Dexamethasone (Dex), an artificially synthetic cortisol substitute, is commonly used as an anti-inflammatory drug, and is also employed to mimic the stress state experimentally. It is well known that chronic stress disturbs the gut microbiota community and digestive functions. However, no relevant studies have been conducted in ruminants.n = 5) was consecutively injected intramuscularly for 21 days to simulate chronic stress in growing goats. Goats were injected with saline (0.2 mg/kg body weight) as the control group . Dex-treated goats showed a higher number of white blood cells and blood glucose levels (p < 0.01), but lower dry matter intake (DMI) and body weight (p < 0.01) than those of saline-injected goats. Plasma cortisol concentration decreased significantly in response to the Dex injection compared to the control (p < 0.05). The Dex treatment did not change most ruminal volatile fatty acid (VFAs) concentrations before the morning feeding after 1–21 days of treatment (p > 0.05); however, ruminal VFA concentrations decreased dramatically 2, 4, 6, and 8 h after the morning feeding on day 21 of the Dex injections. In this study, chronic Dex exposure did not alter the community structure of microbes or methanogenes in the rumen, caecum, or colonic digesta. Only Prevotella increased on days 7 and 14 of Dex treatment, but decreased on day 21, and Methanosphaera was the only genus of methanogene that decreased.In this study, a low dosage of Dex  contains supplementary material, which is available to authorized users. Cortisol is a stress hormone synthesized and released by the pituitary gland under stress conditions . Stress Microbiota play an important role in maintaining gut health and body homeostasis. Microbiota affect mucosal defense and inhibit the ability of enteric pathogens to colonize. Bäckhed et al. and Ley et al. reported that intestinal microbiota are associated with obesity and diabetes in germ-free mice , 13. ManCoprococcus, Pseudobutyrivibrio, and Dorea populations  and pentobarbital . After death, the hind gut mucosal tissues were carefully removed. The content of caecum and colon were collected (approx. 50 mL). 2 mL of the colonic and caecal content were stored at liquid nitrogen were used for DNA extraction. The remaining of colonic and caecal samples were centrifuged at 12000×g for 15 min at 4 °C, supernatant was collected. About 1 mL of freshly prepared 25% metaphosphoric acid was added to 4 mL of supernatant from caecum and colon. Then samples were centrifuged , and the supernatant fluid was stored at − 20 °C prior to the determination of VFAs.Plasma glucose was measured using an automatic biochemical analyzer . Cortisol was detected by using RIA cortisol kit , strictly following the manufacturer’s instructions.The rumen fluid samples were taken out of − 20 °C refrigerator. Then defreeze samples in room temperature. Berthelot (phenol-hypochlorite) reaction was used to determine NH3-N concentration . The disThe methods of DNA extraction and 16S rRNA gene amplicon pyrosequencing were described previously . 2 mL flRaw tags were got by the FLASH (version 1.2.7). Further, high quality clean tags were obtained through strict filtering processing by the Quantitative insights into microbial ecology (Qiime) (version 1.7.0). Effective tags were clustered to the Operational Taxonomic Units (OTUs) by Uparse (version 7.0.1001). The RDP classifier (version 2.2) and GreenGene database were used to species annotation. The unweighted principal component analysis (PCoA) analysis was made by the R (version 2.15.3). Linear discriminant analysis effect size (LEfSe) was used to detect significant changes in relative abundance of microbial taxa (LDA > 2).p < 0.05. The numbers of replicates used for statistics were noted in the figures.Data are presented as means ± SE. The data were tested for normal distribution and analyzed by Student’s unpaired t test using SPSS software packages . Data were considered statistically significant when Additional file 1:Figure S1. Dynamic alteration of VFAs in rumen fluid from goat treated with Dex or Con, at 1, 7, 14, 21 day, after morning feeding. A. Day 1. B. Day 7. C. Day 14. D. Day 21. Means ± SE are plotted; #p < 0.10, *p < 0.05, **p < 0.01 versus Con group. (TIF 12562 kb)Additional file 2:Figure S2. Dynamic alteration of VFAs in feces from goat treated with Dex or Con, at 1, 7, 14, 21 day, after morning feeding. A. Day 1. B. Day 7. C. Day 14. D. Day 21. Means ± SE are plotted; #p < 0.10, *p < 0.05, **p < 0.01 versus Con group. (TIF 424 kb)Additional file 3:Figure S3. Dynamic alteration of urea in rumen from goat treated with Dex or Con, at 1, 7, 14, 21 day, after morning feeding. A. Day 1. B. Day 7. C. Day 14. D. Day 21. Means ± SE are plotted; #p < 0.10, *p < 0.05, **p < 0.01 versus Con group. (TIF 10055 kb)Additional file 4:Figure S4. Dynamic alteration of ammonia in rumen from goat treated with Dex or Con, at 1, 7, 14, 21 day, after morning feeding. A. Day 1. B. Day 7. C. Day 14. D. Day 21. Means ± SE are plotted; #p < 0.10, *p < 0.05, **p < 0.01 versus Con group. (TIF 9728 kb)Additional file 5:Figure S5. Altering compositions of microbial phylum , in rumen, caecum, and colon from goat treated with Dex or Con. A. Day 1 in rumen. B. Day 7 in rumen. C. Day 14 in rumen. D. Day 21 in rumen. E. Content of caecum. F. Content of colon. (TIF 8329 kb)Additional file 6:Figure S6. Altering compositions of methanogenic class , in rumen, caecum, and colon from goat treated with Dex or Con. A. Day 1 in rumen. B. Day 7 in rumen. C. Day 14 in rumen. D. Day 21 in rumen. E. Content of caecum. F. Content of colon. (TIF 13224 kb)


T. wallichiana var. mairei due to the effects of growth meteorological parameters were investigated in this study. The needles of T. wallichiana var. mairei were collected from different months and the contents of taxoids , baccatin III, cephalomannine, 10-deacetyltaxol (10-DAT)), flavones  and polysaccharides were quantified by ultra performance liquid chromatography (UPLC) and the resonance light scattering (RIL) method. The content of taxoids gave the highest level of 1.77 ± 0.38 mg·g−1 in January, and the lowest value of 0.61 ± 0.08 mg·g−1 in September. Unlike taxoids, the content of flavonoids was the highest in August. The content of polysaccharides reached peak value of 28.52 ± 0.57 mg·g−1 in September, which was two times higher than the lowest content of 9.39 ± 0.17 mg·g−1 in January. The contents of paclitaxel, 10-DAB, 10-DAT and polysaccharides significantly depended on meteorological parameters. The mean of minimum temperature (R = −0.61) and length of daylight (R = −0.60) were significantly correlated to 10-DAB content, while 10-DAT level showed significant correlation with length of daylight (R = −0.70) and relative humidity (R = 0.70). In addition, temperature had significantly negative effect on the content of paclitaxel and a significantly positive effect on that of polysaccharides. This study enriched the knowledge on the accumulation pattern of metabolites and could help us to determine the collecting time of T. wallichiana var. mairei for medicinal use.Seasonal variations of the phytochemicals contents in needles of  Taxus wallichiana var. mairei, also known as the Chinese yew belonging to the Taxaceae family, is a protected, valuable and natural anti-cancer plant endemic to China . Li. Li−1 any = 9.04x − 0.03) [−1 and a temperature of 30 °C. The elution program was set as follows: 0–5 min, 20% A; 5–6 min, 20%–30% A; 6–10 min, 30% A; 10–12 min, 30%–40% A; 12–15 min, 40%–60% A; 15–18 min, 85% A; 18–20 min, 100% A; and 20–25 min, 5% A. Detection was conducted at a wavelength of 360 nm (n = 6) in the range of 0.001–0.05, 0.002–0.10 and 0.002–0.20 mg·mL−1 for amentoflavone, quercetin and ginkgetin, respectively, were prepared in methanol, giving linear calibration curves y = 40x − 0.01, R2 = 0.99; y = 80x − 0.11, R2 = 0.99 and y = 40x − 0.02, R2 = 0.99 for them, respectively. The yields of amentoflavone, quercetin and ginkgetin were quantitatively determined by the calibration curves, respectively.The total flavonoids content was determined using a modified colorimetric method, calculated by a rutin standard curve ( − 0.03) . Chromatf 360 nm . A serieIRLS = IRLS − I0RLS, where IRLS and I0RLS were the intensities of the systems with and without polysaccharides [For the experiment, 1 mL of cetylpyridinium chloride (CPC) (1 mM), 0.50 mL NaOH (1 M) and 2 mL polysaccharides were added into a tube and mixed rapidly. Then, the mixed solution was removed from the test tube to a cuvette for RLS. The RLS spectra were obtained by scanning simultaneously excitation and emission monochromators of the spectrofluorometer from 300 to 800 nm. The enhanced RLS intensity of CPC was represented as △charides .p < 0.05. The results of ANOVA showed the significant differences at the 0.05 significance level for multiple comparisons among the different months. Correlation between the content of detected compounds and climatic parameters were determined using Pearson’s correlation analysis.All resulting data were presented as the mean ± standard error of three replications. SPSS version 16.0  was used for statistical analysis. ANOVA was conducted to compare the compound content every month. A correlation was considered to be significant when −1) was found in January. On the contrary, the maximum content of total flavonoids obtained in the spectrophotometry (15.82 ± 1.45 mg·g−1) was observed in August. The polysaccharide accumulation reached a value of 28.52 ± 0.57 mg·g−1 in September. Moreover, the mean of the minimum temperature and length of daylight were significantly negatively correlated to 10-DAB content, while 10-DAT level showed significant correlation with length of daylight (R = −0.70) and relative humidity (R = 0.70). Temperature had a significantly negative effect on paclitaxel content and a significantly positive effect on polysaccharides.The quantitative analysis of UPLC showed that the highest content of taxoids (1.77 ± 0.38 mg·g


