Over 240 million people are chronically infected with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. The quantification of the HBV DNA level is critical for monitoring the efficacy of antiviral treatment of chronic HBV patients. artus HBV QS-RGQ assay to the CAP/CTM v2.0 test, as a reference method, on 142 Moroccan patients. The analytical performance of the artus HBV QS-RGQ assay, such as the limit of detection, quantification, precision, reproducibility, and linearity, was determined using dilution series from 10 to 0.1 log10 IU/mL.  In our study, we compared the performance of the artus HBV QS-RGQ assay were significantly lower than those from the CAP/CTM v2.0 assay . A Bland-Altman plot found a mean difference of (CAP/CTM v2.0 − artus HBV QS − RGQ) = 0.5717 log10 IU/mL, with an average range of -1.13 to 2.31 log10 IU/mL. The two methods demonstrated a high correlation (r = 0.88) for 100 positive samples, a moderate correlation for samples below 2000 IU/mL (r = 0.76), and a very high correlation for the samples above 2000 IU/mL (r = 0.95). Linearity of the artus QS-RGQ test ranged from 1.07 to 7.51 log10 IU/mL.  Detection rates and viral loads quantified by the artus HBV QS-RGQ assay showed a strong correlation, precision, and linearity in comparison with the CAP/CTM v2.0. However, viral loads determined by the artus HBV QS-RGQ assay were lower than those determined by the CAP/CTM v2.0 assay. The  Worldwide, about 240 million people are HBV surface antigen- (HBsAg-) positive chronic carriers . The preA total of 142 HBsAg-positive samples, using an automatic chemiluminescence immunoassay analyzer , were included in the current study. All samples were collected between May 2018 and May 2019 at the molecular biology laboratory at the Institute Pasteur of Morocco . Samples were quantified by the Roche CAP/CTM v2.0 platform, as a routine practice. Analyzed plasma was separated from EDTA whole blood, and the remaining amounts were stored at -20°C and analyzed by the QIAGEN EZ1 Advanced XL/Rotor-Gene Q platform. Serologically testing, all patients had been diagnosed positive for HBsAg.μL of the serum or plasma sample. In our case, we used 650 μL of plasma samples and the nucleic acid was extracted in 45 min using magnetic particle technology. HBV DNA (50 μL) was analyzed by the COBAS TaqMan 48, system for real-time automated amplification, and nucleic acid detection using primers and probes targeting the highly conserved precore and core region. Amplification concerns two targets: HBV DNA and the internal quantitation standard (QS), and results were given in IU/mL with a conversion factor of 5.82 copies/IU of HBV DNA. The CAP/CTM HBV v2.0 system offers a broader dynamic range, from as low as 2 × 101 IU/mL to as high as 1.7 × 108 IU/mL (1.3–8.2 log10 IU/mL), according to the manufacturer and required 150 min to be amplified and detected by the COBAS TaqMan 48.The CAP/CTM HBV v2.0 assay (Roche Molecular Diagnostics) is a fully automated viral load quantitative hepatitis B test used in the management of patients with chronic hepatitis B infection undergoing antiviral therapy. The test treats 650 μL of plasma samples using the EZ1 advanced XL instrument, using the EZ1 DSP virus kit (QIAGEN) based on magnetic bead technology. For each HBV plasma sample, a premix of 7.9 μL internal control, 4 μL of cRNA, and 54.1 μL of AVE were added to the elution tube, resulting in a final volume elution of 60 μL. 20 μL of extracted HBV DNA were added to 30 μL of the master mix, containing all necessary reagents and enzymes for specific amplification of a 134 bp region of the HBV core gene, provided by the artus HBV QS RGQ kit. PCR reaction was insured by the Rotor-Gene Q platform for 107 min. Results were provided in IU/mL with a conversion factor of 8.21 copies/IU. The linear range offered by the artus HBV QS-RGQ assay covers concentrations from 3.16 × 101 IU/mL to 2 × 107 IU/mL (1.5–7.3 log10 IU/mL), and analytical sensitivity (LOD) is 10.21 IU/mL, according to the manufacturer.HBV DNA was extracted in 43 min from 400 1 IU/mL for CAP/CTM HBV v2.0 and 3.16 × 101 IU/mL for artus HBV QS-RGQ, in the current study.Interpretation of the HBV viral load results is based on the limit of detection (LOD) of each assay and was recorded either as target detected or as target not detected, while the quantitative result concern was conducted only to patients with viral loads above 2 × 1010 for further statistical analysis using IMB SPSS Statistics 25 . The Spearman correlation coefficient (r) was calculated to determine the linear relationship between the two assays, and the Bland-Altman analysis was used to assess the agreement between the two methods of viral load measurement , and >1 log10 IU/mL. For all analyses, p values < 0.05 with the two-tailed test referred to statistical significance.All results were converted to IU/mL and transformed to logsurement . The biaartus HBV QS-RGQ and the CAP/CTM HBV v2.0 assays. Twenty samples yielded undetectable loads on both assays. Thirty-seven samples showed undetectable results (<31.6 IU/mL) using the artus HBV QS-RGQ assay, 17 of which generated quantitative results on the CAP/CTM HBV v2.0 (≥20 IU/mL). However, one sample, undetectable on the CAP/CTM HBV v2.0, was measured using the artus HBV QS-RGQ assay and generated a low signal. This result was verified, by double reextraction of the sample followed by its amplification five times. The obtained signals for such sample were between 6.06 IU/mL and 12.7 IU/mL  using the CAP/CTM v2.0. To determine the actual sample concentration, the two samples were diluted 10- and 100-folds and the concentrations were calculated as 1.28 × 1010 IU/mL (10.11 log10 IU/mL) and 1.28 × 109 IU/mL (9.11 log10 IU/mL), respectively. The two samples were tested 3 and 4 times using the artus HBV QS-RGQ assay, respectively. For each sample, two of the runs resulted in no given concentrations. The four missing values were determined using the five HBV quantitation standard graphs (concentration and Ct) which range from 104 IU/mL to 108 IU/mL. The results were 3.01 × 107 IU/mL (7.47 log10 IU/mL), 2.57 × 107 IU/mL (7.41 log10 IU/mL), 3.18 × 107 IU/mL (7.5 log10 IU/mL), and 3.22 × 107 IU/mL (7.51 log10 IU/mL), respectively.Concerning high viral loads, two samples showed concentrations above the maximal value of detection (1.7 × 10artus HBV QS-RGQ assay and 117 samples (82.39%) by the CAP/CTM HBV v2.0 assay. The mean ± SD of the HBV DNA level of detection was 3.34 ± 1.94 log10 IU/mL for the artus HBV QS-RGQ assay and 3.91 ± 2.45 log10 IU/mL for the CAP/CTM HBV v2.0 assay .Among the 142 tested samples, HBV DNA was quantified in 105 samples (73.94%) by the .0 assay . The limartus HBV QS-RGQ assay) and were uniformly distributed between 20 IU/mL and 1.28 × 1010 IU/mL. The Bland-Altman analysis revealed the mean difference between the two assays (CAP/CTM HBV v2.0–artus HBV QS-RGQ) as m = 0.5717 log10 IU/mL, with an average range ± 1.96 SD of -1.13 to 2.31 log10 IU/mL (SD = 0.87) (p < 0.01) (r = 0.88) showed a strong correlation between the CAP/CTM HBV v2.0 and the artus HBV QS-RGQ assays  of intercept: 0.179 to 0.636; slope: 0.7 to 0.8; R2 = 0.90) (p < 0.01) (n = 47) were ≤0.5 log10 IU/mL of difference between the two methods, 20% (n = 20) were between 0.5 and 1 log10 IU/mL, and 33% (n = 33) were >1 log10 IU/mL. Six samples were outliers (6%), three of which read higher on the CAP/CTM HBV v2.0.One hundred samples yielded positive results for both assays (≥20 IU/mL for the CAP/CTM HBV v2.0 and ≥31.6 IU/mL for the  < 0.01) . The Spe < 0.01) . Among tr = 0.76; p < 0.01) and a linear regression's equation as follows: [artus HBV QS − RGQ] = 0.157 + 0.721 [CAP/CTM HBV v2.0]; 95% CI of intercept: -0.234 to 0.548; slope: 0.551 to 0.891; R2 = 0.47 (10 IU/mL with an average range ± 1.96 SD of -1.22 to 2.06 log10 IU/mL (p < 0.01). Conversely, samples with HBV DNA levels above 2000 IU/mL (n = 38) showed a high correlation