± 1.1 × 106 CFU/ml) survive better than dried spores from water (4.6 × 104 ± 7.2 × 103 CFU/ml) (p = 0.001). The same is seen for spores in saline solution where wet spores (5 × 106 ± 8.7 × 105 CFU/ml) survived better than dried spores from saline solution (8.2 × 103 ± 1.2 × 103 CFU/m) (p = 0.000) (3 ± 1.2 × 103 CFU/ml) survive significantly less than spores dried from water (4.6 × 104 ± 7.2 × 103 CFU/ml) (p = 0.000). Loss of pigmentation did not affect resistance to desiccation (0 Gy) (p = 0.6); or radiation resistance (at 500 Gy) when spores were air-dried from saline solution (p = 0.4). However, loss of pigmentation decreased spore survival in irradiated spores dried in water: at 500 Gy, ΔfwnA spores were significantly more sensitive (5.2 × 103 ± 1.2 × 103 CFU/ml) than wild-type spores (4.6 × 104 ± 7.2 × 103 CFU/ml) (p = 0.002).Survivability of air-dried  strains . At 500 = 0.000) . When coA. niger was tested by irradiating spores with helium- and iron-ions. Spores from the wild-type, color mutant (ΔfwnA) and NHEJ mutant (ΔkusA) strains were exposed to up to 500 Gy of helium- and iron-ions. At 500 Gy, wild-type spores survived less when irradiated with iron-ions (4.7 × 102 ± 6.01 × 101 CFU/ml) than when irradiated with X-rays (5 × 106 ± 8.7 × 105 CFU/ml) (p = 0.06), or helium-ions (8.7 × 106 ± 1.9 × 106 CFU/ml) (p = 0.01)  was decreased in comparison to both X-rays (3.4 × 106 ± 5.4 × 105 CFU/ml) (p = 0.004) and helium-ions (1.2 × 107 ± 2.0 × 106 CFU/ml) (p = 0.004) (kusA spores was significantly reduced when irradiated with iron-ions (1.9 × 103 ± 9.9 × 101 CFU/ml) than when irradiated with X-rays (4.7 × 102 ± 6.01 × 101 CFU/ml) (p = 0.02), with no colony formation being observed above 250 Gy of cosmic radiation  in comparison to the wild-type strain, we tested whether pigmentation is involved in protecting the spore from H2O2-induced oxidative-stress by incubating the spores up to 15 min in 5% H2O2  being higher than that of the ΔfwnA (3.1 min)  . The sam= 0.004) . This is= 0.004) . At 250 adiation . Exposurectively , suggest 5% H2O2 . Both th3.1 min) .A. niger spore survivability, spore suspensions with 106 spores/ml (spore monolayer) were exposed to 0–4000 J/m2 UV-C radiation (254 nm). At the highest tested dose of 4000 J/m2, both the wild-type (7.1 × 102 ± 4.3 × 102 CFU/ml) and polar growth mutant (4.2 × 101 ± 2.5 × 101 CFU/ml) demonstrated high survival (kusA at 4000 J/m2. All tested strains were able to cope with 3000 J/m2 of UV-C exposure. At 3000 J/m2 wild-type spores (1.3 × 103 ± 6.8 × 102 CFU/ml) and ΔracA spores (2.3 × 102 ± 1.3 × 102 CFU/ml) displayed high survivability; whereas ΔfwnA spores (6.3 × 101 ± 3.7 × 101 CFU/ml) and ΔkusA spores (1.2 × 102 ± 0.1 × 101 CFU/ml) displayed low survivability. The UV-C dose required to eliminate 90% of the wild-type spores (LD90) was 1038 J/m2. LD90 of ΔracA spores was 826 J/m2, of ΔkusA spores was 580 J/m2, and for ΔfwnA spores it was 512 J/m2. The data also shows that deletion of racA increases survival toward UV-C , a defect in pigmentation (ΔfwnA) decreased biofilm formation by 23% (p = 0.02); a defect in the NHEJ pathway (ΔkusA) decreased biofilm formation by 25% (p = 0.01); and a defect in both NHEJ and polar growth  showed a decrease of 49% (p = 0.007) , 97% reduction in ΔfnwA spores, (p = 0.001), 82% in ΔkusA spores (p = 0.001), and 94% in ΔracAΔkusA spores, (p = 0.005) (When assessing = 0.007) . When as.007)  .A. niger spores have previously been shown to have increased radiation sensitivity when vacuum-dried and irradiated in air compared to wet spores irradiated in air , which is consistent with previous findings concerning the role of pigmentation in fungal survival to low water activity . When irA. niger spore survivability was highly dependent on the NHEJ pathway (ΔkusA). Additionally, ΔkusA spores showed similar survival rates toward both high-LET iron-ion radiation . Nevertheless, this discrepancy in survival might be attributed to the fact that hyphal compartments of N. crassa can contain up to 100 nuclei, which in turn can lower the hit rate of radiation induced DNA damage  or B. subtilis spores (LD90 = 100 J/m2). In a study using UV-C to treat drinking water, A. niger was found to be completely inactivated after exposure to 1920 J/m2 UV-C (90 = 1038 J/m2).UV-derived decontamination methods are commonly used in modern laboratories and healthcare systems , with UV12.5 min . Yet, inard UV-C , where trganisms , and bec/m2 UV-C , which iA. niger spores after exposure to UV-C and X-ray radiation. Interestingly, previous studies with NHEJ mutants of N. crassa and Cryptococcus neoformans did not show differences in survival after UV-C irradiation when compared to the wild-type  demonstrated lower survivability after exposure to UV-C radiation. Interestingly, deficiency in pigmentation did not alter survivability after exposure to ionizing radiation (X-rays or cosmic radiation)  , 2. Thisarcticus . HoweverfwnA spores were more sensitive to hydrogen peroxide than wild-type spores  strain also demonstrated decreased ability of surface-associated growth, both before and after UV-C treatment, which suggests the involvement of pigments in spore adhesion.To address the contamination risks and possible decontamination procedures concerning ormation . Becauser hyphae , 2013. Rild-type . The undA. niger are extremely resistant to space radiation. Spores were able to withstand high doses of X-ray (LD90 = 360 Gy), cosmic radiation , and UV-C radiation (LD90 = 1038 J/m2). A. niger spore resistance to UV-C is particularly interesting, given that it is even higher than that of other radiation-resistant microorganisms . Air-drying the spores made them more susceptible to X-ray radiation. Moreover, wild-type spores treated with 1000 J/m2 UV-C were shown to have decreased biofilm formation ability (81% reduction). It is important to note that the ionizing radiation doses used in this study (up to 1000 Gy) are multiple times higher than doses expected from traveling in interplanetary space. For example, a 360-day round-trip to Mars would yield a dose of 0.66 ± 0.12 Gy (A. niger spores become easily inactivated due to space radiation alone. We thus recommend that current planetary protection guidelines are revisited to address the high resistance of fungal spores in space travel scenarios. In addition, further studies are needed in order to address fungal spore resistance to other space environmental factors such as vacuum, changes in pressure, and extreme temperature fluctuations.This study shows that spores of  0.12 Gy . It is tAll datasets generated for this study are included in the article/MC, AH, and RU performed the experiments, analyzed the data, and wrote the manuscript. VM, TS, RM, and AF contributed to the conception and design of the study, and manuscript preparation.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.