Nigella sativa, as a potential anticancer agent. However, no extensive studies on α-HN have been done as yet, as it was in the case of thymoquinone—the main ingredient of the N.sativa essential oil. To our knowledge, there are also no data available on how α-HN acts on the human cancer ovarian cell line SKOV-3. In this study we attempt to present the cytotoxic influence of α-HN on the SKOV-3 cell line by means of two methods: Real-Time xCELLigence and 3--2,5-diphenyltetrazolium bromide (MTT) assay. The obtained IC50 values are 2.62 ± 0.04 μg/mL and 2.48 ± 0.32 μg/mL, respectively. An induction of apoptosis in SKOV-3 cells was confirmed by staining cellular nuclei with Hoechst 33342 dye and by flow cytometry analysis by binding annexin V to the cell membranes. We found that α-HN induces apoptosis in a dose-dependent manner. In the first stages of apoptosis, the mitochondrial membrane potential was found to decrease. Also, inactivation of anti-apoptotic protein Bcl-2 was observed, as well as the caspase-9 and then caspase-3/7 activation. In addition, the treatment of SKOV-3 cells with α-HN induced the cell cycle arrest of cancer cells in G0/G1 phase. The results of our investigations indicate that α-HN induces apoptosis in the SKOV-3 cell line and that the intrinsic mitochondrial pathway is involved in the programmed cancer cell death.Alpha-hederin (α-HN), a pentacyclic triterpene saponin, has recently been identified as one of the active compounds of  It is also known as -3-[[2-O-(6-deoxy-α-l-mannopyranosyl)-α-l-arabinopyranosyl]oxy]-23-hydroxyolean-12-en-28-oic acid (Alpha-hederin (α-HN) -α-l-arabinopyranoside [As far as an anticancer property of α-HN is concerned, not much research has been done in that regard, and as such, the mechanism of its cytotoxic activity is yet to be established. Lorent et al. claim that α-HN triggers membrane pore formation and it then enters into the cell. presumably through a sterol-dependent pathway ,14. It hrophages . Also, αrophages . Besidesrophages , the genrophages . In laryrophages . α-HN isrophages ,19. Withranoside ,21. Furtranoside . Not only does α-HN possess the potential for numerous biological activities in vitro, but also it is an efficacious agent in many studies in vivo; for instance, in the treatment of asthma in a rat ovalbumin (OVA)-sensitized model of asthma. The results have shown that in a group of rats pretreated with α-HN the IL-17 mRNA levels were decreased and miRNA-133a gene expression was increased in comparison to the OVA-sensitized group of rats .Despite a few studies available on α-HN as an anti-cancer agent on various cell lines, no data can be found about its impact on human ovarian cancer cell line SKOV-3. SKOV-3 (or interchangeably SK-OV-3) lines were first derived from ovarian cancer cells in 1973 from a 64-year-old woman who showed high resistance to numerous cytotoxic agents, including TNF, adriamycin, and cisplatin ,25. AddiThis study investigates the cytotoxic effect of α-HN on the SKOV-3 cell line and examines the type of cell death and mechanism of its action on the cells.N. sativa seeds, on the viability of the cells, was tested by the Real-Time xCELLigence system that works based on electronic impedance that is measured in E-plate wells equipped with sensor electrodes. It allows monitoring the cells continuously and quantitatively [50 value is calculated on the basis of CI at every measuring point of the experiment.The effect of α-HN, the active triterpenoid saponin of tatively . Any alttatively ,29. The 50 value of α-HN by using the RTCA system was 2.62 ± 0.04 µg/mL, based on the sigmoidal dose-response formula (SKOV-3 cells were treated with α-HN (1–70 µg/mL) for 24 h . The obt formula .50 value was 2.48 ± 0.32 µg/mL (v/v)) did not inhibit the cell growth.The MTT assay was performed with α-HN at the concentration range 0.5–50 µg/mL, for 24 h, to confirm the RTCA results of the cytotoxic activity of α-HN on SKOV-3 cells. The obtained IC32 µg/mL . A contrAdditionally, we prepared the same experiments using the non-tumor HaCaT cell line. The results collected from RTCA and MTT assays were 2.71 ± 0.35 and 2.57 ± 0.21 µg/mL, respectively .The obtained results indicate that SKOV-3 and HaCaT cells were sensitive to α-HN and the viability was significantly decreased in both the lines after treatment with the compound. This effect was time and dose-dependent.The changes in chromatin distribution in SKOV-3 cells after treating with α-HN were evaluated by staining the cellular nuclei with Hoechst 33342 dye. The nuclear fragmentation and chromatin condensation were visible in SKOV-3 cells after staining, contrary to the control sample with DMSO .The induction of apoptosis in the SKOV-3 cells was confirmed by a staining test (after cells’ incubation with 0.5–30 µg/mL of α-HN for 24 h) with annexin V/7-AAD and then examined by Muse Cell Aanalyzer. An increase in a dose-dependent manner in the percentage of apoptotic cells was observed. The total percentages, as mean values of three independent experiments, of apoptotic cells (early and late apoptotic) were 15.55% ± 6.51%, 18.50% ± 2.04%, 36.1% ± 0.21%, 46.13% ± 2.09%, 45.23% ± 3.15%, and 52.63% ± 6.12% with α-HN concentrations of 0.5, 2, 10, 13, 17, and 30 µg/mL, respectively, in comparison to the untreated SKOV-3 cells and the cells treated with DMSO: 10.42% ± 2.26%, and 13.6% ± 0.92%, respectively .Mitochondrial loss of inner transmembrane potential, which demonstrates mitochondrial dysfunction, is one of the hallmarks of the early stages of apoptosis. The condition of mitochondrial membranes of SKOV-3 cells after treatment with α-HN was evaluated by flow cytometry. The inner membranes of intact mitochondria accumulated fluorescent dye, which was visible in the control samples. The cells incubated with the increasing concentrations of α-HN depicted declining fluorescence. The mean values of depolarized/live cells after treatment with α-HN were 2.24% ± 0.73%, 2.35% ± 0.74%, 3.11% ± 0.40%, 3.37% ± 0.93%, 41.15% ± 2.57%, 43.51% ± 2.25%, 51.25% ± 2.84%, and 96.79% ± 1.83% for concentrations of 0.5, 2, 5, 10, 13, 17, 20, and 30 μg/mL, respectively . The acqThe total level of Bcl-2, an anti-apoptotic family member, expression was measured according to the Muse Activation Dual Detection Kit, which utilizes a pair of antibodies: One antibody enables the detection of total protein expression and the other allows the detection of the phosphorylated form (activated) of the same target, which gives an overview of whole Bcl-2 activation pathway measured simultaneously in one cell.SKOV-3 cells were exposed to the increasing concentrations of α-HN (0.5–30 µg/mL) for 24 h to trigger cell death and to inhibit the response of the Bcl-2 signaling cascade. The flow cytometry data depict the relative values (in percentage) for each population of cells: Activated, inactivated, and non-expressing Bcl-2. In our study, a significant dose-dependent inactivation of the anti-apoptotic protein was observed, contrary to the control samples with almost 100% of Bcl-2 activation rate. The percentages of Bcl-2 inactivation measured were 61.87% ± 3.35%, 92.65% ± 8.56%, 89.3% ± 11.88%, 86.37% ± 10.26%, 98.03% ± 0.7%, 99.07% ± 0.51%, 98.8% ± 0.84%, and 99.07% ± 0.4% with α-HN concentrations of 0.5, 2, 5, 10, 13, 17, 20, and 30.0 µg/mL, respectively .In order to estimate the activity of caspase-8 and caspase-9, the SKOV-3 cells were treated with 0.5–30 µg/mL of α-HN for 2, 5, and 24 h. In the case of caspase-8 in comparison to the control (DMSO-treated cells), no significant activation was observed. The activity of caspase-8 for the concentrations 0.5, 2, and 5 µg/mL of α-HN was similar to the control ; howeverAs far as caspase-9 is concerned, there was a significant increase in its activation after 5 h of SKOV-3 cells’ treatment with α-HN for the concentrations of 0.5, 2, and 5 µg/mL. The activity of caspase-9 was 1.56, 1.58, and 1.71-fold higher, respectively, in comparison to the DMSO control . SimilarIn order to estimate the activity of caspase-3/7, SKOV-3 cells were treated with increasing concentrations of α-HN for 24 h and then stained with the Muse Caspase-3/7 Kit and analyzed with the Muse Cell Analyzer. The percentages of the cells in different stages of apoptosis were determined based on the activation of caspase-3/7. Four types of cell populations were registered: Live, early apoptotic, late apoptotic, and dead. The total percentages, as mean values of three independent experiments, of apoptotic cells (early and late apoptotic) were 7.93% ± 1.20%, 9.3% ± 0.92%, 16.7% ± 2.41%, 16.0% ± 3.44%, 15.23% ± 1.14%, 16.27% ± 1.93%, 30.12% ± 0.95%, and 48.42% ± 0.95% with α-HN concentrations of 0.5, 2, 5, 10, 13, 17, 20, 30 µg/mL, respectively, in comparison to the untreated SKOV-3 cells and the cells treated with DMSO: 17.13% ± 0.46% and 10.72% ± 1.03%, respectively .The data acquired from cell cycle analysis by the Muse Cell Analyzer depict a crucial dose-dependent increase of SKOV-3 cells in the G0/G1 population, as a result of arresting the cell cycle in this phase after 24 h exposure to the increasing concentrations of α-HN.After 24 h treatment, SKOV-3 cells indicated percentages  of G0/G1 populations: 57.83% ± 4.75%, 56.35% ± 5.94%, 50.35% ± 0.07%, 61.86% ± 8.62%, 63.75% ± 0.35%, 83.5% ± 2.26%, 83% ± 1.13%, 82.65% ± 2.19% for the concentrations of α-HN: 0.5, 2, 5, 10, 13, 17, 20, 30 µg/mL, respectively; compared to the control of untreated cells 55.98% ± 2.24% and the cells treated with DMSO 56.05% ± 2.17% . A signiNigella sativa seeds have been used since ancient times to cure many diseases, as there were believed to have stimulatory, choleretic, cholagogue, carminative, diuretic, and diaphoretic properties. In folk medicine, seeds were meant to be the treatment for and prevention option for dyslipidemia, asthma, and eczema [d eczema . The seed eczema ,36,37,38N. sativa seeds, α-HN (a triterpenoid with hederagenin aglycone), especially as far as anticancer properties are concerned and the mechanisms of that activity. Apart from its scientifically researched anti-inflammatory, antioxidant, antiarthritic, antiparasitic, antifungal, bronchiolytic, and hepatoprotective potential [However, limited attention has been paid to the active saponin of otential , it was otential ,19.To our knowledge, not much information is available about the influence of α-HN on the SKOV-3 ovarian cell line  and alsoPulsatilla koreana roots [The SKOV-3 cell line was tested for cytotoxicity with triterpenoid saponins, but not those with hederagenin aglycone. The lupane and oleane-type saponins were isolated from na roots ,40 and tna roots .In this study, we examined the impact of α-HN on the ovarian cell line, SKOV-3, and estimated the mechanism of its anticancer activity. Our results show that this triterpenoid saponin exhibits crucial cytotoxic activity against SKOV-3 cells. The proliferation of the tested cells assessed through the Real-Time xCELLigence system and MTT assays was strongly inhibited in a dose and time-dependent manner.Our investigations also indicate that the type of cell death induced by α-HN in SKOV-3 cells is apoptosis. This programmed cell death is distinguished by specific biochemical and morphological hallmarks, such as cell shrinkage, formation of condensed nuclei followed by nuclear fragmentation and membrane blebbing . In our Additionally, an early apoptosis hallmark is the loss of plasma membrane asymmetry with the cell membrane phosphatidylserine translocation from the inner to the outer surface. As a result, a dependent phospholipid-binding protein, called annexin V, can bind to the phosphatidylserine ,44,45. OAs far as the main pathways of apoptosis are concerned, there are the extrinsic, via the death receptor pathway, and the intrinsic, via the mitochondrial pathway ,47,48. IThe ability of the Bcl-2 protein to inhibit the apoptosis is regulated by the phosphorylation process. Its phosphorylated form has an anti-apoptotic function and dephosphorylation is required for pro-apoptotic activity ,54. ThisIn our study, a crucial dose-dependent depolarization of the mitochondrial membrane in the SKOV-3 cell line was detected in a few hours after treating the cells with the saponin, together with inactivation of the anti-apoptotic Bcl-2 protein. Moreover, a significant increase in the caspase-9 activity was observed and this led to caspase-3/7 activation in the cells, which confirms the initiation of the intrinsic mitochondrial pathway in SKOV-3 cell line death. Simultaneously, no activation of caspase-8, involved mainly in the extrinsic apoptosis pathway, was observed.In the research performed by Sun J. et al., α-HN also showed strong anticancer-effect on colorectal cancer cells (HCT116 and HCT8) and induced apoptosis via ROS-activated (reactive oxygen species) mitochondrial signaling pathway and