between the two assays  (10 IU/mL with an average range ± 1.96 SD of -1.25 to 3.1 log10 IU/mL (p < 0.01).Eighty-four samples were found positive in at least one assay, with HBV DNA levels < 2000 IU/mL. They were quantified by both assays and showed moderate correlation (Spearman's coefficient: 2 = 0.47 . The Bla = 0.90) . The Bla10 IU/mL in duplicate per each concentration. Each dilution was prepared using 0.1 mL of the sample and 0.9 mL of HBV-negative plasma. Results ranged between 1.07 and 7.51 log10 IU/mL. The straight line and regression statistics were determined using a linear regression of the log10-calculated concentrations with the log10 nominal concentrations: y = −0.03460 + 0.7779 x; R2 = 0.9908 , medium (1 × 107 to 2 × 103 IU/mL), and low (<2 × 103 IU/mL) HBV DNA titers. The SD and the coefficient of variation (CV) were determined. The CV ranged from 0.62% to 81.23% and from 0.00% to 3.35% for the artus HBV QS-RGQ and the CAP/CTM HBV v2.0 assays, respectively  and, during the chronic infection, can vary from undetectable to more than 10sistance . To the r2 = 0.90) was detected. Our results were similar to those reported by Brichler et al. who found a good correlation and agreement of r2 = 0.89 between the two methods [r2 = 0.86, and agreement between the two assays [In our study, a strong overall correlation and agreement between the two assay methods r2 = 0.0 was deto assays .10 IU/mL of difference between the two methods. This difference is not clinically significant and does not affect the results. In this regard, Pawlotsky reported that differences or variations of less than 0.5 log10 IU/mL should not be taken into account, as they may be due to intrinsic or between-patient variability [10 IU/mL of difference, revealing higher HBV DNA loads quantified by the CAP/CTM HBV v2.0 in comparison with the artus HBV QS-RGQ. Those results are close to the results reported by Brichler et al., who found that the difference in 23% of the samples exceeded ±0.5 log10 IU/mL and concluded that HBV DNA loads by the artus HBV QS-RGQ assay were lower than the results by the CAP/CTM HBV v2.0 assay [10 IU/mL. In contrast, the difference in 33% of the samples in the current study was above 1 log10 IU/mL which disagree with Yeh et al. who found such a difference was recorded in only 8.6% of the examined samples [n = 17) yielded detectable results only with the CAP/CTM HBV v2.0, while 3.5% of samples (n = 5) yielded detectable low signals only with the artus HBV QS-RGQ assay. The artus HBV QS-RGQ manufactures report that, while testing their HBV DNA load samples in comparison with the CAP/CTM HBV v2.0, 3 of 189 samples (1.59%) were detected only by the artus HBV QS-RGQ. This difference in HBV DNA load detection might be explained by the low HBV load or mutations in the precore and core promoter regions, which are highly prevalent in Morocco [10 IU/mL [Overall, 47% of samples showed ≤0.5 logiability . However.0 assay . In addi samples . Additio Morocco , 14. Mor Morocco . Other s10 IU/mL . Althoug10 IU/mL , 17, 14.n = 84) and above 2000 IU/mL (n = 38). As results, we found that the HBV DNA levels determined by the artus HBV QS-RGQ assay were lower than those determined by the CAP/CTM HBV v2.0 test, showing a moderate correlation for the low viral loads (<2000 IU/mL) estimated to r = 0.76, higher than the correlation found by Han et al. (r = 0.49), according to Spearman's coefficient [r = 0.71 between the COBAS 4800 HBV test and the CAP/CTM HBV v2.0 assay, comparing samples with HBV DNA levels below 2000 IU/mL [r2 = 0.90. Medium to high viral loads mostly show a good correlation between two methods of measurements. Shin et al. also reported a strong correlation (r = 0.95) between the COBAS 4800 HBV test and the CAP/CTM HBV v2.0 assay for samples with HBV DNA levels above 2000 IU/mL [Hepatitis B infection has a broad virological and clinical spectrum while antiviral therapy is not indicated for all patients. According to EASL 2017 recommendations, patients who have HBV DNA > 2000 IU/ml and, at least, moderate fibrosis may initiate treatment even if alanine aminotransferase (ALT) levels are normal, whereas patients with HBeAg-negative chronic HBV infection and HBV DNA ≥ 2000 IU/mL should be followed with ALT determinations at least every 3 months for the first year and every 6 months for at least 3 years [fficient . Another00 IU/mL . Convers00 IU/mL .artus HBV QS-RGQ assay was about r2 = 0.991, close to that given by the manufacturer (r2 = 0.999).Linearity of HBV DNA loads using the The limitations of the current study included relatively low sample size and inability to genotype the samples.artus HBV QS-RGQ assay were lower than those quantified by the CAP/CTM HBV v2.0 test. However, this study demonstrated the satisfactory fastness and performance of the artus HBV QS-RGQ assay at quantifying viral HBV DNA and managing patients with chronic HBV infection. Other than that, the EZ1 DSP virus kit has the benefit of copurifying both viral RNA and DNA with one chemistry and one purification protocol.In summary, results of viral loads quantified by the


T. thermophilus Mfd-RNAP complex with and without ATPγS. The structures reveal that Mfd undergoes profound conformational changes upon activation, contacts the RNAP β1 domain and its clamp, and pries open the RNAP clamp. These structures provide a foundation for future studies aimed at dissecting the precise mechanism of Mfd-dependent transcription termination and pave the way for rational drug design targeting Mfd for the purpose of tackling the antimicrobial resistance crisis.Mfd-dependent transcription termination plays an important role in transcription-coupled DNA repair, transcription-replication conflict resolution, and antimicrobial resistance development. Despite extensive studies, the molecular mechanism of Mfd-dependent transcription termination in bacteria remains unclear, with several long-standing puzzles. How Mfd is activated by stalled RNA polymerase (RNAP) and how activated Mfd translocates along the DNA are unknown. Here, we report the single-particle cryo-electron microscopy structures of  Mfd is a highly conserved ATP-dependent DNA translocase in bacteria. It recognizes stalled RNA polymerase (RNAP) and removes it from DNA, leading to Mfd-dependent transcription termination. Mfd-dependent transcription termination has several physiological roles.The best-characterized function of Mfd-dependent transcription termination is transcription-coupled DNA repair (TCR), which is a sub-pathway of nucleotide excision repair (NER) ,2. In TCMfd-dependent transcription termination has also been suggested to play important roles other than TCR. For example, replisome and RNA polymerase translocate along the same DNA template, often in opposite directions. These processes routinely interfere with each other and lead to catastrophic effects on genome stability and cell viability . Mfd mayMycobacterium tuberculosis, by increasing the mutation rates , an RNAP interacting domain (RID), a translocation module (TM), and a C-terminal domain (CTD) Figure . BiochemIn the crystal structures of Mfd , the conEscherichia coli RNAP-σ70 holoenzyme was purified and assembled as previously described (T. thermophilus RNAP core enzyme and T. thermophilus RNAP-σA holoenzyme were purified and assembled as reported (escribed . T. therreported . NusG wareported .Escherichia coli strain BL21(DE3)  was transformed with plasmid pET28a-NH-EcoMfd encoding N-hexahistidine-tagged E. coli Mfd under the control of T7 promoter. Single colonies of the resulting transformants were used to inoculate 1 l LB broth containing 50 μg/ml kanamycin, cultures were incubated at 37°C with shaking until OD600 = 0.6. Protein expression was induced by addition of IPTG to 1 mM, and cultures were incubated 4 h at 30°C. Then cells were harvested by centrifugation , resuspended in 20 ml buffer A  and lysed using a JN-02C cell disrupter . The lysate was