C. albicans microcolonies form extensive hyphal structures that enhance surface adherence and penetrate underlying tissues to promote fungal infections. This study examined the environmental conditions that promote microcolony formation and how these signals are relayed, in order to disrupt signaling and reduce pathogenesis. We found that a membrane-localized protein, Sho1, is an upstream regulator of glycolysis and required for Ras1-cAMP signaling. Sho1 controlled the Ras1-dependent expression of core microcolony genes involved in adhesion and virulence. This new regulatory function for Sho1 linking glycolysis to microcolony formation reveals a novel role for this fungal membrane protein. Candida albicans is an opportunistic, dimorphic fungus that causes candidiasis in immunocompromised people. C. albicans forms specialized structures called microcolonies that are important for surface adhesion and virulence. Microcolonies form in response to specific environmental conditions and require glycolytic substrates for optimal growth. However, fungal signaling pathways involved in sensing and transmitting these environmental cues to induce microcolony formation have not been identified. Here, we show that the C. albicans Ras1-cAMP cascade is required for microcolony formation, while the Cek1-MAP kinase pathway is not required, and Hog1 represses microcolony formation. The membrane protein Sho1, known to regulate the Cek1 pathway in yeasts, was indispensable for C. albicans microcolony formation but regulated the Ras1-cAMP pathway instead, based upon diminished intracellular levels of cAMP and reduced expression of core microcolony genes, including HWP1, PGA10, and ECE1, in C. albicanssho1Δ cells. Based upon predicted physical interactions between Sho1 and the glycolytic enzymes Pfk1, Fba1, Pgk1, and Cdc19, we hypothesized that Sho1 regulates Ras1-cAMP by establishing cellular energy levels produced by glycolysis. Indeed, microcolony formation was restored in C. albicanssho1Δ cells by addition of exogenous intermediates of glycolysis, including downstream products of each predicted interacting enzyme . Thus, C. albicans Sho1 is an upstream regulator of the Ras1-cAMP signaling pathway that connects glycolytic metabolism to the formation of pathogenic microcolonies.IMPORTANCEC. albicans microcolonies form extensive hyphal structures that enhance surface adherence and penetrate underlying tissues to promote fungal infections. This study examined the environmental conditions that promote microcolony formation and how these signals are relayed, in order to disrupt signaling and reduce pathogenesis. We found that a membrane-localized protein, Sho1, is an upstream regulator of glycolysis and required for Ras1-cAMP signaling. Sho1 controlled the Ras1-dependent expression of core microcolony genes involved in adhesion and virulence. This new regulatory function for Sho1 linking glycolysis to microcolony formation reveals a novel role for this fungal membrane protein. Candida albicans is an opportunistic fungus and the causative agent of oropharyngeal candidiasis (OPC), although it is typically a harmless commensal resident of the oral cavity , ECE1 , and PGA10 (involved in iron uptake)  is a target of the cAMP-protein kinase A (PKA) pathway  cycle and the Ras1-cAMP signaling pathway  . However uptake) , 7. In t pathway . In C. a pathway  to regulS. cerevisiae and are highly conserved among fungi  pathway counters extracellular osmostress by Hog1 phosphorylation, which induces glycerol production as well as other adaptive responses, including ion transport, metabolism, and cell cycle progression  signaling cascades were first identified in ng fungi , as theyalbicans  and S. cgression . The C.  the gut . In S. cate Hog1 . Howeverss genes , 15. C. ing Cek1 .C. albicans Cek1 MAP kinase (MAPK) pathway also controls hyphal differentiation and virulence  is a stable glucose analogue that cannot be utilized by yeast in glycolysis  parental strains, including SC5314, CAI-4, CAF4-2, and SN250, all formed typical microcolonies characterized by long filamentous hyphae radiating from a dense center , also resulted in severely defective or absent microcolonies (RAS1 OE). Moreover, among the TPK1 catalytic isoforms of the cAMP-dependent PKA, the tpk1Δ mutant formed atypical microcolonies with wispy hyphae without any compact dense center, while deletion of its opposing repressor subunit (tpk2Δ) resulted in typical microcolonies  known to initiate filamentation in response to environmental stimuli might be involved, we examined  pathway  for theie center . We expecolonies , since tcolonies . The impcolonies . Althougand RPMI  and S2;  strains ).10.1128/mSphere.00366-20.1FIG S1C. albicans strain SC5314 and the sho1Δ, ras1Δ, cdc25Δ, cyr1Δ, and efg1Δ mutants were grown in N-acetyl-d-glucosamine and RPMI 1640 at 37°C for 4 h with continuous shaking at 220 rpm. All mutants except cdc25Δ and efg1Δ strains were able to filament in the presence of N-acetyl-d-glucosamine. In RPMI, the wild-type, sho1Δ, ras1Δ, and cyr1Δ strains formed small filaments or pseudohyphal structures. Both the cdc25Δ and efg1Δ mutants were unable to filament when cultured in RPMI. Bar, 10 μm. Download FIG S1, EPS file, 1.8 MB.Copyright © 2020 Kumar et al.2020Kumar et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00366-20.2FIG S2C. albicans SC5314 and the sho1Δ strain were cultured for 12 h at 37°C and then diluted to an OD600 of 0.1 in RPMI alone or RPMI supplemented with 0.1% 2-deoxyglucose (2-DG), 0.2% sodium acetate, and 0.2% sodium acetate with 5 mM fructose 1,6-bisphosphate . Cells were then cultured at 37°C with continuous shaking at 220 rpm for 24 h, and OD600 was measured at various time points. There were no significance differences in growth curves between the sho1Δ and WT strains among the culture media. (B) Percent germination of C. albicans SC5314 and sho1Δ strains was calculated as the number of germinated cells relative to total observed C. albicans cells. C. albicans strains were grown for 12 h in YPD, diluted to an OD600 of 0.3 to 0.4 in RPMI or YNB +1.25% N-acetyl-d-glucosamine , and incubated for 2 h to 4 h at 37°C to induce germination. Both RPMI and YNB+GlcNAc were supplemented with 0.1% 2-DG, 0.2% sodium acetate, or 0.2% sodium acetate with 5 mM F1,6-BP. Images were acquired using bright-field microscopy, and hyphal formation was determined in multiple fields from at least 100 cells. Download FIG S2, EPS file, 0.7 MB.(A) For growth curve analysis, Copyright © 2020 Kumar et al.2020Kumar et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicansmsb2Δ, cst20Δ, hst7Δ, cek1Δ, and cph1Δ mutants all formed microcolonies similar to those of the WT  reduced 3-fold compared to WT cells, while the complemented strain sho1Δ/SHO1 produced cAMP nearly at WT levels , as might be expected due to the direct role of these genes in cAMP production. This result suggests that there is a regulatory link between Sho1 and the Ras1-cAMP pathway. To further assess this possibility, we tested whether addition of cAMP in sho1Δ, ras1Δ, and cyr1Δ deletion mutants would functionally complement microcolony defects. Interestingly, addition of cAMP to WT cells increased the length of hyphae and numbers of WT microcolonies  in sho1Δ, sho1Δ/SHO1, ras1Δ, RAS1 OE, and efg1Δ strains. When normalized to those of WT cells, the expression levels of three genes were significantly reduced (0.3- to 0.01-fold) in sho1Δ cells, and these levels were restored to those of WT cells in the complemented sho1Δ/SHO1 strain  was used to predict C. albicans orthologs for S. cerevisiae genes. Four interacting C. albicans glycolytic enzymes  were predicted to interact with SH3 or transmembrane (TM) domains of C. albicans Sho1  encoded by IRA2 and/or TPK1/TPK2 in the Ras1-cAMP pathway  to bypass Pfk1 function, glyceraldehyde 3-phosphate (G3P) to bypass Fba1 function, 3-phosphoglycerate (3PG) to bypass Pgk1 function, and pyruvate (Pyr) to bypass Cdc19 function  and without evidence of feedback repression. This effect was specific to Sho1, because ras1Δ and cyr1Δ deletion mutants showed no impact on microcolony phenotype with the addition of F1,6-BP or Pyr  and nutrient acquisition (PGA10) as well as maintaining intracellular cAMP levels necessary for Ras1-cAMP signaling.Here, we describe a new regulatory connection between Sho1 and the Ras1-cAMP