autophagic cell death through ROS dependent AMPK/mTOR signaling pathway activation . MoreoveκB and ERK pathways [In this study, the cell cycle arrest in SKOV-3 cells was also observed in G0/G1 phase after treatment for 24 h with increasing concentrations of α-HN. In another study conducted by Sun D. et al., α-HN inhibited the cell cycle in G2/M phase and induced apoptosis in colon cancer cells (SW620) via mitochondrial and caspase-dependent pathway together with disruption of NF-pathways .To sum up, our investigations proved that α-HN induces apoptosis in the SKOV-3 cell line and that the intrinsic mitochondrial pathway plays a significant role in this process. However, further investigation into the programmed death of ovarian cancer cell line SKOV-3 is required, since no more related data can be found.McCoy’s 5A Medium DMEM (Dulbecco’s Modified Eagle’s Medium), streptomycin, penicillin, PBS , DMSO (dimethyl sulfoxide), MTT -2,5-diphenyltetrazolium bromide) and α-HN were purchased from Sigma-Aldrich . α-HN was dissolved in DMSO (99.9%) to the final concentration of 10 mg/mL.The ovarian cancer cell line (SKOV-3) and the human keratinocytes (HaCaT) were obtained from the American Type Culture Collection .v/v) FBS , 100 units/mL of penicillin, and 100 µg/mL of streptomycin. The human keratinocytes (HaCaT) were cultured in DMEM medium supplemented with 10% (v/v) FBS, 100 units/mL of penicillin, 100 µg/mL of streptomycin and 2 mM L-glutamine. All the cultures were kept in a humidified 5% CO2 incubator at 37 °C.The SKOV-3 cells were cultured in McCoy’s medium supplemented with 10% . DMSO concentration in the control did not exceed 0.7% (v/v). The cells were then incubated with α-HN and monitored for 24 h at 37 °C in a 5% CO2 atmosphere. The Real-Time xCELLigence system  is devoted to monitoring cytotoxicity and is based on electronic sensor array technology. All the cells were seeded into E-plate 16 (each well with 100 µL medium) at a density of 2 × 1050 values were taken from the measured cell-electrode impedance that is directly related to the viability and number of cells.The cell index (CI) and IC3 cells/well and then treated for 24 h with α-HN in the concentration range 0.5–50 µg/mL. DMSO was added to the control cells at a concentration of 0.5% (v/v). Next, the medium with α-HN was discarded and a fresh medium with MTT solution (0.5 mg/mL) was added to the cells and incubated for 3 h at 37 °C. Formazan solution’s optical density was measured at 570 nm by Epoch , a plate reader. The results of the three independent experiments  are expressed as IC50 mean values (±SD).The viability of the cells was also determined by means of MTT assay. The cells were seeded in 96-well plates at a density of 2 × 105 cells/well in 6-well plates and then treated with α-HN dissolved in DMSO at a concentration of 0.5, 2, and 10 µg/mL. DMSO concentration did not exceed 0.1% (v/v). Then, 24 h later, SKOV-3 cells were stained with 0.5 µg/mL of the Hoechst dye (dissolved in PBS) for 25 min in a CO2 incubator and observed under a fluorescent microscope .The blue fluorescent Hoechst 33342 dye  was used to analyze the effect of α-HN on nuclei in SKOV-3 cells. The cells were seeded at a density of 5 × 105 cells/well were treated with α-HN at a concentration of 0.5–30 µg/mL for 24 h. The DMSO concentration (control sample) did not exceed 0.3% (v/v). SKOV-3 cells were first harvested, then stained following the Muse Annexin V and Dead Cell Assay Kit , and analyzed using Muse Cell Analyzer (Merck Millipore). The tests were repeated at least three times independently.A test involving the binding of annexin V to cellular phosphatidylserine was performed to assess the induction of apoptosis. The SKOV-3 cells with a density of 1 × 105 cells/well and then treated with α-HN at a concentration of 0.5–30 µg/mL for 3 h. Such a period of incubation time was selected in order to depict the early stage of mitochondrial dysfunction of the SKOV-3 cells. The DMSO concentration (control sample) did not exceed 0.3% (v/v). The cells were harvested after 3 h of exposure and prepared, following the manufacturer’s protocol of Muse Mitopotential Kit (Merck Millipore). The percentage of depolarized/live cells was determined using Muse Cell Analyzer. The trials were repeated at least three times independently.The SKOV-3 cells were seeded at a density of 1 × 105 cells/well and then treated with α-HN at a concentration of 0.5–30 µg/mL for 24 h. DMSO (control sample) concentration did not exceed 0.3% (v/v). The cells were then collected, washed with PBS, and, following the manufacturer’s protocol, the cocktail of specific antibodies was added (a phospho-specific anti-phospho-Bcl-2 (Ser70)-Alexa Fluor 555 and an anti-Bcl-2-PECy5 conjugated antibodies) from the Muse Activation Dual Detection Kit (Merck Millipore). The cells were incubated at room temperature for 30 min in the dark and then the total level of Bcl-2 expression and activated form of this protein was measured using Muse Cell Analyzer. The experiments were repeated minimum three times independently.The SKOV-3 cells were seeded at a density of 5 × 104 cells/well) to α-HN at concentration values of 0.5, 2, 5, 10, 13, 17, 20 and 30 µg/mL. The final concentration of DMSO did not exceed 0.3% (v/v). The cells were then treated with Caspase-Glo 8 or 9 Assay Kits  according to the manufacturer’s protocol. The activity of the caspases was analyzed by Glomax Multi+ Detection System (Promega). The experiment was repeated three times, independently.The activity of caspase-8 and caspase-9 was measured after 2, 5, and 24 h of exposure of cells . After 24 h cells were harvested, stained with Muse reagent, and incubated for 30 min in a CO2 incubator; thereafter they were stained with 7-AAD and incubated at room temperature in the dark for 5 min. In the end, SKOV-3 cells were analyzed by Muse Cell Analyzer. The experiment was repeated three times independently.The caspase-3 and caspase-7 activation was determined by Muse Caspase-3/7 Kit (Merck Millipore). The kit measures two significant parameters of cell health—apoptotic status based on caspase-3/7 activation and cell membrane integrity or cell death. The method allows determining simultaneously the percentage of live, total apoptotic (early and late apoptotic), and dead cells. The cells were seeded at a density of 1 × 105 cells/well in 6-well plates and then treated for 24 h with α-HN dissolved in DMSO at concentration values of 0.5, 2, 10, 20 and 30 µg/mL. DMSO concentration did not exceed 0.3% (v/v). SKOV-3 cells were harvested, stained with Muse Cell Cycle reagent following the manufacturer’s protocol, incubated for 30 min at room temperature, and analyzed by Muse Cell Analyzer. The experiments were repeated three times independently.Propidium iodide (PI), the nuclear DNA intercalating stain, was used to discriminate SKOV-3 cells at various stages of the cell cycle, following the manufacturer’s protocol of the Muse Cell Cycle Assay Kit (Merck Millipore). The cells were seeded at a density of 5 × 10


The results showed that the PM concentrations on the hazardous haze days and unhealthy haze days were 10.7 and 8.0 times higher, respectively, than those on the sunny days. The chemical composition of PM2.5 was highly correlated with the AQI. The concentration and percentage of water-soluble inorganic ions (WSII), which dominated the PM2.5 constituents, as well as the levels of endotoxin were higher on hazardous haze days than on unhealthy haze days and sunny days. Interestingly, the abundances of bacteria and fungi demonstrated the following order: unhealthy haze days> sunny days> hazardous haze days. Most culturable bacteria and fungi were distributed in particles with aerodynamic diameters of 2.1–4.7 μm. Redundancy analysis found total organic carbon explained 30.0% and 27.1% of total variations in bacterial composition and fungal composition at the genera level, respectively. Our results facilitate a better understanding of the biological and chemical composition dynamics of PM in Beijing.Particulate matter (PM), a major air pollutant in Beijing in recent years, poses a formidable public health threat. Even through many studies have documented the chemical and biological characteristics of PM, less is known about these characteristics on hazardous haze days  and the difference with sunny or unhealthy haze day (AQI 151–200) characteristics. Herein, studies were performed during a red alert air pollution event (continuous hazardous haze days) and the first few days following the event  in Beijing from December 19 to 25, 2016. A laser particle counter and an ANDERSEM-6 sampler were used to study the concentration and size distributions dynamics of the PM and the culturable airborne bacteria and fungi, respectively. PMThe online version of this article (10.1007/s11356-018-3355-6) contains supplementary material, which is available to authorized users. From the perspective of scientific research, this event provided a good opportunity to explore whether the heavily polluted air conditions could influence the survival of microorganisms and how the microbial or chemical components attached to the PM2.5 samples were analyzed. The results of this study may offer insight into the biological and chemical composition dynamics of PM on hazardous haze days, unhealthy haze days and sunny days.Here, we studied the concentration and size distribution dynamics of atmospheric PM and culturable airborne bacteria and fungi from December 19 to 25, 2016. Simultaneously, the chemical compositions, bacterial and fungal community structures, and endotoxin levels of PM2.5 sampling was performed in an open-space area without major pollution sources nearby, ∼ 2 m above the ground on the campus of the Beijing Institute of Technology . The PM2.5 samples were collected on 20.32 × 25.4-cm2 Tissuquartz filters  using a high-volume air sampler equipped with a PM2.5 fractionating inlet  at a flow rate of 1000 L/min. Five 23-h sample collections (8:00 AM–7:00 AM of the next day) were performed from December 20 to December 25  and recorded per hour.Meteorological data including AQI, temperature, relative humidity, and wind speed were collected from the China Weather website , very unhealthy (201–300), unhealthy (151–200), unhealthy for sensitive groups (101–150), moderate (51–100), and good (0–50). In this study, days with AQI values below 100 were categorized as sunny days, while haze days had an AQI value greater than or equal to 100.A laser particle counter  was used to monitor the number and size distribution of PM in the ambient air. The counter could measure up to six channels of simultaneous data, namely, 0.3–0.5 μm, 0.5–1 μm, 1–3 μm, 3–5 μm, 5–10 μm, and > 10 μm. Data were recorded every 5 mins. The sampling rate of the laser particle counter was 2.83 L/min, and the air sampling time was 10 s.An international standard ANDERSEN-6 sampler  was used to sample the culturable airborne bacteria and fungi at a flow rate of 28.3 L/min, with Columbia blood agar  and sand fort weak training bases  as the sampling media, respectively. The Columbia blood agar base was composed of special peptone (23 g/L), starch (1 g/L), sodium chloride (5 g/L), agar (10 g/L), and 5% sterile defibrinated blood. The sand fort weak training bases were composed of peptone (10 g/L), glucose (40 g/L), and agar (20 g/L). The sampling time was 35 min. The sampler was sufficiently disinfected with 75% ethyl alcohol