centrifuged , and the supernatant was loaded onto a 2 ml column of Ni-NTA agarose  equilibrated with buffer A. The column was washed with 10 ml buffer A containing 0.04 M imidazole and eluted with 10 ml buffer A containing 0.5 M imidazole. The sample was further purified by anion-exchange chromatography on a Mono Q 10/100 GL column . Fractions containing E. coli Mfd were pooled and stored at –80°C. E. coli Mfd derivatives were expressed and purified in the same way as wild type protein. Yields were ∼10 mg/l, and purities were >95%.Escherichia coli strain BL21(DE3)  was transformed with plasmid pET28a-NH-TthMfd encoding N-hexahistidine-tagged T. thermophilus Mfd under the control of T7 promoter. Single colonies of the resulting transformants were used to inoculate 1 l LB broth containing 50 μg/ml kanamycin, cultures were incubated at 37°C with shaking until OD600 = 0.6. Protein expression was induced by addition of IPTG to 1 mM, and cultures were incubated 4 h at 30°C. Then cells were harvested by centrifugation , resuspended in 20 ml buffer B  and lysed using a JN-02C cell disrupter . The lysate was centrifuged , and the supernatant was loaded onto a 2 ml column of Ni-NTA agarose  equilibrated with buffer B. The column was washed with 10 ml buffer B containing 0.1 M imidazole and eluted with 10 ml buffer B containing 0.5 M imidazole. The eluate was loaded onto a 5 ml column of HiTrap Heparin HP  equilibrated in buffer B and eluted with a 100 ml linear gradient of 0.2–1 M NaCl in buffer B. Fractions containing T. thermophilus Mfd were pooled and stored at –80°C. T. thermophilus Mfd derivatives were expressed and purified in the same way as wild type protein. Yields were ∼2 mg/l, and purities were >95%.T. thermophilus Mfd, 0.4–2 mM ATP, 50 mM Tris–HCl (pH 7.9), 0.1 M KCl, 10 mM MgCl2, 1 mM DTT and 5% glycerol. Reaction mixtures were incubated 60 min at 37°C. 200 μl reagent  was added to terminate the enzyme reaction and generate the colorimetric product. The absorbance at 620 nm was measured using a SpectraMax M5 microplate reader . Phosphate standard solution was used to calculate the extinction coefficient of the colorimetric product. Less than 2% ATP was consumed to make sure initial velocity was measured.ATPase activity assays were performed in a 96-well microplate format using a commercial kit . Reaction mixtures contained (40 μl): 5 μM Template strand DNA oligonucleotide and nontemplate strand DNA oligonucleotide  were annealed at a 1:1 ratio in 10 mM Tris–HCl, pH 7.9, 0.2 M NaCl and stored at −80°C.T. thermophilus RNAP displacement assay was performed in reaction mixtures containing (20 μl): 2 μM RNAP holo enzyme, 0.1 μM DNA scaffold, 2 mM ATP, 0.2 mM UTP, 0.2 mM GTP, 50 mM Tris–HCl (pH 7.9), 0.1 M KCl, 10 mM MgCl2, 1 mM DTT and 5% glycerol. Reaction mixtures were incubated 10 min at 65°C, supplemented with 0.1 mg/ml heparin and 4 μM Mfd or Mfd derivative, incubated 10 min at 65°C.E. coli RNAP displacement assay was performed in reaction mixtures containing (20 μl): 0.1 μM RNAP holo enzyme, 0.13 μM DNA scaffold, 2 mM ATP, 0.2 mM UTP, 0.2 mM GTP, 50 mM Tris–HCl (pH 7.9), 0.1 M KCl, 10 mM MgCl2, 1 mM DTT and 5% glycerol. Reaction mixtures were incubated 10 min at 37°C, supplemented with 0.1 mg/ml heparin and 1 μM Mfd or Mfd derivative, incubated 10 min at 37°C.The reaction mixtures were applied to 5% polyacrylamide slab gels (29:1 acrylamide/bisacrylamide), electrophoresed in 90 mM Tris-borate, pH 8.0 and 0.2 mM EDTA, stained with 4S Red Plus Nucleic Acid Stain  according to the procedure of the manufacturer.ATPγS was prepared in reaction mixtures containing (31 μl): 14 μM T. thermophilus RNAP core enzyme, 16 μM nucleic acid scaffold, 17 μM T. thermophilus Mfd, 5 mM MgCl2 and 2 mM ATPγS. MTCapo was prepared in reaction mixtures containing (31 μl): 18 μM T. thermophilus RNAP core enzyme, 19 μM nucleic acid scaffold, 21 μM T. thermophilus Mfd, 5 mM MgCl2. T. thermophilus RNAP core enzyme was incubated with nucleic acid scaffold for 10 min at 4°C, and incubated with T. thermophilus Mfd for 10 min at 4°C.DNA oligonucleotides and RNA oligonucleotide  were glow-discharged for 60 s at 15 mA prior to the application of 3 μl of the complex, then plunge-frozen in liquid ethane using a Vitrobot  with 95% chamber humidity at 10°C.E. coli Mfd complex with ATPγS were collected, but there is no density for Mfd in the final map. Then we turned to determine the structure of T. thermophilus Mfd complex. The grids were imaged using a 300 kV Titan Krios  equipped with a K2 Summit direct electron detector . Images were recorded with Serial EM  and 1 149 168 particles (MTCapo) were auto-picked, extracted with a box size of 200 pixels, and subjected to 2D classification in RELION (E. coli TEC (EMD-8585) (ATPγS), 24 037 (MTCapo) and 558 003 (TEC).In the preliminary experiment, some data of erial EM  in counttionCor2 . The contionCor2 . From thMD-8585)  low-passT. thermophilus Mfd was generated on Phyre2 server (T. thermophilus RPo (PDB 4G7H) (T. thermophilus Mfd were fitted into the cryo-EM density map using Chimera (The homology model of 2 server . The modDB 4G7H)  and the  Chimera . The mod Chimera . The coo Chimera .2 and 2 mM ATPγS. Following incubation mixtures for 10 min at 25°C, fluorescence emission intensities were measured using a SpectraMax M5 microplate reader . Fluorescence polarization was calculated using:IVV and IVH are fluorescence intensities with the excitation polarizer at the vertical position and the emission polarizer at, respectively, the vertical position and the horizontal position.5′ 6-FAM labeled DNA oligonucleotide  and unmodified DNA oligonucleotide  were annealed at a 1:1 ratio in 10 mM HEPES, pH 7.5, 50 mM KCl. Equilibrium fluorescence polarization assays were performed in a 96-well microplate format. Reaction mixtures contained (100 μl): 0–16 μM Mfd or Mfd derivative, 0.1 μM 6-FAM-labelled DNA scaffold, 10 mM HEPES, pH 7.5, 50 mM KCl, 5 mM MgClKD, were extracted by non-linear regression using the equation:P is the fluorescence polarization at a given concentration of Mfd, Pf is the fluorescence polarization for free 6-FAM-labeled DNA scaffold, Pb is the fluorescence polarization for bound 6-FAM-labeled DNA scaffold, and [M] is the concentration of Mfd or Mfd derivative.Equilibrium dissociation constant, E. coli RNAP and Mfd is transient, the initial attempt to determine the structure of E. coli Mfd-dependent transcription termination complex (MTC) failed. In the preliminary experiment, some data of E. coli MTC with ATPγS were collected, but there is no density for Mfd in the final map. Then we turned to determine the structure of T. thermophilus MTC. The RID and the TM are highly conserved between E. coli Mfd and T. thermophilus Mfd . El. ElE. coled RNAP .T. thermophilus MTC, we modified the scaffold, which has been used for structure determination of a TEC (ATPγS and MTCapo) were determined at 4.1 and 5.0 Å, respectively  motif from TD2 is highly conserved among RecG-like SF2 helicases. Its antiparallel helical hairpin conformation is critical for coupling nucleotide hydrolysis to duplex translocation  and translocation domain 2 (TD2), contains the characteristic motifs that identify Mfd as a RecG-like SF2 helicase . The TRGlocation ,37.E. coli, M. smegmatis, M. tuberculosis and T. thermophilus) have been solved in complex with different partners  using different methods   Figure ,19,38,39ATPγS and 36° in MTCapo at position –16 (E. coli Mfd), which is positioned near the DNA backbone, shows defects in DNA binding assay and RNAP displacement assay , in, in2 , wh, wh2 , EMD-30118 (MTCATPγS), and EMD-30119 (TEC). The accession numbers for the atomic coordinates reported in this paper are Protein Data Bank: 6M6A (MTCapo), 6M6B (MTCATPγS), and 6M6C (TEC).The accession numbers for the cryo-EM density map reported in this paper is Electron Microscopy Data Bank: EMD-30117 (MTCgkaa904_Supplemental_FilesClick here for additional data file.