pathway. Little is known about the function of Sho1 in ynthesis , 17, 38,ynthesis , and as ynthesis . Sho1 isynthesis , similarS. cerevisiae Sho1 is a membrane-bound adaptor protein composed of four transmembrane (TM) domains and cytoplasmic Src homology 3 (SH3) domain at its C terminus. S. cerevisiae Sho1 forms planar oligomers that allow it to assume a scaffold structure that is able to bind with multiple other proteins, including Opy2/Hkr1 (a subbranch of Hog1) and Msb2/Opy2 (a subbranch of Hog1), which results in Hog1  , suggesting signaling through this pathway. Structurally, C. albicans Sho1 also contains four TMs and an SH3 domain  or pyruvate (the enzymatic product of cdc19), while only a 1 mM concentration of other intermediates was needed to produce microcolonies in a sho1Δ deletion mutant. Since both enzymes catalyze glycolysis-committed steps, the absence of Sho1 may alter the kinetics so that a much higher level of substrate is required to drive catalysis  , and we 3 domain  that we 3 domain . To provatalysis . We alsoatalysis , 45. Thehenotype .C. albicans Sho1 in the Hog1 MAPK pathway, there is likely some regulatory role, based on the aberrant microcolony structures observed in deletion mutants of the Hog1 pathway (S. cerevisiae Sho1 and Pbs2 (the kinase activated by osmotic and oxidative stress-regulating Hog1)   . One prefor Hog1 . This is members , also suignaling . Interesosamine) . It is pC. albicans Sho1 in signaling via the Ras1-cAMP pathway. Future studies exploring the functional relevance of protein interactions linking Sho1 to glycolysis may further our understanding of C. albicans microcolonies as a form of infection in the human host.Our results define a new role for C. albicans strains used and their genotypes are listed in C. albicans SC5314 was kindly provided by David Roberts , CAI-4 and the cek1Δ strain were kindly provided by Malcolm Whiteway , the hog1Δ strain was kindly provided by Janet Quinn , and the cyr1Δ, tpk1Δ, tpk2Δ, efg1Δ, ras1Δ, cyr1Δ/CYR1, ras1Δ/RAS1, and ras1-G13V (RAS1 OE) strains were provided by Deborah Hogan (Dartmouth College). All C. albicans strains were cultured for 12 h in yeast extract-peptone-dextrose medium  broth supplemented with 50 μg/ml of uridine (Sigma) at 30°C in an orbital shaker at 220 rpm. C. albicans cells were pelleted by centrifugation at 2,500 × g for 5 min, washed three times in phosphate-buffered saline  and resuspended in RPMI 1640 supplemented with l-glutamine (Corning Cellgro) for experiments.The C. albicans strains were grown for 12 h in YPD at 30°C in an orbital shaker at 220 rpm, washed twice with PBS, and diluted to 103 cells/ml in PBS. A total of 100 C. albicans cells were added to 1 ml RPMI medium  or RPMI no-glucose (RPMI NG) medium (Sigma R1383) supplemented with 0.2% glucose or other glycolysis intermediates per well of a 12-well cell culture plate  and incubated at 37°C for 16 h in 5% CO2. For cAMP experiments, 3′,5′-cyclic AMP sodium salt (Sigma) was added to RPMI medium  before addition to yeast cells. For the assessment of glycolytic pathway components, fructose 1,6-bisphosphate (Sigma), 3-phosphoglyceric acid (Sigma), glyceraldehyde 3-phosphate (Sigma), or sodium pyruvate (Sigma) was added to RPMI medium, and C. albicans cells were tested for the formation of microcolonies. To assess the effect of alternate carbon sources on microcolony production, cells were incubated in RPMI NG supplemented with sodium acetate  (Sigma) as the carbon source, RPMI supplemented with sodium citrate , or RPMI supplemented with 0.1% 2-deoxy-d-glucose (Sigma) and subsequently imaged. The optimum physiological pH was maintained using 0.5 N NaOH. Microcolonies were observed using bright-field microscopy with a Zeiss Axio Observer Z1 microscope at a magnification of ×10.C. albicans strains (wild-type SC5314 and sho1Δ) were cultured for 12 h in YPD at 30°C in an orbital shaker at 220 rpm and washed twice with PBS. The culture was then diluted to an optical density at 600 nm (OD600) of 0.1 in RPMI medium alone or with the addition of 0.1% 2-deoxyglucose (2-DG), 0.2% sodium acetate, or 0.2% sodium acetate with 5 mM F1,6-BP and incubated at 37°C in air with continuous shaking at 220 rpm. Growth of each strain was determined by measuring the OD600 at multiple time points.C. albicans strains were grown for 12 h in YPD, diluted to an OD600 of 0.3 to 0.4 in RPMI or yeast nitrogen base (YNB) containing 1.25% N-acetyl-d-glucosamine , and incubated for 2 to 4 h at 37°C. Both RPMI and YNB+GlcNAc were supplemented with 0.1% 2-deoxyglucose (2-DG), 0.2% sodium acetate, or 0.2% sodium acetate with 5 mM F1,6-BP. Images were acquired using bright-field microscopy with a Zeiss Axio Observer Z1 microscope at a magnification of ×10. Hyphal formation was determined in multiple fields from at least 100 cells, and percent germination was calculated as the number of germinated cells relative to total observed C. albicans cells.To induce hypha formation, C. albicans cells were inoculated in RPMI 1640 medium in 12-well cell culture plates (200 cells/well) and grown at 37°C for 16 h in 5% CO2. The medium was discarded, and 1 ml of 1× PBS was added to the wells. Cells were recovered from each well and pelleted by centrifugation at 2,300 × g for 5 min. The cell pellet was resuspended in 1 ml of TRIzol and vortexed  with 0.45-μm glass beads using a FastPrep-24 5G homogenizer . After vortexing, 200 μl of chloroform was added to the lysed cells, and the cells were mixed vigorously for 15 s and kept for 2 min at room temperature. Lysed cells were centrifuged at 21,000 × g for 10 min at 4°C to separate the upper clear aqueous layer and mixed with 0.5 volume of 100% ethanol to precipitate total RNA from C. albicans cells. Total RNA was further purified using an RNeasy minikit (Qiagen) according to the manufacturer’s instructions. The RNA samples were stored at −80°C.C. albicans strains and the ras1Δ, RAS1 OE, sho1Δ, sho1Δ/SHO1, and efg1Δ strains was isolated from microcolonies as described above to quantitate gene expression of selected core microcolony genes . Total cDNA was synthesized for each sample using an iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s instructions, with equal amounts of RNA (1 μg in a 20-μl reaction mixture). Gene expression was quantified by real-time PCR using the iQ SYBR green Supermix (Bio-Rad),150 nM concentrations of both forward and reverse primers, and 50 ng of cDNA template in a total reaction mixture volume of 20 μl. After an initial denaturing step of 3 min at 95°C, the reactions were cycled 40 times with a CFX-96 Touch real-time PCR system (Bio-Rad) under the following conditions: 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Each qRT-PCR assay included two negative controls, no template and RNA alone (10 ng), and a positive control, genomic DNA (10 ng). Fluorescent data were collected and analyzed with iCycler iQ software. Relative fold changes in gene expression of HWP1, HYR1, ECE1, and PGA10 were calculated from the corresponding standard curves compared to ACT1 as the control. Data shown are the means and standard errors of the means (SEM) for three replicates from at least two independent experiments. Significant differences were determined by one-way analysis of variance (ANOVA).Total RNA from wild-type parental ACT1qRT , ECE1qRT , HWP1qRT , HYR1qRT , and PGA10qRT .The primers used for the study are g. Supernatants were washed five times with water-saturated ether to remove trichloroacetic acid. After trichloroacetic acid neutralization, the supernatant was dried using a Speed Vac in 100 μl of enzyme-linked immunosorbent assay (ELISA) buffer. cAMP was quantified using a cyclic AMP Select ELISA kit  according to the manufacturer’s protocol. In each experiment, a standard curve was generated and used to calculate cAMP concentrations. Experiments were repeated three independent times, and significant differences were determined by one-way ANOVA.Intracellular cyclic AMP (cAMP) was measured as previously described  with theC. albicans Sho1 were predicted based upon the published S. cerevisiae Sho1 protein membrane interactome ; then, the BIOGRID interactome database was applied to these genes to identify any MAPK pathway genes interacting physically or genetically with both S. cerevisiae Sho1 and glycolytic genes. The Candida Genome Database  was used to predict C. albicans orthologs for S. cerevisiae genes identified in this screen.Interacting proteins for eractome . Glycoly