after each sampling. The ANDERSEN-6 sampler had six stages defined by the aerodynamic diameters of the airborne particles, namely, stage 6 (0.65–1.1 μm), stage 5 (1.1–2.1 μm), stage 4 (2.1–3.3 μm), stage 3 (3.3–4.7 μm), stage 2 (4.7–7.0 μm), and stage 1 (> 7.0 μm).AC=CN/(Qs• Ts), where CN is the colony number in the collection plates (CFU), Qs is the sampler volumetric flow rate (L/min), and Ts is the sample collection duration (min).The airborne bacteria collection plates were cultured in 37 °C for 24–48 h, and the airborne fungi collection plates were cultured in 25 °C for 72 h; then, the bacteria and fungi colony numbers were counted on the sample dishes at each stage. The aerosol concentration (AC) of the culturable bacteria or fungi was calculated as: 3−, SO42−, Cl−, NH4+, Mg2+, Na+, Ca2+, K+, et al.) were analyzed by ion chromatography . The total organic carbon (TOC) concentrations were detected using a thermal-optical transmittance aerosol carbon analyzer (Sunset Laboratory Inc.)A quarter of each Tissuquartz filter sample and blank filter were cut into pieces, and the filter treating process was conducted as described in a previous study  and 806R (5′- GGACTACHVGGGTWTCTAAT-3′) were used; for fungal ITS, ITS1 (5’-TCCGTAGGTGAACCTGCGG-3′) and ITS1 (5’-GCTGCGTTCTTCATCGATGC-3′) were used. Q-RT-PCR assays were run on an Applied Biosystems® 7500 Real-Time PCR System .For bacterial and fungal structure analysis, 1/8 of each Tissuquartz filter sample was used for DNA extraction by the MO-BIO PowerSoil DNA isolation kit  according to the manufacturer’s protocols. Quantitative real-time polymerase chain reaction (Q-RT-PCR) was performed to quantify the relative abundance of bacteria and fungi among the five sampling filters. The primers were as follows: for bacterial For bacterial and fungal community structures, the V1–V3 regions of the bacterial 16S ribosomal DNA gene and the ITS region of the fungal rRNA operon were amplified by PCR . The primers were as follows: for bacteria, V1-9F (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGACGAGTTTGATCMTGGCTCAG-3′) and V3-541R (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-barcode-ACWTTACCGCGGCTGCTGG-3′) were used, and for fungi, ITS-3F (5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCACATCGATGAAGAACGCAGC-3′) and ITS-4R (5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-barcode-GCTCCTCCGCTTATTGATATGC-3′) were used. The 20 μL PCR reaction mixture included 2 μL of 2.5 mM dNTPs, 4 μL of 5× FastPfu Buffer, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA dissolved in 12 μL of deionized water . The subsequent DNA purification, quantification, and pyrosequencing processes were the same as described in the previous study  . All the operations were conducted according to product instructions (Pyrotell-®T Multi-Test Kit).p value of less than 0.05 indicates a significant difference between groups. Pearson correlation analyses were used to assess the relationship of AQI with relative humidity, wind speed and chemical compositions. One-way analysis of variances (ANOVA) was used to analyze the difference of PM concentrations on haze days and sunny days, and the concentration difference of endotoxin and microbes was analyzed by independent sample t test analysis.GraphPad Prism version 6.0 was used to perform statistical analyses, and a Relationship between airborne microbial composition and environmental factors  was determined with redundancy analysis (RDA) which was implemented in the R software package. The total water-soluble inorganic ions concentration, the total heavy metal ions concentration, the TOC concentration, and the endotoxin concentration were introduced as explanatory variables. The relative contributions of the 21 bacterial genus-level or 10 fungal genus-level (average relative abundance > 1% in at least one sample) phylogenetic groups were used as response variables. Variance inflation factor analysis was used to analyze the collinearity of environmental factors, and the most colinear factor was deleted until no collinearity existed.r = 0.91) and the relative humidity was found. There was a moderate negative correlation between the wind speed and the AQI (r = − 0.56), indicating that higher humidity and lower wind speed might favor the accumulation of atmospheric pollutants.The study period ranged from December 19 to December 26 in 2016, including hazardous haze days , sunny days  and unhealthy haze days . Paired pictures of hazardous haze days and sunny days are shown in Fig. 2.5 samples were collected and named Filter-1 to Filter-5. As observed in Fig. 3 for Filter-1, Filter-2, Filter-3, Filter-4, and Filter-5, respectively.A total of 5 PM3) and unhealthy days (AVG 697.7 particles/cm3) were significantly higher (10.7 and 8.0 times) than those on sunny days (AVG 87.5 particles/cm3). Figure The PM concentration and size distributions during the study period were monitored by a laser particle counter . The data were recorded every 5 min. As shown in Fig. 2.5 and its chemical components during the study period had almost the same variation trend and were strongly correlated with the AQI.The concentrations of water-soluble inorganic ions (WSII), heavy metal ions, and total organic carbon (TOC) during the study period were analyzed and are shown in Fig. 3−, Cl−, SO42−, NH4+, K+, Na+, Mg2+, and Ca2+ are important parts of atmospheric particles. The total concentrations of WSII on hazardous haze days, sunny days, and unhealthy haze days were 138.41, 9.78, and 48.64 μg/m3, respectively. The percentage of WSII in the PM2.5 on haze days was higher than that on sunny days. The percentage of WSII in the PM2.5 was 83.7% on hazardous haze days, 77.1% on unhealthy haze days, and 65.2% on sunny days. SO42−, NH4+, and NO3− dominated the WSII, accounting for 71.9 ± 9.7% of the WSII concentration. The concentrations of SO42−, NH4+, and NO3− were 16.8 and 5.9 times higher on hazardous haze days and unhealthy haze days, respectively, than those on sunny days.Water-soluble inorganic ions (WSII) including NO2.5 in the urban atmosphere. The TOC concentrations were higher on haze days than that on sunny days, with values of 26.9, 5.2 and 14.4 μg/m3 on hazardous haze days, sunny days and unhealthy haze days, respectively. However, the TOC percentage in the PM2.5 on haze days was lower than that on sunny days, with values of 16.3, 34.8 and 22.9 on hazardous haze days, sunny days, and unhealthy haze days, respectively.Carbonaceous fractions were important components of the PM3, accounting for 0.92%, 1.56%, and 1.35% of the PM2.5 mass on hazardous haze days, sunny days, and unhealthy haze days, respectively. The concentrations of As and Cr during the hazardous haze days surpassed the PM2.5 Chinese class II standards (0.006 and 0.000025 μg/ m3).The total concentrations of the measured heavy metal ions  were 1.5, 0.23 and 0.85 μg/m3, respectively.The concentration of airborne endotoxin over the study period is shown in Fig. 3, 237.67 CFU/m3, 310.67 CFU/m3, 364.00 CFU/m3, and 518.00 CFU/m3; the average concentrations of fungi for the five different sampling days, in order, were 101.33 CFU/m3, 121.33 CFU/m3, 180 CFU/m3, 183.33 CFU/m3, and 257.33 CFU/m3  and at least at the sixth level 0.65–1.1 mm). The highest distribution rates at level 4 were 32.7 and 32.3%; the lowest distribution rates at level 6 were 3.0 and 2.1%. The distribution of bacteria and fungi at levels 3 and 4 accounted for more than 50% of the total CFU , Microbispora (6.78 ± 0.48%), Paracoccus (4.11 ± 1.58%), and Skermanella (3.75 ± 0.94%). The predominant fungal genera were Alternaria (31.42 ± 3.63%), Cladosporium (18.48 ± 2.66%), Phoma (6.25 ± 2.12%), and Aspergillus (4.71 ± 3.06%). The hierarchical distribution maps and heatmaps of the bacterial and fungal class community structures with different abundances are shown in Fig. In addition, the bacterial and fungal genus community structures of the five PMRubellimicrobium, Paracoccus, Clostridium, Aspergillus, Cryptococcus, and Torula, were positively correlated with TOC, while some other microbes, such as Sphingomonas, Methylobacterium, Acinetobacter, Cladosporium, Davidiella, and Aureobasidium, were negatively correlated with TOC.Relationship between airborne microbial composition and environmental factors  was determined with redundancy analysis. However, the four environmental factors showed strong collinearity. Only TOC was left after the procedure of collinearity elimination. 30.0% of total variations in bacterial composition at the genera level and 27.1% of total variations in fungal composition at the genera level were related to TOC. The first principal component contributed to 6.31% and 2.71% of total variations in bacterial and fungal composition, respectively Fig. . Some mi2.5 can penetrate deep into the lower respiratory tract, and the components attached to PM2.5 might cause toxicological effects and many other diseases associated with the respiratory and cardiovascular system   and the first few days following the event  in Beijing. The concentration of PM and its chemical components were proportional to the AQI. The percentage of particles with aerodynamic diameters of 0.5 μm–3 μm was significantly higher on haze days than on sunny days. The abundances of bacteria and fungi were higher on early haze days than on sunny days, and decreased once the haze episode had progressed for a few days. The endotoxin levels were higher on haze days than on sunny days. Most culturable bacteria and fungi were distributed in the 3rd and 4th stages (2.1–4.7 μm) of the ANDERSEM-6 sampler. Our results facilitate a better understanding of the biological and chemical composition dynamics of PM in Beijing and provide useful data for health impact evaluations during haze episodes.Figure S1Paired pictures of hazardous haze days and sunny days (DOCX 227 kb)Figure S2Heatmap of bacterial and fungal community structures (classes) with different abundances in the air samples. (A) and (C) represent the relative abundances of bacteria and fungi (classes), respectively. (B) and (D) represent the heatmaps of bacteria and fungi (classes), respectively. The air samples were collected during hazardous haze (Filter 1 and 2), unhealthy haze (Filter 4 and 5) and sunny days (Filter 3). The text on the right side of the heatmaps lists every bacterial or fungal class name. (DOCX 306 kb)Figure S3Plot of redundancy analysis (RDA) of the airborne bacterial composition (A) and fungal composition (B) at the genus level relative to total organic carbon (TOC). Only taxa with an average relative abundance ≥1% in at least one sample were involved. Constrained explanatory variable (TOC) was indicated by blue arrow. (PNG 199 kb)High resolution image (TIF 421 kb)Table S1Levels of the chemical compositions measured in the five air samples during the sampling campaign (DOCX 17 kb)


Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli. However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.The Mag1 and Tpa1 proteins from budding yeast ( A. Pathways for repair of alkylated bases in many organisms, including Escherichia coli and humans, include base excision repair (BER)N6-ethenoadenine (ϵA) in E. coli can be initiated by the DNA glycosylase AlkA and completed by other enzymes in the BER pathway, or DRR can be catalyzed by the DNA dealkylase AlkB in a reaction requiring ferrous iron, oxygen, and 2-oxoglutarate (2OG) . Other alkylated bases are typically repaired by either BER or DRR. Interestingly, it has been suggested that some bacteria and archaea that appear to lack an AlkB-family oxidative dealkylase instead use BER with an expanded substrate repertoire to repair alkylation damage - and 2OG-dependent dioxygenase superfamily  and Tpa1  to the repair of alkylation damage. The results reveal that the mag1Δ strain is ∼500-fold more sensitive to 0.3% MMS after 1-h exposure than is WT  is widely used to generate DNA damage to investigate DNA repair pathways. MMS methylates DNA primarily at the N-7 position of G (7mG), the N-3 and N-1 positions of A (3mA and 1mA), and the N-3 position of C (3mC) . We consan is WT , as has an is WT . This isan is WT . Howeverin alone .mag1Δ, but not with tpa1Δ, agree qualitatively with previous observations in E. coli where both the alkA and alkB genes were initially discovered in screens for mutants with increased sensitivity to MMS /2OG-dependent dioxygenase superfamily other than Tpa1 have been identified as possible AlkB family oxidative dealkylases in S. cerevisiae  . Single-turnover kinetic measurements ([Mag1] > [substrate]) for each substrate showed that Mag1 has much higher kmax values for excision of 3mC, ϵC, and ϵA than for excision of 1mA (S1). Furthermore, although kmax values for excision of 3mC, ϵC, and ϵA are very similar .We considered the possibility that yeast, like some bacteria and archaea, utilize DNA glycosylases to remove alkylated bases that are more commonly considered to be substrates for DRR by AlkB and related DNA dealkylases –3. To te–Fig. 1A) –24. The A) –; we usedn of 1mA  and S1. 26 min−1 . Excisiokcat/mK values for different substrates provides a better measure of substrate specificity. These apparent second-order rate constants, commonly referred to as specificity constants, report on all of the steps up to and including the first irreversible step . Compared with the previously reported Mag1 substrate, ϵA, both 3mC and ϵC have larger relative kcat/mK values, and 1mA has a relative kcat/mK value within 2-fold of the relative kcat/mK value for ϵA  and 2OG. Whereas AlkB completely repaired ϵA, 1mA, 3mC, and ϵC embedded in either ssDNA or dsDNA within 1 h, Tpa1 showed no repair activity on any of these eight substrates even after 29 h (A and S4).Following our finding that the ter 29 h A and S4.Table S2 and Fig. S5). Notably, N-terminal prolyl hydroxylase domains (P4Hc and EGL9) are conserved in Tpa1 and its close homologs, which extend to humans, consistent with crystallographic studies (–X(D/E)…H Fe(II)-binding triad and hence is unlikely to have oxygenase activity /2OG-dependent dioxygenase superfamily from human, fruit fly, and fission yeast using the Conserved Domain Database from the NCBI  (Table S studies –10. A distudies –. In addiactivity –10. Thesctivity ––10.B). The RPS23 peptide contains Pro-62, the residue determined to be hydroxylated by OGFOD1, the human homolog of yeast Tpa1, in previous studies  and competition assays  with AlkA, as described above for Mag1, to compare the preferences of the two glycosylases for the known AlkB substrates in Fig. 1A.AlkA is a DNA glycosylase from  of Mag1 , 5. BecaE. coli AlkA excises ϵA from dsDNA  as has been reported previously  and of 3mC from 3mC-25mer , although excision of these bases from dsDNA by AlkA was not detected in previous studies , so only limits for its kmax/K1/2 and relative kcat/mK values are reported in We confirmed that eviously . We also studies , 29. No kmax, K1/2, and kmax/K1/2 are more favorable for Mag1 than for AlkA for all substrates tested  and transformed into each of the three strains by the lithium acetate procedure . To construct the mag1Δ::LEU2 tpa1Δ::kanMX double knockout, the LEU2 cassette was amplified from pRS425 by PCR using the primers in Table S4 and transformed into the tpa1Δ::kanMX strain. Integrants were selected on media lacking leucine. Correct integration was verified by PCR using primers listed in Table S4 .BY4741, llection . Derivat methods  and are rocedure . Integra600) from overnight cultures and grown in yeast extract/peptone/dextrose (YPD) medium to OD600 = 0.6. The culture was diluted to OD600 = 0.2 with fresh YPD. This cell suspension was further diluted to OD600 = 0.1 with YPD (for −MMS control cultures) or 0.6% MMS (v/v) in YPD (for +0.3% MMS cultures). Cell suspensions were incubated at room temperature with rotation for 1 h before diluted aliquots were spread on YPD plates. Colonies were counted after 3–5 days at 30 °C to determine cell survival.Yeast cultures were inoculated to an optical density of 0.1 at 600 nm . Cells were grown overnight in LB broth to inoculate 500-ml terrific broth cultures. These cultures were grown at 37 °C until OD600 reached 1.0, and then the temperature was reduced to 18 °C for 1 h prior to induction with 200 μm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested by centrifugation, and each pellet was resuspended in 100 ml of lysis buffer  glycerol, pH 7.4). Cells were lysed by two passes through an Emulsiflex C3 homogenizer  at 18,000 p.s.i. gauge and centrifuged to clarify. The supernatant was stirred at 4 °C while polyethyleneimine was added to 0.05% (w/v), and then centrifugation was repeated. The protein was bound to a nickel-charged HisTrap HP column  and eluted over an imidazole gradient (15–400 mm). Peak fractions were pooled, and the fusion protein was digested by incubation with ULP1 overnight. The protein was then bound to a HiTrap Q HP column  and eluted over a sodium chloride gradient. Peak fractions were pooled and concentrated using a centrifugal ultrafiltration cell (Amicon) to a final concentration of 188 μm as determined by the absorbance at 280 nm using the predicted extinction coefficient of 98,780 m−1 cm−1.iae Mag1  and E. coli AlkA  were expE. coli AlkB  glycerol, 5 mm 2-mercaptoethanol, pH 7.4) per pellet. Lysis and nickel-affinity chromatography were performed as described for Tpa1. The tag was removed by incubation with PreScission protease for 1 h. The protein was then bound to a HiTrap SP HP column  and eluted over a salt gradient. Peak fractions were concentrated via centrifugal ultrafiltration and then loaded onto a Superdex 200 column equilibrated with storage buffer . Protein was concentrated to 650 μm as determined by the absorbance at 280 nm using the predicted extinction coefficient of 32,430 m−1 cm−1. An SDS-polyacrylamide gel of the purified proteins used in this study is shown in Fig. S10.A pET19-derived expression construct for oli AlkB  was tranat 18 °C . Cells wTable S1. Single-strand DNA concentrations were determined from the absorbance at 260 nm using calculated extinction coefficients. For oligonucleotides containing ϵA, the extinction coefficient was calculated for the same sequence with an A in place of ϵA and corrected by subtracting 9400 m−1 cm−1 to account for the weaker absorbance of ϵA . Reactions were initiated by adding a small volume of DNA substrate to the remaining reaction components in a final volume of 20–40 μl. Aliquots were withdrawn at various times and quenched with sodium hydroxide to give a final concentration of 0.2 m. Abasic sites were converted into single-strand breaks for most quenched samples by heating at 70 °C for 10 min; ϵC degrades under the standard hydrolysis conditions, so samples containing ϵC were subjected to milder hydrolysis at room temperature for 1 h. The milder hydrolysis gives a mixture of two products, the first from β-elimination on the 3′ side of the abasic site to give an oligonucleotide terminated by an α,β-unsaturated aldehyde fragment and the second from δ-elimination of the aldehyde fragment remaining on the first , and emission was measured with a 520BP40 filter following excitation of the fluorescein label at 488 nm. Fluorescence intensities of gel bands were quantified using ImageQuant TL  and corrected for the amount of background signal. The data were converted to fraction product (P) and then fit by the single exponential shown in Reactions were carried out at 25 °C in 50 mhe first . Only thTable S1). Each substrate was mixed with the reference substrate at varying ratios for total DNA concentrations of 0.8–1.8 μm. Reactions were carried out at 25 °C in 50 mm MOPS, pH 7.5, 100 mm NaCl, 0.1 mg/ml BSA, 1 mm EDTA, 1 mm TCEP. Mag1 or AlkA was added to a final concentration of 50 nm, and the glycosylase activity was followed up to 10% depletion of either substrate. Samples were quenched, hydrolyzed, and analyzed by PAGE as described above. Initial velocities are proportional to the relative kcat/mK values as described in Relative specificity for Mag1 and AlkA substrates was determined by competition assays  with them each enzyme with 100 nm ss or duplex versions of the 25mer DNA substrates shown in Table S1. Reactions were carried out at 37 °C in a 10–20-μl reaction mixture containing 50 mm HEPES pH 7.5, 150 mm NaCl, 0.1 mg/ml BSA, 1 mm TCEP, 2 mm sodium l-ascorbate, 1 mm 2OG, 50 μm ammonium iron (II) sulfate. Repair reactions were quenched with 10 mm EDTA in a solution that contained a 10-fold excess of Mag1 or Bacillus subtilis AAG (B. subtilis AAG are active in EDTA). Sodium hydroxide was then added to a final concentration of 0.2 m, samples were heated at 70 °C for 10 min to convert abasic sites into single-strand breaks, and the hydrolyzed samples were analyzed by denaturing PAGE on 20% (w/v) gels as described above. This glycosylase treatment converts unrepaired 25mer DNA into 12mer DNA, but 25mers that have been repaired by AlkB or Tpa1 remain intact and appear as slower migrating bands on gels (B. subtilis AAG was used instead of Mag1 to treat 1mA-containing DNA samples because of its faster kinetics with this substrate under these conditions (data not shown). Mock DNA repair reactions were performed for each DNA substrate in the absence of Tpa1 or AlkB as a positive control for the DNA glycosylase activity of Mag1 and B. subtilis AAG and to reveal the maximum amount of repairable DNA in each initial sample. As noted above, end points for both ϵA-25mer and ϵC-25mer were 0.95, whereas end points for 1mA-25mer and 3mC-25mer substrates were 0.60 . Reaction mixtures contained 50 mm HEPES, pH 7.5, 100 μm sodium l-ascorbate, 20 μm 2OG, 50 μm ammonium iron (II) sulfate. Concentrated stocks of 2OG, sodium l-ascorbate, and RPS2347–84 were prepared in 50 mm HEPES, pH 7.5; a 400 mm stock of ammonium iron (II) sulfate was prepared in 20 mm HCl, and dilutions were made in Milli-Q water. The reaction was initiated by addition of enzyme, incubated for 10 min at room temperature, and quenched with formic acid . Hydroxylation of the RPS23 peptide was monitored by MALDI-TOF MS on a Bruker Autoflex Speed instrument in positive reflectron mode. Assay mixture (1 μl) was mixed with α-cyano-4-hydroxycinnamic acid matrix  and applied onto a ground steel target.The hydroxylation activity of Tpa1 was assessed by incubating 2 μS. J. A., D. E. E., C. J. S., and P. J. O. conceptualization; S. J. A., D. E. E., C. J. S., and P. J. O. formal analysis; S. J. A., D. E. E., M. R. B., and P. J. O. validation; S. J. A., D. E. E., M. R. B., E. M. B., C. T. L., and P. J. O. investigation; S. J. A. and P. J. O. writing-original draft; S. J. A., D. E. E., M. R. B., E. M. B., C. T. L., C. J. S., and P. J. O. writing-review and editing; C. T. L., C. J. S., and P. J. O. funding acquisition; C. J. S. and P. J. O. supervision; C. J. S. and P. J. O. project administration.