Yeasts are important microorganisms used for ethanol production; however, they are not equally efficient in the amount of ethanol production under different environmental conditions. It is, therefore, necessary to screen for elite strains to utilize them for commercial production of these commodities. In this study, yeasts were isolated from different Ethiopian traditional fermented alcoholic beverages , milk and ergo, teff and maize dough, soil and compost, flowers, and fruits to evaluate their potential use for ethanol fermentation process. Isolates were screened for efficient ethanol production and the selected ones were identified using phenotypic and genetic characters using D1/D2 region of LSU rDNA sequence analysis. The yeast isolates were evaluated based on their growth and fermentation of different carbon sources. Response surface methodology (RSM) was applied to optimize temperature, pH and incubation time using central composite design (CCD) in Design-Expert 7.0.0. A total of 211 yeasts colonies were isolated of which 60% were ethanologenic yeasts (ethanol producers) and 40% were non-ethanol producers. The yeast population detected from various sources was in the range of 105CFU from traditional foods and beverages to that of 103CFU from fruits and soil samples. The data also showed that the number of colony types (diversity) did not correlate with population density. The highly fermentative isolates were taxonomically characterized into four genera, of which 65% of the isolates  were categorized under Saccharomyces cerevisiae, and the remaining were Pichia fermentans ETP22, Kluyveromyces marxianus ETP87, and Candida humilis ETP122. The S. cerevisiae isolates produced ethanol (7.6-9.0 g/L) similar with K. marxianus ETP87 producing 7.97 g/L; comparable to the ethanol produced from commercial baker's yeast (8.43 g/L) from 20 g/L dextrose; whereas C. humilis ETP122 and P. fermentans ETP22 produced 5.37 g/L and 6.43 g/L ethanol, respectively. S. cerevisiae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122 tolerated 10% extraneous ethanol but the percentage of ethanol tolerance considerably decreased upon 15%. S. cerevisiae ETP53 produced ethanol optimally at pH 5.0, 60 h, and 34oC. pH 4.8, temperature 36oC, and 65 h of time were optimal growth conditions of ethanol fermentation by K. marxianus ETP87. The ethanol fermentation conditions of P. fermentans ETP22 was similar to S. cerevisiae ETP53 though the ethanol titer of S. cerevisiae ETP53 was higher than P. fermentans ETP22. Therefore, S. cerevisiae ETP53, K. marxianus and P. fermentans ETP22 are good candidates for ethanol production. •Among 211 yeast isolates, 126 were able to ferment dextrose.•Saccharomyces cerevisiae, Pichia fermentans, Kluyveromyces marxianus, and Candida humilis were isolated from traditional alcoholic drinks, compost, ergo, and enjera dough, respectively.•Kluyveromyces marxianus ETP87 can be used to produce ethanol from whey.•Saccharomyces cerevisiae, Kluyveromyces marxianus and Pichia fermentans are good candidate for ethanol production. Mohd AzPlants are the preferred habitats for yeasts and they include nectars, flowers, fruits, decaying tissues, and tree saps ,4. TheseKluyveromyces (46.8%), Sporobolomyces (31.5%), Candida (12.5%), Torulopsis (6%) and Leucosporidium (3.2%)  RNase A solution (Qiagen), 10 mM potassium phosphate, 10 mM EDTA, 0.8 M NaCl, pH 7.0), and incubated at 37 °C for 1.5 h. Approximately, 50 μL of Φ 0.8 mm glass beads and 67 μL of SDS/ProK solution , 5 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, pH 8.0) were added to each tube. The tube was vortex-mixed for 1.5 min, and incubated at 60 °C for 10 min. After this, 87 μl of 3 M sodium acetate (pH 5.2) solution was added, vortex-mixed, and chilled on ice.The tube was centrifuged at 15,000 rpm for 5 min at 4 °C from which 70 μL of the supernatant was transferred to a well of AcroPrep™ 96 Multi-Well Filter Plate with 3.0 μM glass fiber media/0.2 μM Bio-Inert® membrane, natural housing (PALL Life Science), to which 110 μL of isopropanol was added and mixed well by pipetting. After incubation for 3 min at room temperature (15–20 °C), the filter plate was vacuumed with a vacuum manifold device. The well was rinsed with 200 μL of 70% [w/v] ethanol twice in vacuum. After the filter plate was air-dried, 60 μL of TE buffer  was poured in the well, and incubated for 3 min. The filter plate was placed onto a new 96-well plastic plate. Two plates were centrifuged at 3000 rpm for 5 min at room temperature. The DNA was resuspended with TE buffer, centrifuged twice, and kept at −20 °C for further use.2.5.2xl Genetic Analyzer (Applied Biosystems) following the manufacturer's instruction. Sequence data were corrected by manual inspection whenever needed, and aligned using BioEdit Sequence Alignment Editor version 7.1.3.0   3] also S. cerevisiae NS G-48, S. cerevisiae NC007, S. cerevisiae CBS 2984, and S. cerevisiae CBS 5493 from NCBI data base; they were also similar with S. cerevisiae CBS 6308 (CBS data base) with 100% confidentiality. Based on sequence analysis of the first 600 nucleotides from 5’ end of LSU of rRNA and comparison from NBCI blast, ETP87 was found to be 100% alike with K. marxianus U-MF11, K. marxianus Y12, K. marxianus CBS 5672 and K. marxianus IMAU6Y146 (DX9-2). D1/D2 region of ETP22 matched 100% with P. fermentans A5, P. fermentans KDLYH2-3, and P. fermentans CBS 5662 (from NCBI). The overlapping between ETP22 and P. fermentans CBS 6662 and CBS 5663 (CBS database) was 100 and 99.825% respectively. The same region sequence analysis of ETP122 exhibited that the sequence was 100% identical with C. humilis H17 and C. humilis IMAU Y10085 that were accessed from NCBI. The resemblance of ETP122 and C. milleri CBS 6897 from CBS data base was 99.825%.D1/D2 domain of 26 S rRNA of ETP37, ETP50, ETP53, ETP89, and ETP94 was 100% similar with 3.4Saccharomyces cerevisiae containing four isolates; ETP37, ETP50, ETP53, ETP89 isolated from beverages tella, teji, and shamita and ETP94 isolated from flower sample. The others were identified as Kluyveromyces marxianus, ETP87 isolated from the milk product, ergo, Pichia fermentans, ETP22 isolated from compost, and Candida humilis (milleri), ETP122 isolated from enjera dough. These isolates produced ethanol ranging from 5.37 g/L (ETP122) to 9.0 g/L (ETP53) . Thus, the Saccharomyces cerevisiae isolates were dominant both in terms of number (65%) and production of alcohol (7.67g/L-9.0 g/L). It is interesting to note that the non-Saccharomyces yeast, K. marxianus was as equally efficient in alcohol production (7.97gL) as that of S. cerevisiae isolates.The different isolates were screened for efficient ethanol production, of which 8 isolates were selected and characterized . These y (ETP53) . Thus, t3.5Almost all of the selected isolates were capable of growing and vigorously fermenting glucose, fructose and galactose, and majority of them utilized mannose, raffinose, maltose and sucrose . One or K. marxianus ETP87 was capable of utilizing the maximum number of sugars (78%) and vigorously fermented glucose, galactose, fructose, and raffinose, lactose and sucrose unlike the other isolates that were limited to grow on and ferment fewer substrates. The different S. cerevisiae strains were consistent in their growth and fermentation of the majority of the sugars (50%–60%).Out of the isolates, K. marxianus ETP87 was the only yeast that ferments lactose besides glucose, galactose, fructose, maltose and sucrose within 24 h. It could be good candidate for ethanol production from whey. K. marxianus would only ferment dextrose and assimilate dextrose and xylose among carbon sources tested  al alK. marP. fermentans ETP22 and Candida humilis (milleri) ETP122 were capable of growing on the majority of the sugar substrates (60–65%), but fermented fewer (20–30%) of the substrates indicting that they were not efficient alcohol fermenters. On the other hand, however, P. fermentans ETP22 which was isolated from compost, fermented glucose, mannose and fructose though dextrose fermentation was rarely reported before  al alSaccharevisiae ,20.Saccharomyces yeasts for large scale production of ethanol. However, De la Torre-González et al.  re rePichia5S. cerevisiae ETP53, K. marxianus ETP87, and P. fermentans ETP22 showed similar trend in ethanol production to the commercial baker's yeasts. The high survival rate (40–70%) of the non-Saccharomyces yeasts was similar to the S. cerevisiae strain, under 10% ethanol shock treatment which is a desirable characteristic of high fermenting yeasts. Their ability to grow and ferment different monosaccharaides , and disaccharides could make them good candidates for ethanol production from different carbon sources, whey, and lignocellulosic hydrolysates. It is particularly interesting to note that the non-Saccharomyces yeast, K. marxianus ETP87 utilized and vigorously fermented more sugars than even Saccharomyces cerevisiae strain ETP53, and was as equally efficient in ethanol production as the latter, and the only isolate that grew and vigorously ferment lactose could be singled out for further studies under optimum conditions.This study revealed that the local strains of S. cerevisiae ETP53 at 100 g/L dextrose with 5.5S. cerevisiae ETP53, K. marxianus ETP87 and P. fermentans ETP22 were 4.5–5.5, 28–30, and 48–76; 3.8–5.2, 35–40 and 48–76; and 4.5–6.0, 30–32, 48–80, respectively. The optimization, in this study, was performed using YPD. It is more appropriate if it is going to be done in the actual media in which ethanol is produced in industry.Optimal ethanol was produced by