This topsoil exhibited twin AOP peaks (1 and 2 mM ammonium) at 10°C, but not at 20°C. Six and ten operational taxonomic units (OTUs) were identified for ammonia-oxidizing bacteria (AOB) and archaea (AOA) amoA, respectively. AOB were classified into Nitrosospira; the two dominant OTUs corresponded to the Mount Everest cluster. AOA were classified into three clusters; Nitrososphaera and Nitrosocosmicus were the two dominant clusters.The maximum ammonia oxidation potential (AOP) of a topsoil in Langhovde, East Antarctica was 22.1±2.4 ng N g Soils formed in these areas are permafrost-affected soils (Gelisols)  in soils. We investigated the AOPs of East Antarctic soils, including the substrate dependency and genetic classification of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these soils.Ice-free areas constitute 0.4% of the Antarctic continent (elisols) ; 2008 anelisols) . Researcn=3) during the field expedition were measured at approximately 10°C, whereas those for RL (n=3) were measured at 10 and 20°C in April 2017 in Japan. Further measurements of AOP using combinations of eight substrate concentrations  and two temperatures (10 and 20°C) were conducted on the topsoil of Y2 (depth of 0–3 cm), which exhibited the maximum AOP from the standard assay. ANOVA and Tukey’s multiple comparison tests (α=0.05) were performed for the measured AOPs using SAS Add-In for Microsoft Office (SAS Institute). We performed total DNA extraction, quantification, a pyrosequencing analysis, and sequence analysis with phylogenetic assignments of the ammonia monooxygenase subunit A (amoA) genes of AOB and AOA. Further methodological details are outlined in Supplementary Information.Soil samples were collected at two plots (Y1 and Y2) in January 2017 in Yukidori-zawa, located in Langhovde, Dronning Maud Land , and at one plot (RL) in February 2017 near Mt. Riiser-Larsen, Enderby Land . Each plot was covered with lichen and moss. AOP was defined as the nitrite production rate by an aerobic incubation with the addition of a substrate solution , whereas–1 dry soil h–1 in the Y1, Y2, and RL plots, respectively   (P<0.001) (P<0.001); however, these peaks were not present at 20°C. Meanwhile, AOP at 0.1 mM ammonium and 20°C was significantly higher than that at 10°C (P<0.05). Soil pH was nearly neutral, ranging between 6.1 and 6.6 : 5.8±2.1 (mean±SD), 22.1±2.4, and 4.3±0.3 ng N gectively . These mP<0.001) . The twi and 6.6 . Soil toic soils . AOB weramoA: all were classified into Nitrosospira  were identified for AOB-osospira . OTU1 anosospira  and grouosospira  .Supplementary Material


The (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA has a uniform face-centered cubic plus body-centered cubic solid solution structure and a fine grain-sized microstructure with a size of about 2 microns. The HEA possesses an excellent strain hardening rate and high strain rate sensitivity at a high strain rate. The Johnson–Cook plastic model was used to describe the dynamic flow behavior. Hat-shaped specimens with different nominal strain levels were used to investigate forced shear localization. After dynamic deformation, a thin and short shear band was generated in the designed shear zone and then the specimen quickly fractured along the shear band.The dynamic mechanical properties and microstructure of the (Al The crystal structure of the AlxCoCrFeNi alloys transforms from face-centered cubic (FCC) to body-centered cubic (BCC) with an increase in Al content [0.5CoCrFeNi alloys aged at different temperatures and found that an FCC + BCC solid solution structure with optimal hardness appeared when the alloys were aged at 350–800 °C. Niu et al. [0.5CoCrFeNi HEA and found that the nano-sized BCC phase particles reinforced the Al0.5CoCrFeNi HEA. Wang et al. [0.5CoCrFeNi HEA was 1044 K, and the tensile strength increased after phase transformation. Therefore, previous works showed that the existence of the FCC + BCC solid solution structure is beneficial for improving the mechanical properties of the Al0.5CoCrFeNi alloy. Regarding the effects of the addition of Mo and C elements to HEAs, the addition of Mo could cause solution strengthening because of the large atomic size [0.5CoCrFeNi HEA could enhance the formation of a -rich σ phase and adjust the mechanical properties of the Al0.5CoCrFeNiMox HEAs, including hardness, strength, and ductility. Jian et al. [0.5CoCrFeNi HEA reinforced by Mo and C elements.High entropy alloys (HEAs) contain at least five elements in equiatomic or near-equiatomic ratios and have excellent properties ,2,3,4. T content ,7,8. Mea content ,7. Lin e content  investigu et al.  studied g et al.  discovermic size ,13,14,15mic size  found thn et al.  suggesten et al.  also fou0.1CrFeCoNi HEA at a high strain rate is the same as that of low stacking fault energy materials. Dirras et al. [20Hf20Zr20Ta20Nb20 HEA was much higher under dynamic loading conditions than under quasi-static conditions and observed a shear band in the dynamic regime. Wang et al. [−4 s−1 to 1 × 10−1 s−1 and from 1 × 103 s−1 to 3 × 103 s−1. Additionally, the dynamic mechanical properties of the as-cast NiCrFeCoMn HEA were investigated by Wang et al. [2 HEA and found a linear relationship between the dynamic yield strength and strain rate. Shear localization, or shear band, is the main failure mechanism for the materials deformed under high velocity loading. Li et al. [0.3CoCrFeNi HEA was unable to form a shear band [0.5CoCrFeNi HEA could significantly increase the tensile strength under quasi-static loading. However, the dynamic mechanical properties of Al0.5CoCrFeNi reinforced by Mo and C are not clear yet.Many materials researchers are paying more and more attention to the dynamic mechanical properties of HEAs for industry application. Kumar et al.  reached s et al.  found thg et al. . Ma et ag et al.  studied i et al.  studied ear band , and theear band  found th0.5CoCrFeNi)0.95Mo0.025C0.025 HEA prepared by powder extrusion and explored the forced shear localization of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA.In this paper, we investigated the microstructure and mechanical behavior of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 (at.%) were melted in an induction heated vacuum furnace. Then, the melt was drop through a ceramic tube and atomized by high purity Ar with a pressure of 4 MPa. After cooling down the gas-atomized powder in the atomization chamber, a stainless can with dimensions of φ 60 mm × 150 mm, which was degassed for 12 h at a temperature of 500 °C and sealed in a vacuum, was used to fill the as-prepared HEA powder. The encapsulated powder was then pre-heated for 1 h at a temperature of 1150 °C and immediately hot extruded into bars with an extrusion ratio of 6. After hot extrusion, the bar was cooled in the air. The content of oxygen in the gas-atomized powder was 750 ppm, and the chemical compositions of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA are listed in High purity Al, Co, Cr, Fe, Ni, Mo, and C powders of a nominal composition ΔSmix0.5CoCrFeNi)0.95Mo0.025C0.025 HEA, the configurational entropy ΔmixS can be calculated as 1.725R, which satisfies the definition of HEA [By substituting the actual compositions of (Aln of HEA .0.5CoCrFeNi)0.95Mo0.025C0.025 HEA was 25 mL hydrochloric acid + 25 mL ethyl alcohol + 5 g CuSO4·5H2O. An X-ray diffraction (XRD) test was also performed on a Rigaku 2550 diffractometer  using Cu radiation to analyze the structure of the as-received (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA.The produced material was observed using a POLYVAR-MET optical microscope  and a JXA-8230 electron probe microanalyzer . The etchant for the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA, cylinder specimens were used for quasi-static compression tests and dynamic load tests. The cylinder specimens were cut from the as-received HEA bars along the extrusion direction (ED) with dimensions of φ 4 mm × 5.6 mm. The quasi-static compression tests were performed on INSTRON 8802  with strain rates of 1 × 10−1, 1 × 10−2, and 1 × 10−3 s−1. The dynamic load tests were performed on a split Hopkinson pressure bar  with strain rates of 550, 1300, 2200, and 3000 s−1. Hat-shaped specimens cut along the ED were used to induce forced shear localization, as shown in nom. = 2 × (fbefore − fafter)/(bbefore − abefore). After dynamic deformation, hat-shaped specimens were observed using a Quanta-200 scanning electron microscope .To obtain the mechanical properties of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA. The HEA is composed of fine grains with a size of about 2 microns. 