To evaluate two different techniques of cross-linking: standard epithelium-off (CXL epi-off) versus transepithelial (CXL epi-on) cross-linking in patient with progressive keratoconus. 2.  Forty eyes from 32 patients with progressive keratoconus were prospectively enrolled from June 2014 to June 2015 in this nonblinded, randomized comparative study. Twenty eyes were treated by CXL epi-off and 20 by CLX epi-on, randomly assigned, and followed for 2 years. All patients underwent a complete ophthalmologic testing that included uncorrected and best corrected visual acuity, central and peripheral corneal thickness, corneal astigmatism, simulated maximum, minimum, and average keratometry, corneal confocal microscopy, Schirmer I and break-up time (BUT) tests, and the Ocular Surface Disease Index. Intra- and postoperative complications were recorded. The solution used for CXL epi-off comprised riboflavin 0.1% and dextran 20.0% (Ricrolin), whereas the solution for CXL epi-on (Ricrolin TE) comprised riboflavin 0.1%, dextran 15.0%, trometamol (Tris), and ethylenediaminetetraacetic acid. Ultraviolet-A treatment was performed with a UV-X system at 3 mW/cmp=0.01; Group 2: baseline 0.32 ± 0.18 logMAR, 2-year follow-up 0.27 ± 0.19 logMAR, p=0.01) was recorded. Keratometry remained unchanged in both groups. The mean corneal thickness showed a significant reduction . One-month after treatment, OSDI© reached 13.56 ± 2.15 in Group 1 (p=0.03) and 11.26 ± 2.12 in Group 2 (p=0.04). At confocal microscopy, abnormal corneal nerve alterations were found in both groups. Fibrotic reaction (43.75%) and activated keratocyte (62.6%) were more commonly recorded in Group 1 than in Group 2 (25.0% and 18.75%), with p=0.668 and 0.356, respectively.  In both groups, a significant improvement in visual function (Group 1: baseline 0.36 ± 0.16 logMAR, two-year follow-up 0.22 ± 0.17 logMAR,  Our findings demonstrate that both procedures are able to slow keratoconus progression. Both treatment modalities are equivalent in terms of results and related complications. CXL epi-on technique is preferable to CXL epi-off since it preserves the corneal thickness and improves visual acuity, also reducing the postoperative ocular discomfort during the study period. Corneal collagen cross-linking (CXL) has acquired nowadays popularity for the treatment of progressive corneal ectasia. This technique, stabilizing the progression of keratoconus, delays the need for keratoplasty and, thus, decreases the chance of corneal transplantation , throughIontophoresis is a novel noninvasive system aimed at enhancing the delivery of charged molecules into tissues using small electric current . RiboflaThe aim of this study has been to compare these two techniques and to evaluate the efficacy and safety of the two treatments.Forty eyes from 32 patients with progressive keratoconus, followed at the University of Molise, Italy, from June 2014 to June 2015, were included in this nonblinded, randomized comparative study. The patients were randomly assigned to one of the two treatment groups . Progression of keratoconus was documented through a clinical and instrumental  worsening in the previous 6 months of observation. Inclusion criteria were patients with evolving keratoconus, aged between 18 and 40 years, and with no evidence of corneal scarring. Exclusion criteria were patients with central and paracentral corneal opacities, Vogt's striae, previous intraocular surgery, history of herpetic keratitis, severe dry eye, and concomitant autoimmune diseases.All patients underwent a complete ophthalmologic testing that included uncorrected and best corrected visual acuity (BCVA), central and peripheral corneal thickness, corneal astigmatism, simulated maximum, minimum, and average keratometry, corneal confocal microscopy, Schirmer I and break-up time (BUT) tests, and the Ocular Surface Disease Index. All intra and postoperative adverse events were recorded.K flat, K steep, and mean K were evaluated with Sirius  and Pentacam®  topographs. Fibrotic reaction, corneal alteration of nerves, activated keratocytes, and corneal opacities were evaluated through confocal microscopy HRT III .BCVA was determined using Snellen's chart and was converted to logarithm of the minimum angle of resolution (logMAR). Central and peripheral corneal thickness, μm  to subjectively estimate the pre- and postoperative anterior stromal cell density [© .Cornea was examined for anterior thinning, the presence of inflammatory cells associated with the lenticule, and activation of corneal keratocytes, which may indicate the development of fibrosis , 22. Ima density . Lastly,2. At the end of the procedure, ofloxacin and cyclopentolate eye drops were administered, and therapeutic contact lens (LAC ACUVUE-etafilcon A) was then applied and was removed 3 days after surgery. Topical tobramycin (four times daily for 1 week) and dexamethasone phosphate 0.1% (four times daily for 2 weeks) were prescribed. The therapeutic contact lens was removed three days later. Lubricating eye drops were prescribed for the following three months.Epi-off CXL technique was performed after instilling 4% lidocaine for topical anesthesia and 1.0% pilocarpine to reduce the risk for ultraviolet light exposure. A 9.0 mm of corneal epithelium was mechanically removed. Riboflavin  was administered topically every 2 minutes for 30 minutes. The administration was continued every 2 minutes during UVA exposure. The cornea was exposed to UVA 370 nm light  for 30 minutes at an irradiance of 3.0 mW/cmIn the epi-on CXL group, corneal epithelial was not removed. Corneal imbibition was obtained with 0.1% riboflavin–15% dextran solution supplemented with Tris-hydroxymethylaminomethane and sodium ethylenediaminetetraacetic acid  applied every 5 minutes for 30 minutes. One drop of 1% pilocarpine was administered 30 minutes before treatment to reduce the risk for UVA exposure. Ten minutes later, a single dose of 4% lidocaine eye drops was administered to anaesthetize the cornea. Postoperatively, topical tobramycin (four times daily for 1 week) was prescribed. All patients were operated by the same surgeons (F.C. and M.M.). The patients were checked at day 1, 3, 7, and 15 and then after 1, 6, 12, and 24 months.NCT01350323), and each patient gave their written informed consent after a detailed description of the procedure used and of the aim of the work.The study was approved by the Institutional Review Board . In Group 2, the mean age was 31 ± 10 years .p=0.01), whereas in Group 2, the values progressed from 0.32 ± 0.18 logMAR to 0.27 ± 0.19 logMAR (p=0.01). At the end of the follow-up, the difference between the two groups was also significant (p=0.01).In Group I, BCVA at the baseline was 0.36 ± 0.16 logMAR and improved to 0.22 ± 0.17 logMAR in postoperative 2 years ; these two values in the postoperative period of 2 years remained unchanged: 46.16 ± 3.15 D (p=0.57) (Group 1) and 47.82 ± 4.06 D (Group 2) (p=0.10). In addition, the differences between the two values were not significant (p=0.08).Mean K steep and K flat at the baseline in Group 1 were, respectively, 47.75 ± 3.20 D and 44.62 ± 2.63 D and in Group 2 were 48.86 ± 3.27 D and 45.84 ± 2.53 D. Two years after treatment, K steep and K flat of Group 1 reached 47.76 ± 3.47 D (p=0.10) and 44.71 ± 3.03 D (p=0.33), whereas in Group 2, they were 49.75 ± 3.47 D (p=0.60) and 46.44 ± 3.67 D (p=0.25). On the contrary, at the end of the follow-up, the difference between the two groups was significant for both parameters (p=0.01).μm to 501.41 ± 21.91 μm (p=0.01) and from 565.41 ± 31.91 μm to 495.45 ± 43.16 μm (p=0.01), resp.), but the difference between groups was not significant (p=0.10).Mean corneal thickness after 2 years significantly change in both groups . After one month, the score increased to 13.56 ± 2.15 in Group 1 (p=0.01) and 11.26 ± 2.12 in Group 2 (p=0.04). The difference between the two groups was also significant (p=0.02).At the baseline, the OSDIp=0.001 in both).Confocal microscopy data in both groups revealed corneal nerve alterations: 93.8% in the epi-off group (Group 1) and 87.5% in the epi-on group (Group 2). Activated keratocyte and fibrotic reaction in Group 1 represented 62.5% and 43.75%, respectively, whereas in Group 2, they were recorded in a significantly lowest percentage  and one in Group 2 (Vogt's striae and in the same eye follicular conjunctivitis). Schirmer and BUT tests did not reveal lacrimation defects in both groups.© (Ocular Surface Disease Index) difference in patients who underwent CXL. Through the OSDI©, we evaluated the degree of ocular discomfort in the patient treated with the two different methods. The results show that the score was lower in patients of Group 2 (p < 0.05). At the baseline, the score was 4.85 ± 1.18 and 4.98 ± 1.32 OSDI©, respectively. After treatment, the score increased to 13.56 ± 2.15 OSDI© in Group 1 and 11.26 ± 2.12 OSDI© in Group 2. Despite the use of topical anesthetics, the greater mean postoperative pain in the epi-off CXL group compared to the epi-on CXL group probably depends on the exposure of the corneal nerves and the release of inflammatory mediators, especially prostaglandins and neuropeptides after epithelium removal and related healing processes [Our findings report for the first time OSDIrocesses  (Tables μm to 501.41 ± 21.91 μm (p=0.01) and from 565.41 ± 31.91 μm to 495.45 ± 43.16 μm (p=0.01), resp.), as already reported [Statistical analyses show that the mean corneal thickness, two years later, change significantly in both groups . However, the epi-on group exhibits a better improvement compared to the epi-off group at the end of the follow-up (p=001) (A significant increase in BCVA compared to the baseline was recorded in both groups ( (p=001) . Our res (p=001)  and 5.The activated keratocyte and fibrotic reaction are more frequent in Group 1 patients. This might be due to a rearrangement of the corneal epithelium secondary to the treatment or more likely to the much deeper cross-linking activity in the epi-off group , 32.© questionnaire.Few side effects occurred in our study: in Group 1, two patients had complications  and in Group 2, only one eye developed Vogt's striae at the apex of keratoconus. On the contrary, several complications have been reported in other previous series, especially after epi-off CXL, such as clinically significant corneal haze, endothelial damage, and sterile infiltrate infections , 34. Las©.The limit of this study is the small number of patients in each group; the strengths are the prospective design, the long term follow-up (two years), and the evaluation of OSDIDespite the different penetration stroma demonstrated in other studies, the clinical outcomes after CLX epi-off and epi-on procedures show that keratoconus was relatively stable after 24 months, and no differences were observed comparing the two procedures. Moreover, our findings demonstrate that the CXL epi-on technique is preferable to CXL epi-off since it reduces postoperative ocular discomfort, maintaining the same profile of safety and efficacy.