Hedychium flavum Roxb., a medicinal, edible, and ornamental plant, is widely cultivated throughout China, India, and Southeast Asia. The rhizome from this plant has been used for food flavoring and in traditional Chinese medicine to treat diverse diseases, but the detailed constituents and bioactivities are still limited known. Therefore, phytochemical analysis by GC-MS and UHPLC-Q-Orbitrap-MS, and antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities tests have been conducted in the current study. Based on the GC-MS results, the essential oil (EO) of rhizome was mainly composed of coronarin E (20.3%), β-pinene (16.8%), E-nerolidol (11.8%), and linalool (8.5%). Among them, coronarin E was reported in H. flavum EO firstly. Furthermore, the spectrophotometric indicated rhizome had high total phenolic content  and total flavonoid content , no matter in water extract (WE) or in 70% ethanol extract (EE). UHPLC-Q-Orbitrap-MS was applied to further characterize composition, and 86 compounds were putatively identified from WE and EE, including 13 phenolic components. For the bioactivities, both WE and EE showed remarkable antioxidant activity by DPPH and ABTS tests, being superior to the positive control . EO revealed significant antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Proteus vulgaris with DIZ (10.34–24.43 mm), MIC (78.13–312.50 μg/mL), and MBC (156.25–625.00 μg/mL). Moreover, EO exhibited a considerable selectivity to human tumor cell K562 (IC50 = 27.16 μg/mL), and its toxicity was more than 3.5-fold different from that of non-cancerous MRC-5 cell (IC50 = 95.96 μg/mL) and L929 cell (IC50 = 129.91 μg/mL). A series of apoptosis analysis demonstrated that EO induced apoptosis against K562 cells in a dose-dependent manner. In enzyme inhibitory effect assays, WE and EE showed strong α-glucosidase inhibition activity, being superior to the positive control (acarbose). Besides, the EO, WE, and EE didn’t show a promising inhibition on tyrosinase (19.30–32.51 mg KAEs/g sample) and exhibited a weak inhibitory effect on cholinesterase. Based on the current results, H. flavum could be considered as a source of bioactive compounds and has high exploitation potential in the cosmetics, food, and pharmaceutical industries. Hedychium genus (Zingiberaceae), commonly known as ginger lily, includes approximately 87 species, which are mainly distributed in China, India, and Southeast Asia  and as a medicinal, edible, and ornamental plant in China, India, and Southeast Asia , β-pinene (21.8–24.77%), 1,8-cineole (8.90–28.3%), and linalool (6.29–17.5%) (β-pinene (22.5%), α-humulene (15.7%), and β-caryophyllene (10.4%), as well as the main EO components in stem were α-humulene (18.9%), β-caryophyllene (11.8%), and β-pinene (11.2%) (H. flavum were β-pinene (49.6%), β-caryophyllene (26.9%), α-pinene (5.9%), sabinene (4.2%), and α-humulene (2.3%)  . The maj (11.2%) . Besidese (2.3%) . The EO tivities .H. flavum has been developed as an industrial crop with medicinal, ornamental, and spice values. However, in addition to few reports on the chemical components, antifungal and insecticidal activities of EO, there is no detailed study on the chemical composition and bioactivities of H. flavum, which could impede its exploitation in industry. Therefore, phytochemical analysis by GC-MS and UHPLC-Q-Orbitrap-MS, and antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities tests of the rhizome from H. flavum were conducted in this work. To our knowledge, the antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities of EO, as well as the chemical composition and bioactivities of extracts from H. flavum rhizome were reported for the first time.8–C22) were from Merck . For UPLC-MS analysis, LC/MS-grade acetonitrile, formic acid, and ultrapure water were supplied from Merck . All other chemicals and solvents were of analytical grade and purchased from Aladdin .Gallic acid, rutin, streptomycin, cisplatin, acarbose, kojic acid, galanthamine, resazurin, and Folin-Ciocalteu reagent were obtained from Solarbio Life Sciences . Ascorbic acid, butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), acridine orange (AO), ethidium bromide (EB), α-glucosidase, tyrosinase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), p-Nitrophenyl-α-D-glucopyranoside (p-NPG), L-tyrosine, acetylthiocholine iodide (ATCI), and butyrylthiocholine chloride (BTCl) were purchased from Sigma-Aldrich (Germany). Hoechst 33258 and Annexin V-FITC/PI Apoptosis Detection Kit were from Beyotime . For GC-MS analysis, analytical standard n-alkanes .2SO4, filtered and stored at 4°C.To produce EO, the fresh, finely chopped rhizomes (1.5 kg) were placed into a Clevenger-type apparatus and hydrodistilled (4 h). EO was dried over anhydrous NaWE and EE were prepared by reflux extraction method. The fresh, finely chopped rhizomes (0.5 kg) were placed into a round bottom flask (2 L), mixed with distilled water or 70% ethanol, boiled under reflux for 2 h, and then filtered. The reflux extraction was repeated twice. The combined filtrate was concentrated with a rotary evaporator and dried in a freeze-drier. Then, WE and EE were weighed and stored at 4°C.m/z 29 to 500) and EI mode (70 eV). The interface temperature and ion source temperature were 280°C and 230°C, respectively. The relative percentage of chemical constituents was determined by the peak area. The retention index (RI) is determined by referring to a series of n-alkanes (C8–C22). The identification of chemical constituents was based on comparison of their retention index and mass spectrum with those in Wiley 275 and NIST 2017 databases.The EO was analyzed by an Agilent 6890 gas chromatograph equipped with a flame ionization detector (FID). Capillary column: HP-5MS . The flow rate of carrier gas helium was set at 1 mL/min and the split ratio was 1:20. The following GC oven temperature was used with the injection volume of 1 μL: held at 70°C (2 min), 2°C per min to 180°C (55 min), 10°C per min to 310°C (13 min), and kept at 310°C (12 min). The GC-MS analysis was performed using an Agilent 6890 gas chromatograph equipped with an Agilent 5975C mass selective detector . GC column and parameters were the same as in GC-FID. The mass spectra operated in the mass range  was added and incubated at room temperature for 60 min. The sample absorbance was read using a UV-5200 spectrophotometer  at 760 nm. TPC was expressed as equivalents of gallic acid (mg GAEs/g extract).The TPC was evaluated according to Folin-Ciocalteu method  with mar2-Al(NO3)3-NaOH colorimetry (2 (0.4 mL) and incubated for 6 min. Subsequently, Al(NO3)3 solution  was added and incubated for 6 min. Then, NaOH solution  was added up to a total volume of 10 mL with distilled water. The absorbance was read at 510 nm after 15 min incubation at room temperature. TFC was expressed as rutin equivalents (mg REs/g extract).The TFC was assayed by NaNOorimetry  with minThe chemical components of WE and EE were determined using ultra-high-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS). Chromatographic separation was carried out using a Dionex Ultimate 3000 UPLC System , with a Hypersil GOLD aQ  column. Mobile phases A and B were formic acid water  and formic acid acetonitrile , respectively. The flow rate was 0.3 mL/min. The gradient was as follows: 0–2 min, 5% B; 2–42 min, 5–95% B; 42–47 min, 95% B; 47.1 min, 5% B; 47.1–50 min, 5% B. The injection volume was 5 μL and the column temperature was held at 40°C.2, scanned