0.5CoCrFeNi)0.95Mo0.025C0.025 HEA includes the FFC phase and the BCC phase. The chemical compositions of the phases were identified by an electron probe technique, as shown in 0.5CoCrFeNi)0.95Mo0.025C0.025 HEA consists of a light gray FCC matrix phase (marked as point 1), a black -rich BCC phase (marked as point 2), and a gray -rich M23C6 carbide phase (marked as point 3). This shows that the C element exists in the M23C6 carbide phase and the Mo element exists in the FCC matrix phase and M23C6 phase, and the MoC phase can also be identified in the Mo-rich M23C6 carbide phase. 23C6 phases in the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA are uniform.0.5CoCrFeNi)0.95Mo0.025C0.025 HEA tested at strain rates of 1 × 10−1, 1 × 10−2, and 1 × 10−3 s−1. The cylinder specimen exhibited yield strengths of 702, 703, and 716 MPa at strain rates of 1 × 10−3, 1 × 10−2, and 1 × 10−1 s−1, respectively. Therefore, with the increase of strain rates under quasi-static compression tests, the yield strength of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA increased. It also can be observed from 0.5CoCrFeNi)0.95Mo0.025C0.025 HEA deformed at strain rates of 550, 1300, 2200, and 3000 s−1. The yield strengths of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA were 754, 878, 958, and 1057 MPa at strain rates of 550, 1300, 2200 and 3000 s−1, respectively. The saw-like curves are much serious in dynamic compression strain–stress curves. As we observed from the optical micrograph and EPMA maps, the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA has small -rich BCC phase particles and M23C6 phase particles. The saw-like curves could be attributed to the interaction between the dislocation and -rich BCC phase particles plus the M23C6 phase particles.0.5CoCrFeNi)0.95Mo0.025C0.025 HEA at quasi-static and dynamic loading tests is presented in m can be defined as follows:The trend of the yield strength of the 0.95Mo0.025C0.025 HEA at high strain rates.The value of strain rate sensitivity under quasi-static testing conditions was 4.29 × 100.5CoCrFeNi)0.95Mo0.025C0.025 HEA. The equivalent flow stress σ can be expressed by the following equation:−3 s−1. Using the Matlab program  to fit the digital matrix of the stress–strain–strain rate, the parameter values and the Johnson–Cook physical equation for the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA were obtained. The Johnson–Cook model can describe the plastic deformation of the HEA at high strain rates well, as shown in The Johnson–Cook model, which is considered to have a huge strain, high strain rate, and high temperature, was used in this work to describe the plastic deformation of the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA at the strain rate of 1 × 10−3 s−1 along with the stress–strain curve is shown in xCoCrFeNi HEA, as shown in xCoCrFeNi HEA.The strain hardening rate (dσ/dε) curve for the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA. The hat-shaped specimen with a nominal strain of 2.53 was plastically deformed after dynamic deformation, and a small crack was generated at the upper part of the specimen, as shown in The formation of the shear band can be attributed to the instability of thermal viscoplastic behavior. The critical condition of the constitutive instability can be expressed as0.3CoCrFeNi HEA and found the value of strain hardening was greater than the value of thermal softening, which explains the failure of the Al0.3CoCrFeNi HEA to form a shear band. In our work, with the addition of the Mo and C elements, the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA had a much higher strain hardening rate than that of the Al0.3CoCrFeNi HEA. However, the shear band was generated in the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA. This was due to the dissolved Mo atoms in the matrix phase, which hinder the heat diffusion, thus decreasing the thermal conductivity. The decreasing of thermal conductivity can increase the value of thermal softening, therefore promoting the generation of shear bands. Because of the high strain hardening rate and low interfacial strength [0.5CoCrFeNi)0.95Mo0.025C0.025 HEA under high velocity loading.The strain hardening 0.95Mo0.025C0.025 HEA prepared by powder extrusion has a uniform FCC + BCC solid solution structure and consists of the FCC matrix phase, the -rich BCC phase, and the -rich M23C6 carbide phase. The MoC phase can also be identified in the Mo-rich M23C6 carbide phase. The (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA shows a strong strain rate sensitivity at high strain rates, and the yield strength of the HEA increases from 754 to 1057 MPa as the strain rate increases from 550 to 3000 s−1. The Johnson–Cook model was used to describe the dynamic flow behavior. The addition of Mo and C elements can improve the strain hardening rate of the AlxCoCrFeNi HEA. A short shear band of about 10 microns in width was generated in the designed shear zone and then the specimen quickly fractured along the shear band, which indicates that the shear band can coordinate deformation in the (Al0.5CoCrFeNi)0.95Mo0.025C0.025 HEA under high velocity loading.The (Al


Panax ginseng C.A. Meyer, which is a traditional Korean medicinal plant used to treat for muscle weakness. Skeletal muscle progenitor cell-based screening identified three compounds that enhance cell viability, of which 20(R)-ginsenoside Rh2 showed the most robust response. 20(R)-ginsenoside Rh2 increased viability in myoblasts and cardiomyocytes, but not fibroblasts or disease-related cells. The cellular mechanism was identified as downregulation of cyclin-dependent kinase inhibitor 1B (p27Kip1) via upregulation of Akt1/PKB phosphorylation at serine 473, with the orientation of the 20 carbon epimer being crucially important for biological activity. In zebrafish and mammalian models, 20(R)-ginsenoside Rh2 enhanced muscle cell proliferation and accelerated recovery from degeneration. Thus, we have identified 20(R)-ginsenoside Rh2 as a p27Kip1 inhibitor that may be developed as a natural therapeutic for muscle degeneration.Aging is associated with increased prevalence of skeletal and cardiac muscle disorders, such as sarcopenia and cardiac infarction. In this study, we constructed a compendium of purified ginsenoside compounds from  Panax ginseng C.A. Meyer (P. ginseng) in Korean traditional medicine to treat reduced strength and cardiovascular disorders3. This medicinal plant was considered to be of high value in East Asia and has been associated with numerous therapeutic effects on the human body3. Detailed guidelines concerning the use of P. ginseng in Korean traditional medicine were recorded in the SohoDang miscellany of Taekyoung Kim (1850–1927) and Samjung-Yolam (1908)4. There has been much research effort to identify the active components that give P. ginseng its biological and pharmacological efficacy, but progress has fallen short of completely characterizing these compounds5.There is a long history of the use of 7. For example, sarcopenia is the aging related degeneration of skeletal muscle mass (0.5–1% loss per year after the age of 50) that results from a deterioration in the proliferative capacity of resident muscle stem cells (termed satellite cells)8. Patients become progressively weaker, have greater propensity to fall over and may lose their ability to live independently. Aging is also a risk factor for cardiovascular diseases. For example, myocardial infarction (heart attack) is a leading cause of death, irrespective of race or ethnicity9. In contrast to skeletal muscle, cardiac muscle has little capacity for regeneration after degeneration10. Fibrous scar tissue is produced that can compromise heart function and lead to clinical heart failure11. Due to the historical use of P. ginseng to treat general weakness and disorders of the circulation3, we investigated whether it was possible to isolate an active compound that mediated these therapeutic effects. A library of 39 purified ginsenosides was screened in skeletal muscle progenitor cells (myoblasts) to detect compounds that increased cell proliferation, which is an indicator of the potential to enhance muscle regeneration12. 