Tobacco stalk (TS), a major agricultural waste abundant in pectin, has resulted in concerns about the need for its reuse. The nicotine in TS is considered a chemical that is to\xic and hazardous to the environment.Bacillus tequilensis CAS-MEI-2-33 was isolated from cigar wrappers to produce alkaline pectinase using TS. Subsequently, the medium and fermentation conditions for the production of pectinase by B. tequilensis CAS-MEI-2-33 were optimized. The optimal fermentation period, pH of the initial fermentation medium, concentration of TS, and inoculum amount for B. tequilensis CAS-MEI-2-33 were 40 h, 40 g/L, 7.0, and 3%, respectively. Under optimal conditions, the pectinase activity was 1370 U/mL. Then, the enzymatic properties, such as the optimum pH, reaction temperature, temperature stability, and effects of metal ions, were studied. The optimal pH was determined to be 10.0, indicating that the enzyme was an alkaline pectinase. The optimal temperature was 40 °C, and pectinase activity was stable at 40 °C. The Ag+ metal ions were shown to remarkably promote enzyme activity. The pectinase was partly purified by ammonium sulfate precipitation, ion exchange chromatography, and Sephacryl S-100 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and LC-MS/MS analyses were utilized to analyze the pectinase.In this study, This study provided a new alkaline pectinase candidate and a new strategy for the use of TS.The online version of this article (10.1186/s12896-019-0526-6) contains supplementary material, which is available to authorized users. Extremely large quantities of agro-industrial waste residues are generated from the processing of raw materials, such as tobacco stalk (TS), which is an abundant crop residue in China. It has been estimated that more than one hundred thousand metric tons of TS are discarded annually in China  #1136). 6. 3] #12Pectinase is widely used in food  #1172), 2, 7] #10Based on these criteria, we screened bacteria that could produce pectinase using TS, which is abundant in pectin (approximately 10%) ( #1141). B. tequilensis CAS-MEI-2-33, and further characterization and identification were carried out.Pectinase has many applications in various industrial processes  with EI-2-33 ( #1309). B. tequilensis CAS-MEI-2-33 was less affected under the experimental conditions . 5. B. teqp = 0.05).After the determination of pectinase activity with different concentrations of TS in the fermentation medium, it was found that at the concentration of 40 g/L TS and the optimized pH, pectinase activity was the highest .+, Li+, Cu2+, Ca2+, Ba2+, and Mn2+ ions increased enzyme activity; in particular, Ag+ ions increased pectinase activity by 193.95%, which was approximately 1.94 times higher than that of the control. K+, Co2+, Ni2+, Mg2+, Zn2+, Cd2+, and Fe3+ ions, especially Zn2+, inhibited pectinase activity. Figure B. tequilensis CAS-MEI-2-33. However, pectinase activity decreased when the cell-free supernatant was placed at 60 °C. Pectinase activity was stable when the cell-free supernatant was incubated at 40 °C; however, pectinase could not tolerate the high temperature for a long time.The results of the pectinase property analysis are shown in Fig. Under the optimal fermentation conditions, 2.30 L supernatant was obtained by centrifuging the bacteria in a 3.0 L fermentation flask. The pectinase activity reached 1771 U/mL, and the total pectinase activity was 4,074,513 U  as described by Mehrnoush et al.  #1306). . 6d. TheB. subtilis , Baterium ( #1152),  oryzae ( #1147),  oryzae ( #1101), ilensis ( #1307).B. tequilensis CAS-MEI-2-33 during optimization experiments. The one-factor-at-a-time method was implemented to optimize the components of the medium and conditions, especial the pH of medium. The optimum pH for the growth of and pectinase production by other bacterial strains, such as B. subtilis  , hubtilis ( #1193) aubtilis  #1231) h hB. tequiliveri ( #1307), Bacillus sp. was 5.0 ([B. tequilensis strain to produce pectinase using TS and provides a direction for further investigation of the possible applications of TS.The properties of pectinase are important in the application of this enzyme ( #1170); was 5.0 ( #1221). B. tequilensis CAS-MEI-2-33 strain will not only aid in addressing TS waste but also increase the possible sources of alkaline pectinase. The medium and culture conditions for this strain were optimized in this study. Based on the optimized conditions , the highest enzymatic activity of the strain was 1370 U/mL. The enzymatic properties were studied, including the pH of the reaction buffer, reaction temperature, effects of metal ions, and thermal stability. The alkaline pectinase was relatively stable at 40 °C, and the molecular weight was approximately 45.4 kDa. This is the first report of the ability of the B. tequilensis strain to produce pectinase using TS and provides a direction for further investigation of the possible applications of TS.This study successfully determined that the strain isolated from the cigar wrapper was a potential candidate organism that was useful in the production of pectinase using TS. The 2HPO4, 0.3% (NH4)2SO4, 0.001% Fe2SO4, 0.1% MgSO4, and 1.8% agar powder. Inoculated plates were incubated for 48 h at 37 °C to obtain bacterial colonies. Colonies were picked, inoculated on fresh PAPs, and incubated at 37 °C for 48 h to obtain pure cultures. The pure cultures were maintained on Luria–Bertani (LB) agar plates at 4 °C and stored at − 80 °C with 15% glycerol for further use.Samples collected from the cigar wrapper leaf from the Haikou Cigar Research Institute, Hainan Provincial Branch of the China National Tobacco Corporation, were diluted with 0.9% sterile NaCl solution and then plated onto pectin agar plates (PAPs) containing 0.5% pectin , 0.1% KThe bacterial isolates from the primary screening were tested for their ability to produce pectinases. The isolates were spot inoculated on PAPs in triplicate and incubated at 37 °C for 36–48 h. Depending on the zone of clearance surrounding the colonies  using Congo red  ( #1101), 2O. The genomic DNA of B. tequilensis CAS-MEI-2-33 was extracted with a TIANamp Bacteria DNA Kit  according to the manufacturer’s instructions. The PCR products were detected using 1.0% agarose gel electrophoresis. The product of the appropriate size (1.5 kbp) was sequenced , and the sequence was compared with the NCBI nucleotide sequence database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and deposited under accession number MH806356.1. The phylogenetic tree was constructed using MEGA (version 7)  gene. Morphological characteristics of the isolated bacterial strain, e.g., colony morphology  and cell morphology (shape and Gram staining), were studied ( #921). TPectinase activity production was assessed using the 3,5-dinitrosalicylic acid (DNS) method . The tubes were boiled for 5 min and cooled. A blank sample was prepared with distilled water instead of D-galacturonic acid. One unit (U) of polygalacturonase activity was defined as the amount of enzyme that generated 1 μmol galacturonic acid per min under the assay conditions. Pectinase activity was calculated using the following formula:B. tequilensis CAS-MEI-2-33 during optimization experiments. We took samples and detected the pectinase activity of the fermentation medium every 4 h to confirm the maximum activity of pectinase during fermentation. The initial fermentation medium contained 20 g/L TS, 0.5 g/L MgSO4, 3 g/L (NH4)2SO4, and 0.01 g/L Fe2SO4 at pH 7.0. Fermentation was performed in shake-flask experiments using 500 mL shake flasks containing 100 mL fermentation medium incubated with the strain B. tequilensis CAS-MEI-2-33. The culture was grown at 37 °C and 180 rpm. All experiments were performed in triplicate. The reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.It is critical to determine the fermentation period of B. tequilensis CAS-MEI-2-33 incubated for 8 h at 37 °C in LB. The OD600 of B. tequilensis CAS-MEI-2-33 was approximately 4.52, and the inoculum volume was 1% of the fermentation volume. All experiments were performed in triplicate. One-way ANOVA with Tukey’s test was used to determine any significant differences (p < 0.05) between treatments using SAS .Inocula were prepared using the culture of B. tequilensis CAS-MEI-2-33, growth of this strain in LB containing nicotine was evaluated in shake-flask experiments using 150 mL shake flasks containing 50 mL medium that was incubated with 1% (v/v) inocula. Nicotine was added to the LB medium at final concentrations of 0, 0.5, 1.0, and 2.0 g/L. The optical density of the incubation was monitored at 600 nm every two hours.To test the toxicity of nicotine to Optimization of the pH was performed in a similar manner for the selected isolate. The initial fermentation medium was prepared with pH ranging from 6.0–8.0 using NaOH and the initial medium prior to sterilization at 121 °C for 20 min. The medium was incubated at 37 °C and 180 rpm for 40 h.TS provided the carbon and nitrogen sources in the medium and was important during fermentation. The fermentation medium was prepared with TS concentrations ranging from 10 to 50 g/L at the optimal pH. The medium was incubated at 37 °C and 180 rpm for 40 h.Effect of the inoculum size on enzyme production was studied with different concentrations of inocula. Different amounts, i.e., 1, 3, 5, 7, and 9% (v/v), were inoculated into the fermentation medium with 40 g/L TS at pH 7.0. The medium was incubated at 37 °C and 180 rpm for 40 h.+, K+, Li+, Cu2+, Ca2+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Zn2+, Cd2+, and Fe3+) on pectinase activity were assayed at a concentration of 1.0 mM in the reaction mixture. Thermal stability was investigated by measuring pectinase activity every 20 min following incubation of the enzyme solution at 40 °C, 50 °C, and 60 °C.The enzyme activity was highly influenced by the pH, temperature, and metal ions. The pH and temperature are important characteristics of a biocatalyst for use in industrial applications. The optimal pH of pectinase in the cell-free supernatant was assayed using citrate–phosphate (pH 6.0), phosphate (pH 7.0–8.0), and glycine–NaOH (pH 9.0–11.0) as buffers (50 mmol/L) and 0.2% pectin  as a substrate at 40 °C. The optimal temperature for pectinase activity was determined by incubating the reaction mixture from 30 °C to 70 °C at the optimal pH. The effects of metal ions ). Then, the column was equilibrated with Tris-HCl buffer (pH 8.0) and eluted with a linear salt gradient . The eluent was collected automatically at 1 min/tube.Sephacryl S-100 was equilibrated with ultrapure water, sampled, and eluted with pH 7.2 PBS buffer, with a flow rate of 0.8 mL/min, and the effluent was detected by an online UV detector at a wavelength of 280 nm to record the ultraviolet absorption peak curve. The components were collected, and the enzyme activity and protein content were tested.The protein content was determined using a BCA  standard curve, and SDS-PAGE ( #1297) wB. tequilensis database in UniProt (https://www.uniprot.org/).Protein bands were cut from the SDS-PAGE gel and subjected to LC-MS/MS (liquid chromatographic-tandem mass spectrometric) analysis by Shanghai Applied Protein Technology . Additional file 1:Figure S1. The transparent zone around the strain of B. tequilensis CAS-MEI-2-33 on PAPs. (DOCX 65 kb)Additional file 2:Figure S2. Protein peak detected by IC-MS/MS. The protein was isolated from a gel. (DOCX 78 kb)