from m/z 100 to 1,500 with a resolution of 70,000 in full scan MS1, followed by data-dependent MS2 acquisition at 17,500 resolution with stepped normalized collision energy (NCE) . Maximum injection time (IT) for MS1 and MS2 was separately set at 100 and 50 ms and the target values of automatic gain control (AGC) were 1e6 and 2e5, respectively. Loop count was 3, dynamic exclusion time was 5 s, and quadrupole isolation window (isolation width) was 1.5 m/z. The mass spectrum data of the chemical composition was processed with CD3.0 (Thermo Fisher Scientific) and identified by comparing mzVault, mzCloud, and ChemSpider databases. The allowable calculation error was set as 5 ppm.The Thermo Scientific Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometry (Q-Orbitrap-MS) equipped with heated electrospray ionization (HESI-II) source was used for MS data collection. HESI-II source was operated in negative and positive ionization modes with spray voltages of 2.5 and 3.0 kV, respectively. HESI-II source parameters were set as follows: capillary temperature 320°C, vaporizer temperature 350°C, sweep gas pressure 0 arb, auxiliary gas pressure 10 arb, sheath gas pressure 35 arb, RF lens amplitude (S-lens) 60. The mass spectrometer analysis was executed in the mode of full mass/dd-MS50 values and as equivalents of ascorbic acid (mg AEs/g sample).The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was evaluated according to the method with slight modification . Two mil+ solution was generated by reacting 2.45 mM K2S2O8 solution with and 7 mM ABTS solution incubated in the dark at room temperature for 12 h. Before experiments, to obtain an absorbance of 0.70 ± 0.02 at 734 nm, the ABTS•+ solution was diluted with methanol. The diluted ABTS•+ solution (4 mL) and sample solution (0.4 mL) were mixed and the absorbance was read at 734 nm after 10 min incubation in the dark at room temperature. Ascorbic acid and BHT were used as positive controls. The results were expressed using IC50 values and as equivalents of ascorbic acid (mg AEs/g sample).The 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging capacity was assayed according to the method with marginal modification . The ABTProteus vulgaris (CMCC (B) 49027), Escherichia coli (ATCC 25922), Bacillus subtilis (CMCC (B) 63501), Pseudomonas aeruginosa (CMCC (B) 10104), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 6538P).The antibacterial capacity of EO, WE, and EE was evaluated performed against the following bacterial strains: 6 CFU/mL) was evenly spread on the Mueller-Hinton agar medium. Filter paper discs (diameter 6 mm) containing pure essential oil, WE, EE, and streptomycin solution (20 μL) were added and the diameter of inhibition zone (DIZ) was recorded after 24 h incubation at 37°C.The inhibition zone diameter was assayed according to the agar well diffusion method with slight modification . Briefly5 CFU/mL. The 96-well plates were incubated for 24 h at 37°C. Subsequently, the resazurin aqueous solution  was added to each well and incubated for 2 h at 37°C in the dark. The MIC was determined as the minimum sample concentration without color change. For the determination of MBC value, the sample (10 μL) from the wells without color change was subcultured in Mueller Hinton agar plate for 24 h at 37°C. The MBC was defined as the minimum sample concentration showing no bacterial growth  and minimal bactericidal concentration (MBC) values were determined by the broth microdilution method with marginal modification . Brieflyl growth .3 cells per well and incubated for 24 h. Subsequently, 20 μL of the diluted sample solution was added and cultured for 72 h. After that, the MTT solution  was added and incubated for 4 h. Then, 150 μL of DMSO was added to each well to dissolve formazan crystal after removing the medium. The absorbance was recorded using an iMark microplate reader  at 490 nm. The results were expressed using IC50 values.The cytotoxic effect was assayed according to our previously published MTT method  against 6 cells per well) and incubated for 24 h. Subsequently, the cells were treated with varying concentrations of EO  for 48 h. Morphological changes of the treated cells were recorded using an inverted microscope .The K562 cell suspension was added to a 6-well plate (1×10For Acridine orange/ethidium bromide (AO/EB) staining assay, the cells were treated with EO as described above. The treated cell suspensions (1 mL) were collected, centrifuged at 500 g for 10 min, and resuspended in 50 μL PBS, then stained with AO (10 µg/mL) and EB (10 µg/mL) for 10 min. After that, the stained cell suspension (10 μL) was placed onto a glass slide and photographed using an inverted fluorescence microscope .For Hoechst 33258 staining assay, the cells were treated with EO as described above. The treated cell suspensions (1 mL) were collected, centrifuged, and resuspended in 1 mL PBS. After removing the PBS, the cells were fixed in 4% paraformaldehyde (10 μL) for 10 min. After two PBS washes, the cells were stained with 10 μL of Hoechst 33258  for 10 min. After that, the stained cell suspension (10 μL) was placed onto a glass slide and analyzed by an inverted fluorescence microscope.5/well, and treated with different concentrations of EO  for 48 h. Subsequently, the cells were collected and stained with Annexin V-FITC/PI as described by the manufacturer using Annexin V-FITC/PI Apoptosis Detection Kit . The labeled cells were analyzed using a BD FACSCalibur flow cytometer .Annexin V-FITC and propidium iodide (PI) staining was performed using flow cytometry to quantify EO-induced apoptosis in K562 cell. The K562 cells were seeded in 6-well plates at 2×102CO3 solution , and the absorbance was recorded at 405 nm. Acarbose was used as positive control. The α-glucosidase inhibitory effect was expressed using IC50 values and as equivalents of acarbose (mmoL ACEs/g sample).The α-glucosidase inhibitory activity was evaluated according to the method with slight modification . Sample 50 values and as equivalents of kojic acid (mg KAEs/g sample).The inhibition of tyrosinase was assayed according to the method with L-tyrosine as substrate . Sample 50 values and as equivalents of galanthamine (mg GALAEs/g sample).The inhibitory effects of cholinesterase including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were evaluated according to Ellman’s method with slight modification . Sample P < 0.05).All data were repeated at least three times and expressed as mean ± standard deviation (SD). SPSS software (version 19.0) was used for statistical analysis. The significant difference between the two groups was compared using the two-tailed unpaired t-test (H. flavum fresh rhizomes yielded EO at 0.56% (w/w) on fresh weight basis. Seventy-seven chemical constituents of EO were identified by GC-MS/FID, representing 96.5% of the composition (H. flavum EO were coronarin E (20.3%), β-pinene (16.8%), E-nerolidol (11.8%), linalool (8.5%), 1,8-cineole (5.5%), α-pinene (5.4%), α-terpineol (3.8%), α-terpinyl acetate (2.4%), and α-curcumene (2.0%), which were presented in H. flavum rhizome EO were α-pinene (4.3–12.1%), β-pinene (21.8–24.77%), 1,8-cineole (8.90–28.3%), and linalool (6.29–17.5%) (β-pinene (22.5%), α-humulene (15.7%) and β-caryophyllene (10.4%), as well as the major EO compounds of stem were α-humulene (18.9%), β-caryophyllene (11.8%), and β-pinene (11.2%) (H. flavum were β-pinene (49.6%), β-caryophyllene (26.9%), α-pinene (5.9%), sabinene (4.2%), and α-humulene (2.3%)  . Further (11.2%) . The maie (2.3%) . Howeverronarium , H. acumcumnatum  and H. gnerianum . To our H. flavum rhizome WE and EE were shown in p< 0.01) than that of WE. Plant polyphenols and flavonoids have been widely used in various medicines and food products due to their wide range of pharmacological activities, such as antioxidants, antimicrobial, anticancer, anti-inflammatory, hepatoprotective, antiviral, antiatherosclerotic, antispasmodic, and enzyme inhibitory activities  on fresh weight basis than the EE (1.37%). The TPC and TFC of tivities . The H. 1/MS2) and comparative analysis using mzCloud, ChemSpider, and mzCloud databases, a total of 86 compounds were putatively identified (p-coumaric acid (12), cycloolivil (13), ferulaldehyde (16), 1,7-bis(4-hydroxyphenyl)-3,5-heptanediol (17), (-)-5’-desmethylyatein (20), 2-methoxyestradiol (25), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (42), carnosol (44), 5--1,3-benzenediol (60), ginkgolic acid (C13:0) (62), 2,2’-methylenebis(4-methyl-6-tert-butylphenol) (70), and 