20(R)-ginsenoside Rh2 (CPP531) was identified as an enhancer of myoblast proliferation. Further experiments showed that CPP531 treatment improved recovery in animal models of skeletal and cardiac muscle degeneration. CPP531 treatment in vitro increased Akt1/PKB activation and repressed expression of cyclin-dependent kinase inhibitor 1B (p27Kip1).The increasing lifespan of human society is linked with a greater prevalence of degenerative diseases12. A compendium of 39 ginsenosides purified from P. ginseng was screened in C2C12 murine myoblasts -ginsenoside Rh2 and ‘CPP533’ for ginsenoside Rk2 and ‘CPP534’ for isoginsenoside Rh3) -ginsenoside Rh2 (CPP531) produced a concentration-dependent effect on proliferation  Fig. . 1H and To further confirm that CPP531 increases proliferation in myoblasts, treated cells were immuno-stained for BrdU incorporation. It was observed that CPP531 increased BrdU incorporation into the nuclei of murine myoblasts Fig. . To asse14. Examples include cancer-associated fibroblasts (CAF) and cardiac fibroblasts (CdF)16. Treatment with CPP531 deceased the proliferation of CdF . Certain types of activated fibroblasts (termed myofibroblasts) contribute to degenerative diseases via enhanced proliferationCdF Fig.  and show17. Using western blotting analysis, CPP531 treatment increased the expression of Akt, although this increase did not reach statistical significance  transgenic zebrafish larvae, which produce fluorescence from cardiomyocytes, were treated with 5 µg/mL CPP531 until 48 hpf. EdU staining and confocal microscopy assessment indicated that CPP531 treatment enhanced cardiomyocyte proliferation in the zebrafish  was used to assess the effect of CPP531 on degenerated cardiac tissue. Functional recovery of the heart muscle was measured using echocardiography. It was observed that CPP531 treatment improved cardiac output parameters  Fig. . CPP531 27 Fig. . Immunos28. One week after injury, mean fiber diameter was higher in the gastrocnemius muscle of CPP531 treated mice  as an enhancer of proliferation, but not the epimer, 20(S)-ginsenoside Rh2 . To further investigate the potential of CPP531 to treat muscle diseases, it may be interesting to test this small molecule in models of chronic muscle disease, such as the dexamethasone-induced mouse model of skeletal muscle wasting37 or the mouse hypertension model38. Additionally, the potential application of CPP531 as a drug for genetic muscle wasting disorders, such as Duchenne muscular dystrophy, could be assessed in the mouse mdx model39.Compared to CPP532, there are relatively few publications reporting the biological activities of CPP531. Kang, 40. Our data shows that CPP531 treatment increases the activity of Akt/PKB, which is a major ‘hub’ for transducing signals from activated receptors  and p57Kip2  were purchased from Santa Cruz Biotechnology, Dallas, USA. BIO -6-Bromoindirubin-3′-oxime) was kindly provided by Professor Yong-Chul Kim, Laboratory of Drug Discovery, Gwangju Institute of Science and Technology, Korea.Barium chloride and collagenase type 2 solution (210 U/mL), were purchased from Sigma-Aldrich, St. Louis, USA. Pancreatin solution (0.6 mg/mL) was purchased from Thermo Fisher Scientific, Waltham, USA. An antibody for Akt  was purchased from Cell Signaling Technology, Inc., Danvers, USA. Antibodies for Ser 473 phosphorylated Akt1 , an antibody BrdU  p27HCT116 human colorectal carcinoma cells, HeLa human cervical cancer cells, H9C2 murine cardiomyoblasts and NIH/3T3 murine fibroblasts, were purchased from the Korean Cell Line Bank. C2C12 murine myoblasts were purchased from Koram Biotech Corp., Seoul, Korea. Each cell type was cultured in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 µg/mL streptomycin (PenStrep).YD-10B human oral squamous cell carcinoma (OSCC) cells were purchased from the Korean Cell Line Bank. Seoul, Korea. CAF4 OSCC-associated fibroblasts were a generous gift from Professor Jin Kim, Yonsei University, Korea. YD-10B and CAF4 cells were cultured in DMEM and F-12 media (3:1 ratio) supplemented with 10% FBS and PenStrep.Cardiomyocytes and cardiac fibroblasts were purified from 2 d-old Sprague-Dawley rats. In brief, the heart ventricles were dissected from the euthanized rats and washed in 4 °C PBS, sliced into small pieces with a scalpel and digested in 0.1% collagenase type 2 solution and 0.6 mg/mL pancreatin for 30 min with mild stirring. The supernatant was collected and centrifuged through a Percoll gradient (Sigma-Aldrich) at 1000 rpm for 5 min. The cardiac cell layer was harvested and cultured with DMEM supplemented with 10% FBS for 1 h. The non-adherent cardiomyocytes were then removed from the adherent cardiac fibroblasts and used for experiments. The proliferation culture for cardiac fibroblasts was DMEM supplemented with 10% FBS and 1% PenStrep. The fibroblasts were used for experiments at passage 2 or passage 3.Human primary skeletal muscle myoblasts were isolated from a muscle biopsy taken from the gluteus maximus muscle of a male (29 years old) during knee surgery . The experimental protocols were approved by the Institutional Review Board of Chonnam National University Medical School, Korea. All methods were carried out in accordance with the relevant guidelines and regulations of the Institutional Review Board of Chonnam National University Medical School, Korea, and informed consent was obtained from the participant.The muscle biopsy was placed in sterile tube containing autoclaved PBS and 1% PenStrep. The connective tissue was dissected using a stereo microscope. The muscle tissue was then sliced into 1–2 cm pieces and each piece was transferred to a 10 cm tissue culture dish containing primary cell growth media  for 5 days. Floating muscle tissues were removed and the adherent cells were sub-cultured every 2 d. The trypsinized cells were placed in a culture dish for 15 min, allowing sufficient time for the fibroblasts to attach. The media with floating myoblasts was harvested and transferred to another 10 cm culture dish. This process was repeated five times to remove contaminating fibroblasts.For cell-based experiments, stocks of CPP531 were prepared at 2.5, 5, 10 and 20 mg/mL in the DMSO vehicle to keep the final concentration of DMSO at 0.1% for cell treatment.3 cells/well. 24 h later, the culture media was replenished and cells were treated with 5 µg/mL test ginsenoside for 72 h. Each ginsenoside was treated in triplicate. The effect of screened ginsenosides on myoblast proliferation was assessed using the MTT assay.A compendium of 39 ginsenosides was assembled by Professor Won Keun Oh, Department of Pharmacy, Seoul National University, Korea, as described below and listed in Table 46. After compound incubation, the culture media was changed to 1X MTT solution (0.5 mg/mL in PBS) and the plate was incubated at 37 °C for 2 h. The MTT solution was carefully discarded. 80 µL DMSO was added to each well and the MTT absorbance was read at 540 nm using a microplate reader .Cell proliferation was measured using the MTT -2,5-diphenyltetrazolium bromide) assay, as previously describedP. ginseng resulted in the isolation of 39 dammarane triterpenes and established as a compendium of Korean ginseng saponins. By comparison with previously reported spectroscopic information including NMR data, their structures were determined and identified as followed Extensive studies about ed Table . A GilsoP. ginseng were purchased from Kumsan Ginseng Town, Chungcheongnam-do, Korea and authenticated by Professor W. K. Oh at Seoul National University. A voucher specimen (SNU2010-10) was deposited in the herbarium of the college of Pharmacy, at Seoul National University, Korea.The dried leaves of P. ginseng (10 kg) were extracted with 80% EtOH  using a sonicator. The combined extract was concentrated by an evaporator to yield a dried residue (850 g). Dried extract was suspended in 40% EtOH and then subjected to Diaion HP-20 macroporous resin  open column chromatography (CC) (11 × 55 cm) and eluted with 40% EtOH, EtOH, and washed with acetone. Partial EtOH fraction was set aside for production of sapogenin by a series of chemical reaction. To obtain saponin rich fraction, remained EtOH