AbstractGanodermatropicum is described as from Chiang Rai Province, Thailand. The fruiting body was collected on the base of a living Dipterocarpus tree. The sample is described on the basis of morphological characteristics and phylogenetic analyses, and compared with closely related taxa. Multigene phylogenetic analyses of LSU, ITS, and RPB2 highly support the placement of the G.tropicum group with isolates from China and Taiwan . The optimal media, pH, and temperature for mycelial growth of the G.tropicum strain KUMCC18-0046 was also investigated and is reported as: PDA, MEA, and YPD, at pH 7–8 and 25–28 °C, respectively. This is the first report on the successful growing conditions for mycelial production, but unfortunately fruiting could not be achieved.In this study a new record of  Ganoderma P. Karst. was established as a white rot fungus  Fr. is the original type species of the genus  and 384 records of taxa in MycoBank .t fungus , showingt fungus . Ganoderorldwide , and the tropics . Ganoder pillars . Polyporhe genus . There aGanoderma produces a high number of natural bioactive compounds, such as polysaccharides, triterpenoids, sterols, and secondary metabolites , which can be used to remedy a wide range of diseases  and B (2), as well as compounds with antimicrobial activity  and incubated at 25 °C, for 21 days, under dark conditions (HKAS), Yunnan Province, China (voucher number HKAS 97486).In October 2017, a single fresh basidiocarp of nditions . After iQ = L/W] and calculated considering the mean value of the lengths and widths in side view. The calculation was done by using at least 50 basidiospores from each basidiomata . The PCR amplifications were performed in a total volume of 25 μL of PCR mixtures containing 9.5 μL ddH2O, 12.5 μL of PCR master mix, 1 μL of DNA template, and 1 μL of each primer (10 μM). PCR amplification was carried out using primer pairs LROR/LR5 for the nuclear ribosomal large subunit 28S rDNA gene (LSU), ITS5/ITS4 for internal transcribed spacer rDNA region , and fRPB2-5F/fRPB2-7cR for the partial RNA polymerase second largest subunit region (RPB2)  of the fungi was amplified and the sequence was deposited in GenBank to obtain the accession number.Dried internal tissues of the basidiocarp were ground and total DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux). The ITS, n (RPB2) . The PCRAmaurodermacalcitum D.H. Costa Rezende & E.R. Drechsler-Santos (FLOR:50931) (MP) analysis was performed with PAUP v. 4.0b10 (ML) were executed on the CIPRES webportal (PP) with GTR+I+G model was used for each partition. Bayesian Markov Chain Monte Carlo (MCMC) analyses were conducted in MrBayes v. 3.2.2 (PP) in the majority rule consensus tree   was usedR:50931) , and manR:50931) . Alignme. 4.0b10 . Maximumebportal , performebportal , and carebportal . The besebportal . Bayesiav. 3.2.2 . The numsus tree .ML) and Maximum parsimony (MP) bootstrap values, equal to or greater than 70%, and Bayesian Posterior Probabilities (BP) equal to or greater than 0.95, are presented above each node , malt extract agar (MEA), potato dextrose agar (PDA), rose Bengal agar (RBA), yeast extract agar (YEA), yeast malt extract agar (YMA), and yeast extract peptone dextrose agar (YPD). The media formulae used are shown in Table Seven different solid culture media were evaluated to determine the optimal media for the mycelial growth of the The optimal media shown for mycelial growth was then used to determine the optimal pH for mycelial growth. pH was adjusted to 4, 5, 6, 7, 8, and 9 with 1N HCl and 1N NaOH. The optimal temperature for mycelial growth was determined by using the highest growth rates of media and pH conditions under different dark conditions; including 15 °C, 20 °C, 25 °C, 28 °C, 30 °C, and 35 °C. After 10 days of incubation, five replicates of colony diameter were measured and calculated. The colony diameter was measured as described above.SPSS) with five replicates (n = 5). All data were compared to obtain a mean separation using Tukey’s test (p < 0.05) followed by post-hoc tests. The results are expressed in a one-way analysis of variance (ANOVA) analysis using the SPSS program .Data analysis was carried out using statistical programs .Phylogenetic analyses were inferred from the combined Taxon classificationFungiPolyporalesGanodermataceae(Jungh.) Bres., Annales Mycologici 8(6): 586 (1910)FOF number: FoF 05068Basidiome. Sessile, dimidiate. Pileus shape. Semicircular to dimidiate or conks, up to 7–12 cm in length and 4–8 cm in width, up to 1.5 cm thick. Pileus surface. Dark brown (9F5) at the base, slightly brownish red (10C8) at center, reddish gray (10B8) extending to the margin, light yellow (1A5) to yellow (2A6) under basidiocarp with grayish yellow (4C7) to brown (6D7 to 6F6) close to tube layer on upper surface of pileus glabrous, weakly to strongly laccate, glossy and shiny, smooth, spathulate, shallow sulcate several layers thick, consistency furrows, thicker at the base than the margin, covered by a thin and hard crust, and light in weight when dried. Context trimitic, irregular cuticle cells, mostly light yellow (1A5) to yellow (2A6) close to crust, grayish yellow (4C7), brown (6D7 to 6F6) to dark brown (9F5), near the tubes, dense context layer, thick near the base, tough to break when dried. Hymenophore. Grayish yellow (4C7). Basidiospores. Mostly oblong ellipsoid and broadly ellipsoid with double wall (ganodermoid) at maturity, (7.3–)7.6–10.1(10.8) × (10.1)10.6–13.3(13.9) μm  (including myxosporium), (5.4–)5.6–8.3(8.6) × (8.3)8.4–12.5(12.9) μm  μm (excluding outer myxosporium), light brown (6D6–6D8), reddish brown (9F6) to dark brown (9F8), usually with one end tapering, and usually overlaid by a hyaline myxosporium. Tubes. 2–7 mm long, up to 80–170 µm wide, and sulcate at different levels. Stipe. Lateral, up to 1.5 cm thick, dark brown (9F5). Margin. reddish gray (10B8), up to 0.3–0.7 cm thick, round, tough and hard, thicker towards the margin. Pore. Angular, 4–7 per mm; pore diameter up to 65–120 µm. Pore surface Pale yellow (2A3) to light yellow (2A5) and brown (6D7) to dark brown (6F6) when touched. Hyphal system Generative hyphae up to 0.80–2.85 μm  in diameter, colorless, thin-walled, some thick-walled, branched, with clamp connections; binding hyphae 1.00–3.10 µm , colorless, thin-walled, much-branched, clamped; skeletal hyphae up to 1.45–4.25 μm , colorless, thick-walled, unbranched or with a few branches in the distal end. Culture characteristics. Initially white (4A1) to yellowish white (4A2), pale yellow (4A3) when growing, become orange white (5A2), pale orange (5A3), light orange (5A4–6A5) and some reddish yellow (4A6) to dark brown (9F8) around the plugged circle of active mycelium after incubation for 3 weeks. Odor. Distinctive odor when fresh and dried.Ganodermatropicum is diagnosed as having a distinctly dimidiate, smooth, spathulate pileus, with a laccate or glabrous dark brown slightly brownish red upper surface, usually tough when dried; margin usually has a reddish gray surface, round and hard; pore surface pale yellow when young, light yellow when mature, and becoming brown or dark brown when bruised; basidiospores are described as ellipsoid, with size range of (7.3–)7.6–10.1(10.8) × (10.1)10.6–13.3(13.9) μm (including myxosporium), (5.4–)5.6–8.3(8.6) × (8.3)8.4–12.5(12.9) μm (excluding outer myxosporium); context trimitic, abundant generative hyphae with branches; thin-walled, binding hyphae; and skeletal hyphae with clamp connections.Dipterocarpus species in deciduous forests.Solitary on living 19°48'24"N, 100°03'54"E, 836 m, October, 2017.Thailand, Chiang Rai Province, G.tropicum , growth rates (mm/day), and mycelial density were screened as an indication of favorable growth of Thai um Table . After 1G.tropicum growth on CZA medium were expressed as a very scanty, cotton colony  massive cottony colony with orange white to pale orange (6A1–6A3) colony  white cotton colony . This finding is consistent with those of G.tropicum forms a sister clade with G.multipileum, exhibiting macro-morphological characteristics of strongly echinulate basidiospores 7.6–8.2–10.1(–10.8) × (10.1–)10.6–11.7–13.3(–13.9) μm , and (5.4–)5.6–7.1–8.3(–8.6) × (8.3)8.4–10.8–12.5(–12.9) μm  mm (excluding outer myxosporium); the pore surface is pale yellow (2A3) with pore are 4–7 per mm, and the tubes are 2–7 mm long with a light yellow to dark brown context. The original description of G.tropicum has the basidiospores fasciculate, 7–9 × 10–12 µm with 4–5 µm of hymenia hyphae . These characteristics are in accordance with the basidiospore sizes we recorded for the Thai strain of G.tropicum. The strain of G.tropicum from South America shares much in common with the Thai strain; however, notable differences in the South American strain include light brown ovoid basidiospores, a pileus of dark to black coloring at the base, and a blunt to slightly round margin. Our results of G.tropicum are in accordance with the description of G.tropicum collected from Hainan Province. This Hainan strain had the following characteristics: pileus size 4–8 × 2.5–6 cm, up to 1 cm thick at the base, basidiospores were described as (10.8–)11.2–12.1–12.8(–13.1) × (8.3–)9.6–10.1–10.8(11.1) μm (with myxosporium) and (7.9–)8.8–9.1–10.2(–10.8) × (5.8–)6.4–7.3–7.8(–9.8) μm (without myxosporium), with shared white to orange pore surface.Our morphological analyses show that the Thai PDA, MEA, and YPD media. These three media are composed of high concentrations of dextrose as a carbon source, while various forms of carbon sources have been reported as affecting fungal mycelial growth . The optimal PDA media at pH 7, incubated within the temperature range at 25–28 °C, were found to be the most suitable for G.tropicum mycelial growth, while temperatures lower than 15 °C or higher than 30 °C are not suitable for mycelial growth. G.australe grew well on PDA media, at a pH of 7–8 and at temperature range of 25–30 °C. This study revealed similar mycelial growth conditions with other Ganoderma species that have been previously evaluated (G.tropicum has a high potential for commercial production. Further studies will evaluate the best method to optimize the mushroom spawn and growing substrate for bringing Thai G.tropicum cultivation into high-yield production, and also establish whether other Ganoderma species remain to be discovered in Thailand.The optimal conditions for mycelial growth were investigated based on medium, pH, and temperature. The best growth rates were obtained using l growth . Althougvaluated . In lighGanodermatropicum from Northern Thailand based on morphological characteristics together with phylogenetic analyses. The optimal conditions for promoting the mycelial growth of G.tropicum were investigated and the best media and pH for mycelia growth were found to be PDA, MEA, and YPD media at pH 7–8, respectively. The optimal temperature was found to be 25–30 °C.This study confirmed the new record of