2,7-dihydroxycadalene (76). All 86 compounds were identified from H. flavum for the first time.The WE and EE compounds were investigated by UHPLC-Q-Orbitrap-MS using negative and positive ion mode scanning. According to the primary and secondary high-resolution mass spectrometry (MS50: 15.36 ± 0.15 μg/mL) and EE (IC50: 16.24 ± 0.61 μg/mL) exhibited significantly stronger DPPH free radical scavenging capacity than BHT (IC50: 109.40 ± 9.68 μg/mL), but lower than ascorbic acid (IC50: 9.39 ± 0.79 μg/mL) (p < 0.05). Base on ABTS analysis, the WE (IC50: 16.83 ± 0.45 μg/mL) and EE (IC50: 12.37 ± 0.13 μg/mL) showed significantly stronger antioxidant ability than both BHT (IC50: 18.86 ± 0.51 μg/mL), and ascorbic acid (IC50: 29.98 ± 1.77μg/mL) (p < 0.05). The free radical scavenging capacity of WE and EE was higher in ABTS assay  than in DPPH assay , expressed as ascorbic acid equivalents per gram of extract. Previous studies have shown that the DPPH and ABTS free radical scavenging capabilities of plant extracts were positively correlated with their TPC and TFC  and quantitatively evaluated by MIC and MBC values using streptomycin as positive control Table 3.activity . Hence, 50 = 72.86 ± 6.39 μg/mL), NCI-H1299 (IC50 = 70.74 ± 9.56 μg/mL), PC-3 (IC50 = 63.16 ± 9.20 μg/mL), and K562 (IC50 = 27.16 ± 2.18 μg/mL) human tumor cell lines. Additionally, the EO displayed a considerable selectivity to human tumor cell K562, and its toxicity was more than 3.5-fold different from that of non-cancerous MRC-5 cell (IC50 = 95.96 ± 4.37μg/mL) and L929 cell (IC50 = 129.91 ± 5.27 μg/mL). According to previous studies, the coronarin E, as the most predominant constituent in H. flavum EO, had higher cytotoxicity to human tumor cell lines  (IC50: 31.21–53.26 μM) compared to non-cancerous Vero cell (IC50: 150.45 μM), with selective cytotoxicity against tumor cells , human normal cell line (MRC-5), and murine fibroblast cell line (L929) using the MTT method with cisplatin as positive control Table 4.or cells . The cyt reports . TherefoMorphology assay indicated that EO triggered obvious morphological changes with typical characteristics of apoptosis, such as cell shrinkage and fragmentation in K562 cells  were investigated and the results are shown in The inhibitory effect of 50 = 0.064 ± 0.004 mg/mL) > EE (IC50 = 0.19 ± 0.01 mg/mL) > acarbose (IC50 = 0.25 ± 0.02 mg/mL) > EO (IC50 = 4.15 ± 0.26 mg/mL) (p < 0.05), which were presented in H. flavum rhizome WE (6.02 ± 0.79 mmoL ACEs/g sample) and EE (2.04 ± 0.20 mmoL ACEs/g sample) showed higher α-glucosidase inhibitory capacity than EO (0.094 ± 0.002 ACEs/g sample), expressed as acarbose equivalents per gram of extract. Interesting findings in this study are that WE and EE had higher α-glucosidase inhibitory activity than acarbose. Hence, H. flavum rhizome WE and EE could be a promising α-glucosidase inhibitor, which could be developed as a therapeutic product for type 2 diabetes.It is well known that α-glucosidase inhibitors reduce post-meal blood glucose and insulin levels by delaying the absorption of carbohydrates in the small intestine. Therefore, α-glucosidase inhibitors are used to treat type 2 diabetes . In this50 = 6.12 ± 0.16 mg/mL, 32.51 ± 0.82 mg KAEs/g sample) followed by the EE  and WE . However, compared to positive control kojic acid (IC50 = 0.20 ± 0.01 mg/mL), all of EO, WE, and EE didn’t show a promising inhibition on tyrosinase.Tyrosinase is a key enzyme involved in the catalysis of mammalian melanogenesis and the enzymatic browning reaction in fruits and vegetables. Therefore, tyrosinase inhibitors are used to control enzymatic browning in foods and for treatment of hyperpigmentation in human skin . As show50 = 2.27 ± 0.13 mg/mL, 0.108 ± 0.011 mg GALAEs/g sample) and BChE . Compared with the positive control drug galanthamine, H. flavum rhizome EO, WE, and EE showed weak cholinesterase inhibitory activity.Cholinesterase inhibitors have become the most effective treatment strategy for Alzheimer’s disease by inhibiting the breakdown of acetylcholine to enhance cholinergic neurotransmission . As show50 values of α-glucosidase inhibitory effect of 1,8-cineole and α-pinene were 1.118 and 1.420 μg/mL, respectively  (IC50 = 0.66 μg/mL) was much higher than positive control arbutin (IC50 > 100 μg/mL), and p-Coumaric acid showed a moderate α-glucosidase inhibitory activity . Ginkgolic acid (C13:0) (62) revealed higher α-glucosidase inhibitory activity (IC50 = 3.5 ± 0.13 μg/mL) than the positive control quercetin (IC50 = 3.8 ± 0.12 μg/mL) (Previous studies have indicated that phenolic compounds play an important role in enzyme inhibitory activity . In thisactivity . Accordi2 μg/mL) .H. flavum rhizome were reported for the first time. The coronarin E was the most main constituent in EO and was first detected in H. flavum. The WE and EE exhibited high TPC and TFC values. Moreover, 86 compounds were putatively identified from WE and EE using UHPLC-Q-Orbitrap-MS, and all the compounds were reported in H. flavum for the first time. In addition to the low antioxidant effect of EO and the weak inhibitory activity of EO and extracts on tyrosinase and cholinesterase, EO demonstrated significant antibacterial activity against P. vulgaris, P. aeruginosa, B. subtilis, and S. aureus, and showed significant α-glucosidase inhibitory effect. Besides, EO exhibited a considerable selectivity to human tumor cell K562, and its toxicity was more than 3.5-fold different from that of non-cancerous MRC-5 and L929 cells. A series of apoptosis analysis suggested that EO triggered apoptosis against K562 cells in a dose-dependent manner. The very low inhibition against the tumor and non-cancerous cell lines suggested that WE and EE were less-toxic. Interesting findings in this study are that WE and EE displayed remarkable antioxidant and α-glucosidase inhibition activities, being superior to their respective positive control drugs BHT and acarbose. Hence, H. flavum could be considered as a source of bioactive compounds and has high exploitation potential in the cosmetics, food, and pharmaceutical industries.To our knowledge, the antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities of essential oil (EO), as well as the chemical composition and bioactivities of water extract (WE) and 70% ethanol extract (EE) from The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.YZ and MT conceived and designed the experiments. MT and XW performed the experiments. TL, XZ, and FW analyzed the data. MT and GD drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The metAFLP technique revealed sequence changes in roots of all analyzed triticale lines and few mutations in leaves. MSAP showed that demethylation of CCGG sites reached approximately 3.97% and 3.75% for T and NT lines, respectively, and was more abundant than de novo methylation, which was observed only in two tolerant lines affected by Al stress. Three of the MSAP fragments showed similarity to genes involved in abiotic stress.Abiotic stressors such as drought, salinity, and exposure to heavy metals can induce epigenetic changes in plants. In this study, liquid chromatography (RP-HPLC), methylation amplified fragment length polymorphisms (metAFLP), and methylation-sensitive amplification polymorphisms (MSAP) analysis was used to investigate the effects of aluminum (Al) stress on DNA methylation levels in the crop species triticale. RP-HPLC, but not metAFLP or MSAP, revealed significant differences in methylation between Al-tolerant (T) and non-tolerant (NT) triticale lines. The direction of methylation change was dependent on phenotype and organ. Al treatment increased the level of global DNA methylation in roots of T lines by approximately 0.6%, whereas demethylation of approximately 1.0% was observed in NT lines. DNA methylation in leaves was not affected by Al stress. The metAFLP and MSAP approaches identified DNA alterations induced by AlThe online version of this article (10.1007/s13353-018-0459-0) contains supplementary material, which is available to authorized users. Poa annua population under Antarctic climatic conditions differed from its founding European counterpart mostly at the DNA methylation level, while sequence changes were relatively small  stress frequently occurs in plants growing in soils under acidic conditions Kochian . SeveralMany techniques have been developed for the estimation of 5mC content  throughout the experiment. After 24 h of incubation in the medium containing Al, seedlings were thoroughly washed for 2–3 min in running water and then