and acetone fractions were combined (245 g) and subjected to silica gel CC  (20 × 60 cm) and eluted with gradient system of EtOAc/MeOH from 10:1 to 0:1 to yield five fractions (F.1 – F.8).The dried leaves of 3/MeOH to afford eight subfractions (F.1.1 – F.1.8). F.1.4 was preprocessed by reversed-phase CC  with gradient system from 25% to 100% MeOH to give twenty two subfractions. F.1.4.13 was subjected to semi-preparative (semi-prep) HPLC with a MeOH/H2O , resulting in the isolation of 6α,20(S)-dihydroxydammar-24-ene-3,12-dione (3.0 mg). F.1.4.15 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 3-deoxy-3-oxo-20(S)-protopanaxatriol (3.8 mg). F.1.6 was subjected to reversed-phase CC with gradient system from 33% to 100% MeOH to give nine subfractions. F.1.6.9 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 6α,20(S),25-trihydroxydammar-23-ene-3,12-dione (2.8 mg). F.1.8 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 6α,20(S),24(S)-trihydroxydammar-25-ene-3,12-dione (2.0 mg).F.1 (10.7 g) was subjected seilica gel CC with solvent system of CHCl3/MeOH to afford eight subfractions (F.2.1 – F.2.8). F.2.5 was subjected to reversed-phase CC with gradient system from 25% to 100% MeOH to give ten subfractions. F.2.5.8 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 3β,6α,12β-trihydroxy-27-nordammar-- 20(22),23-diene-25-one (5.6 mg). Ginsenoside Rk2 (24.3 mg) and isoginsenoside Rh3 (24.4 mg) were isolated from F.2.5.10. 3β,20(S)-dihydroxydammar-24-en-12β,23β-epoxy-20-O-β-d-glucopyranoside (3.8 mg) was purified from F.2.6.7. F.2.7 was subjected to reversed-phase CC with gradient system from 25% to 100% MeOH to give fourteen subfractions. F.2.7.8 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 20(R)-ginsenoside Rh1 (10.5 mg). F.2.7.14.5 was subjected to semi-prep HPLC with a MeCN/H2O , resulting in the isolation of compound K (2.8 mg), 20(S)-ginsenoside Rh2 (5 mg) and 20(R)-ginsenoside Rh2 (9.2 mg).F.2 (15.3 g) was subjected to seilica gel CC with solvent system of CHCl3/MeOH  to afford ten subfractions (F.3.1 – F.3.10). F.3.8 was subjected to semi-prep HPLC with a MeCN/H2O , resulting in the isolation of ginsenoside Rh4 (6.3 mg). F.3.9 was subjected to reversed-phase CC with gradient system from 25% to 100% MeOH to give twenty-one subfractions. F.3.9.9 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of ginsenoside F5 (35.0 mg). ginsenoside Rg6 (200.4 mg) and compound Mc (27.1 mg) were isolated from F.3.9.13 and F.3.9.21, respectively.F.3 (30.5 g) was subjected to seilica gel CC with solvent system of CHCl3/MeOH  to afford fifteen subfractions (F.4.1 – F.4.15). F.4.11 was subjected to reversed-phase CC with gradient system from 25% to 100% MeOH to give thirty-two subfractions. F.4.11.32 was subjected to reversed-phase MPLC with gradient system from 33% to 100% MeOH to give eight subfractions. compound Y (12.0 mg) and ginsenoside Rg5 (24.3 mg) were isolated from F.4.11.32.7.F.4 (70 g) was subjected to seilica gel CC with solvent system of CHCl2O , resulting in the isolation of ginsenoside F3 (12.5 mg), 20(S)-ginsenoside Rg2 (32.1 mg), and 20(R)-ginsenoside Rg2 (21.2 mg). F.5.3.11 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 20(E)-ginsenoside F4 (21.0 mg). F.5.6 was subjected to Sephadex-20 (3 × 30 cm) with 100% MeOH to obtain six subfractions (F.5.6.1 – F5.6.6). ginsenoside Rh18 (10.5 mg) was purified from F5.6.5 using semi-prep HPLC with a MeOH/H2O . F.5.6.6 was subjected to semi-prep HPLC with a MeCN/H2O , resulting in the isolation of ginsenoside Re4 (28.6 mg). F.5.7 was subjected to reversed-phase CC (5 × 30 cm) with gradient system from 33% to 100% MeOH, resulting in the isolation of ginsenoside Re (111.6 mg) and ginsenoside Rc (5.0 mg).F.5 (30.5 g) was subjected to Sephadex LH-20  (7 × 30 cm) with 100% MeOH to obtain seven subfractions (F.5.1 – F5.7). F.5.3 was subjected to reversed-phase CC (5 × 30 cm) with gradient system from 33% to 100% MeOH to give eleven subfractions. F.5.3.8 was subjected to semi-prep HPLC with a MeOH/H3 and MeOH to afford nine subfractions (F.6.1 – F.6.9). F.6.5 (3 g) was subjected to reversed-phase MPLC (5 × 20 cm) with gradient system from 25% to 100% MeOH to give five subfractions. F.6.5.2 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 20(S)-ginsenoside Rg1 (25.3 mg). F.6.6 (3.1 g) was subjected to reversed-phase CC (6 × 50 cm) with gradient system from 25% to 100% MeOH to give twenty-three subfractions. F.6.6.23 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of notoginsenoside Fe (33.0 mg). F.6.7 was subjected to reversed-phase CC with gradient system of MeOH/H2O to give twelve subfractions. Vinaginsenoside R4 (75.6 mg) was isolated from F.6.7.12 by semi-prep HPLC with a MeOH/H2O . F.6.8 was subjected to reversed-phase CC with gradient system of MeOH/H2O to give nineteen subfractions. F.6.8.10 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of bipinnatifidusoside F1 (80 mg). F.6.8.18 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of gypenoside XVII (70.4 mg). F.6.8.19 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of gypenoside IX (49.7 mg). F.6.9 was subjected to reversed-phase CC (5 × 60 cm) with gradient system from 25% to 100% MeOH to give eighteen subfractions. F.6.9.18 was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of ginsenoside Rb1 (10 mg), ginsenoside Rb2 (38.2 mg), and ginsenoside Rb3 (16 mg).F.6 (100 g) was subjected to seilica gel CC with solvent system of CHClet al., 1995; Cui et al., 1994). Final reacted fraction was subjected to silica gel CC with solvent system of n-hexane and EtOAc from 7:1 to 1:5 to obtain five subfractions (F.1′ – F.5′). F.2′ was subjected to semi-prep HPLC with a MeCN/H2O , resulting in the isolation of dehydroprotopanaxatriol I (2.0 mg) and dehydroprotopanaxatriol II (3.1 mg). F.4′ was subjected to semi-prep HPLC with a MeOH/H2O , resulting in the isolation of 20(S)-protopanaxadiol (11.3 mg) and 20(R)-protopanaxadiol (10.8 mg) were isolated by semi-prep HPLC with a MeOH/H2O .Meanwhile, the methanol fraction which was kept for sapogenin production was subjected to the alkaline hydrolysis with the slight modification of the previous reports (Im R)-ginsenoside Rh2 with the highest amount, reaction conditions were optimized as followed. Saponins rich fraction mentioned above was dissolved in D.W. to make 10% solution (w/v), to which viscozyme®, one of β-glucosidase, was added and subjected to reaction in a shaking incubator at 50 °C for 5 days. After incubation, resultant solution was autoclaved two times at 120 °C for 2 h each. The solution was subjected to LC/MS analysis for content evaluations  was added to the cells, followed by incubation for 2 h. The culture plate was gently shaken for 1 min before absorbance was read at 450 nm using a microplate reader.5 cells/well. CPP531 was added 24 h later. The cells were treated with 10 µM BrdU 24 h before staining, After BrdU incubation, the media was discarded, the cells fixed by incubation with 10% formaldehyde for 15 min, and then permeabilized with 0.5 % Triton X-100 for 10 min. DNA was denatured by treatment with 2 N HCl for 10 min at 37 °C, followed by neutralization with 0.1 M boric acid pH 8.5 for 5 min at room temperature. Blocking was carried out using 5% bovine serum albumin (BSA) in PBST (0.2% TWEEN 20) for 1 h at RT. Cells were incubated with 1:500 BrdU antibody in 1% BSA diluted in PBST for 1 h at 37 °C, followed by washing with 1X PBS. Cells were then incubated with 1:1000 488 Alexa dye-conjugated anti mouse IgG (Thermo Fisher Scientific) in 1% BSA diluted in PBST for 1 hour at 37 °C, followed by washing with 1X PBS. The stained cells were mounted with Fluoromount™ solution including DAPI (Sigma-Aldrich)). BrdU staining was detected using an inverted fluorescence microscope .Cells were seeded in 6 well plates at a density of 1.5 × 10Cells were lysed with the CelLytic™ reagent (Sigma-Aldrich) and protein was quantified using the Bradford reagent . 50 µg protein samples were loaded onto 10% polyacrylamide gels, and transferred to nitrocellulose membranes after electrophoresis. Expression signals were detected with an ECL solution . Band intensity was quantified with ImageJ 1.45 s software .5 cells/well for compound treatment. RNA was isolated using the TRI-Solution™ (Thermo Fisher Scientific), following the manufacturer’s instructions. RT-PCR was performed using the AccuPower® RT PreMix . 1 µg of mRNA was used for the template. The primer sequences used were as follows: p27 forward 5′-GAG TCA GCG CAA GTG GAA TTT C-3′ reverse 5′-GCG AAG AAG AAT CTT CTG CAG C-3′ and p57 forward 5′-GCC GGT CGA GGA GCA GAA TG-3′ reverse 5′-CCT GGA GGG ACG TCG TTC GA-3′ and GAPDH forward 5′-TGA TGA CAT CAA GAA GGT GAA G-3′ reverse 5′-TCC TTG GAG GCC ATG TAG GCC AT-3′.Cell were seeded in 6 well culture plates at a density of 1.5 × 104 cells/well. 24 h later, myoblasts were treated with CPP531 for 72 h. Myoblasts were then harvested using trypsin-EDTA and stained with 2 μg/mL propidium iodide for 40 min. Flow cytometry was carried out using phycoerythrin (575 nm) detection in a BD FACSCanto II flow cytometer .C2C12 myoblasts were seeded in 60 mm × 15 mm culture dishes at a density of 2 × 1068. Embryos from mated Tg(cmlc2:GFP) transgenic zebrafish were grown in distilled water supplemented with 0.06 g/L sea salt. At 20 hours post fertilization (hpf), embryos were treated with 5 µg/mL CPP531 or vehicle until 40 hpf. After treatment, the compound solution was exchanged to egg water and the zebrafish were maintained until 72 hpf. For staining with 5-ethynyl-2′-deoxyuridine (EdU), zebrafish were treated with 10 mM EdU with 1% DMSO in 1X E3 buffer for 1 h at 28.5 °C, using a fish incubator. The fish were euthanized and fixed in 4% paraformaldehyde for 2 h. Fish larvae were then washed 3 times with PBDT buffer , followed by dehydration in 100% methanol for 1 h at −20 °C. Larvae were then sequentially rehydrated in a graded methanol series: 75%, 50%, 25% for 20 min each, with washing in PBDT for 20 min. The larvae were then digested with 10 µg/mL proteinase K or 20 min. After three further washes with PBDT, larvae were re-fixed in 4% paraformaldehyde for 20 min, followed by washing with PBDT and incubation with 12. 2 N HCl for 1 h at RT, 3 washes with PBDT, and incubation with 10% normal goat serum in PBDT for 1 h. EdU staining was carried out using a EdU imaging kit . Larvae were then incubated with anti-EdU IgG in blocking serum overnight at 4 °C, washed three times with PBDT and incubated in goat anti-mouse IgG conjugated to Alexa-fluor 594 for 5 h in the dark. After washing with PBDT, the stained larvae were mounted and imaged using confocal microscopy .Wild type zebrafish were purchased from Lotte Mart, Gwangju, Korea, and maintained following standard protocolsad libitum before surgery preparation and during drug treatment. Surgeries were performed under general anesthesia induced by intraperitoneal injection of 70 mg/kg ketamine  and 15 mg/kg xylazine  dissolved in saline. CPP531 was dissolved in saline containing 20% PEG400 and 0.2% TWEEN 80. PEG400 is a low-molecular-weight grade of polyethylene glycol that possesses low toxicity and TWEEN 80 is an excipient that is used to stabilize aqueous formulations of drugs for injection into recipient animals. Animals were treated with 2 mg/kg CPP531 delivered by intraperitoneal (IP) injection every 24 h. The untreated control animals were treated with IP injection of vehicle alone. The injection volume was 1 mL for the rats and 100 µL for the mice.Animal studies were carried out in accordance with guidelines established by the Animal Care and Ethics Committees of the Gwangju Institute of Science and Technology. The studies were approved by the Gwangju Institute of Science and Technology Animal Care and Use Committee . Adult male 8-week-old Sprague Dawley rats (weight range 230–250 g) and 8-week-old male C57BL/6 mice (weight range 23–25 g) were used for the myocardial infarction model and muscle degeneration model, respectively . Animals were housed at a constant temperature of 28 °C with a 12 h/12 h light/dark cycle. Food and water were available 69. A 4-gauge polyethylene catheter was used for intubation and ventilated with room air using a rodent ventilator . The coronary artery was ligated with a 5-0 silk suture and 4-0 sutures were used to close the chest. The rats were then placed on a warm pad in the animal facility recovery room. CPP531 treatment commenced 24 h after surgery and animals were sacrificed after 7d.Myocardial infarction (MI) was induced in the rats by ligation of the anterior descending coronary artery, as previously describedCardiac functional indices were obtained using echocardiography . Before taking echocardiographs, anaesthetized rats were left undisturbed for 5 min to stabilize the heartbeat. The following cardiac parameters were obtained: IVSTD , LVIDd , LVIDs , EF (ejection fraction), FS  and ESV (end systolic volume).28. The right gastrocnemius muscle of anaesthetized mice was injected with 1.5% barium chloride dissolved in 50 µL saline using a 30-gauge syringe. Mice were transferred to a warm pad until they regained consciousness. After 7d CPP531 or vehicle treatment, the forced swimming capacity test was used to assess muscle function, as previously described70, with the following modifications: a glass beaker was used in place of an acrylic tank and a steel washer was not require to increase exercise burden. Animals were then sacrificed and the gastrocnemius muscle harvested. In a separate experiment, mice were treated with CPP531 or vehicle alone for 14 d, after which the animals were sacrificed and the gastrocnemius muscle harvested.Intramuscular injection of barium chloride was used to induce muscle fiber degeneration, as previously described71. Microscopic images were obtained at 25× magnification and the thickness of the left ventricle was measured using Image J 1.48 v software .Dissected hearts were sliced horizontally in half, embedded in the OCT compound (Leica), and frozen at −80 °C. 1 d after freezing, cryosections were taken at a 7 µm thickness (Leica CM1850). Masson’s trichrome staining was performed using the previously published protocol72. In brief, the sections were immersed in acetone for 15 min, washed twice with PBS, incubated in 0.1% triton-X solution for 10 min and washed twice with PBS. Sections were then placed in 1 X citrate buffer and boiled for 40 sec in a microwave oven. Blocking was performed by incubation with 5% BSA  and 5% normal goat serum  overnight at 4 °C. Sections were then incubated with primary antibody  and Ki-67  diluted at 1:100 with 0.05% triton-X, overnight at 4 °C. Sections were then incubated with secondary antibody solution ) and goat anti-mouse IgG-heavy and light chain antibody DyLight® 594 conjugated (Bethyl lab A 90-116D4) diluted 1:200 in 1X PBS) at 37 °C for 1 h. Sections were then treated for 30 min with sudan black B solution, immersed twice in a 1X MHB solution for 15 min, and stained with DAPI for 3 min. Fluorescence images were acquired at random by fluorescence microscopy (Leica DMI 3000B).To quantify proliferating cardiomyocytes in the heart tissues, immunofluorescence was performed as previously describedDissected gastrocnemius muscles were frozen in isopentane pre-cooled by insertion into liquid nitrogen, embedded in OCT compound and stored at −80 °C. 1 d after freezing, 7 µm cryosections were taken and the sections stained with Masson’s trichrome or hematoxylin and eosin using a kit (Sigma-Aldrich). Microscopic images were obtained at 25x magnification to assess tissue degeneration and repair (Leica DM 2500). The ratio of damaged area to whole muscle tissue was obtained by analyzing Masson’s trichrome stained muscles using Image J 1.48 v software. The degenerated area was designated as purple staining and the non-damaged area was designated as red staining. To calculate the inflammatory score, 5 micrographs were taken at random from H&E stained sections of each treated muscle at 100x magnification (Leica DM 2500). Inflammatory cells were counted as lightly-stained cells with dark purple nuclei, which had infiltrated the muscle tissue between the darker red-stained muscle fibers. To analyze muscle fiber diameter, 5 images of the H&E stained muscles were selected at random using 200x magnification. Entire muscle fiber diameters present within the micrograph were measured with the Image J 1.48 v software.t-test was used to determine statistical significance in Figs. 4p value < 0.05 was considered to be significant. Unless otherwise stated, all cell-based data are generated from three independent experimental repeats and the error bars for the graphs are standard deviation.The Student’s Supplementary information.