transferred to the nutrient solution without Al for 48 h. Seedlings were then removed from the control and Al stress nutrient solutions, and root regrowth of Al-treated seedlings was evaluated. Leaves (3 cm from the top parts) and root tips (3–4 mm long) from 7-day-old seedlings were used for genomic DNA isolation. Eriochrome cyanine R staining for visualization of Al-damaged regions, as described by Anioł . Lines were selected based on their tolerance to Al stress  and Al-stressed (S) seedlings using a Plant DNeasyMiniKit . DNA quantity was assessed spectrophotometrically, and integrity and purity were checked with 1.0% agarose gel electrophoresis. DNA samples extracted from the same seedling pools were used for all experiments.4 and 10 μl of 1.0 U ml−1 P1 nuclease in 30-mM NaOAc (pH 5.4). After nuclease digestion, 10 μl of 0.5-M tris pH 8.3 and 10 μl of 10 U ml−1 alkaline phosphatase in 2.5-M (NH4)2SO4 were added, followed by incubation at 37 °C for 2 h, then centrifugation at 12,000 rpm for 5 min.DNA samples (5 μg) were denatured by heating to 100 °C and then hydrolyzed for 17 h at 37 °C in a solution containing 5 μl of 10-mM ZnSO2PO4 (v/v); and eluent B, 10% methanol in 10-mM KH2PO4. A linear gradient of 10 min 100% A and 100% B, and 15 min 100% B and 100% A, with 5 min of total running time, was used. The percentage of deoxycytidine methylation in relation to the total content of cytidine was calculated according to the following equation: 5mdC% = [5mdC/ (5mdC + dC)] × 100, where 5mdC and dC represent 5-methyldeoxycytidine and deoxycytidine, respectively. Three RP-HPLC technical replicates were conducted for each DNA sample.Reversed-phase HPLC (RP-HPLC) quantification of overall DNA methylation was performed as described by Johnston et al.  using a Acc65I/MseI and KpnI/MseI endonucleases. Reactions were performed in 15-μl volumes containing 10-U MseI, 3.0-U Acc65I or KpnI, 1× ligation buffer, 50-nM NaCl, and 0.5-mg/ml BSA. The Acc65I and MseI neoschizomers, MseI endonuclease, BSA, and ligation buffer were purchased from New England Biolabs, Beverly, MA, USA. Digestions were incubated for 3 h at 37 °C followed by 15 min at 70 °C. After enzymatic digestion of the DNA samples, adaptors were ligated to the released ends. The ligation mixture (25 μl) consisted of 1× ligation buffer, 50-mM NaCl, 0.5-mg/ml BSA, 1.5-pM MseI adapters, 0.15-pM Acc65I (or KpnI) adapters, and 120-U T4 DNA ligase (New England Biolabs). The reaction was run for 12 h at 20 °C following dilution (1:3 in MiliQ H2O). A pre-amplification step was conducted in a final volume of 25 μl  using 20 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min. The pre-amplified products were then diluted 1:20 . Selective amplification was conducted in a final volume of 10 μl . Fifteen primer pairs were used (Table Genomic DNA (0.5 μg) for metAFLP analysis was subjected to two separate digestions with ed Table . The folHpaII and MspI endonucleases used instead of Acc65I and KpnI, as described by Xiong et al. (HpaII/EcoRI and MspI/EcoRI endonucleases (New England Biolabs). Digestion mixes contained 5.0-U HpaII, 5.0-U EcoRI, 1× ligation buffer, 50-mM NaCl, and 0.5-mg/ml BSA. Digestion, adapter ligation, and preselective and selective PCR were performed as described for metAFLP. Denatured PCR products were separated on a 7.0% denaturing polyacrylamide gel and exposed to X-ray film overnight at − 70 °C. The sequences of EcoRI and HpaII/MspI adapters and preselective and selective primers were as described by Xiong et al. . Sequencing was performed using a SequiTerm Excel™ II DNA sequencing kit  with respective end-radiolabeled  × 100%. Similarly, the number of fully methylated sites (FM) was estimated as follows: FM = [(III + IV) / (I + II + III + IV)] × 100%. Hemi-methylation (HM) and non-methylation (NM) quantitative characteristics were determined using HM = [(II) / (I + II + III + IV)] × 100% and NM = [(I) / (I + II + III + IV)] × 100%, respectively. Such calculations were carried out for each genotype.To evaluate the changes in methylation pattern resulting from Al stress, MSAP profile types were compared for each given marker between the same C and S inbred lines. Transitions encoded as I-I , II-II, III-III, and IV-IV indicated no change in methylation status (LCMS). Similarly, I-II, I-III, II-III, I-IV, II-IV, and III-IV transitions were assigned to de novo methylation (DNM). Transitions II-I, III-I, IV-I, III-II, IV-II, and IV-III were assigned to demethylation (DM). The percentages of each transition type were evaluated as follows: LCMS% = 100 × LCMS / (LCMS + DNM + DM); DNM% = 100 × DNM / (LCMS + DNM + DM); and DM% = 100 × DM / (LCMS + DNM + DM).One-way analysis of variance (ANOVA) with Tukey’s contrast was performed in Statistica 12  failed to grow under Al treatment in a liquid medium.The inbred lines used in this experiment were tested for Al tolerance over several years. All lines demonstrated stable inheritance of the trait. Root regrowth after Al exposure was recorded only in the case of T lines , with maximum regrowth of 2.8, 1.7, and 0.8 cm for plants treated with 15, 20 and 30 ppm AlRP-HPLC analysis estimated total genomic DNA methylation of T and NT triticale genotypes grown under control conditions to be 22.08–22.71% and 22.95–23.90% for root and leaves, respectively . DNA methylation levels in roots of T lines exposed to 15, 20, and 30 ppm Al3+ increased by approximately 0.65%  compared with control plants not exposed to stress. No statistically significant differences in methylation were observed in leaves of lines grown under stress and control conditions regardless of Al-tolerance genotype  total methylated bands (Type II + III + IV bands) were evaluated for control conditions (370) than for stressed conditions (361). Under control conditions, the total methylation of CCGG sequences was approximately 84%, with 36% of sites fully methylated (Type III + IV bands) and 49% hemi-methylated (Type II bands) , from DNA from roots of NT and T lines under control and stressed conditions, respectively . Demethylation of CCGG sites due to Al stress  in NT and T lines equaled 3.75% and 3.97% . De novo methylation (I-IV transition) was observed at 0.23% in two T lines (L195 and L198) . No changes in DNA methylation quantitative characteristics of leaves of NT and T lines due to Al stress were observed (not shown).Seven different banding patterns distinguishing the control and Al stress conditions were observed in MSAP gels Table . The traAcc65I/MseI and KpnI/MseI digestion, resulting in 567 and 571 reproducible fragments, respectively. On average, approximately 30 and 31 polymorphic fragments were shared between NT and T lines, respectively. The same lines under Al stress allowed for the amplification of 571 (NT) and 568 (T) fragments with 29 and 30 polymorphisms, respectively. Similarly, 571 (NT) and 569 (T) signals were detected when leaves of control and stressed lines were used, with 29 (NT) and 31 (T) polymorphic signals. The majority of polymorphisms were common between roots and leaves of NT and T lines with the exception of two fragments present in the roots of NT lines.DNA isolated from roots of NT and T lines grown under control conditions was amplified with 15 selective primer pairs followed by 1111 were the most frequent in all genotypes for roots, with 536 and 538 profiles identified for T and NT plants, respectively. The number of polymorphisms was 23–43 for T and 32–39 for NT lines, depending on the selective primer pair used. The 0011 pattern was observed 28 and 29 times in roots of five T and five NT lines, respectively. Similarly, for T and NT lines, respectively, the 0100 profile was detected 5 and 4 times; 1010, 7 and 10 times; 1000, 3 and 3 times; 1100, 7 and 11 times; and 0101, 10 and 11 times. In leaves, the 1111 pattern was identified in 537 cases. The 0011 code was found 26 times in T and NT lines, and 1100 was observed 7 (T) and 11 (NT) times. The 1010, 1000, and 0101 profiles were observed exclusively in T lines, with 3, 1, and 4 observations, respectively. Example of metAFLP patterns are shown in Fig. The metAFLP results were converted into four-digit codes. Profiles classified as F = 12.3, p < 0.01). There were no de novo methylation (DNMV) or demethylation (DMV) events in roots and leaves of NT and T plants between control and stressed conditions . Genome methylation (GM) values, reflecting methylation within sequences recognized by the Acc65I/KpnI isoschizomers, were approximately 5.0% regardless of organ and line  in roots of NT and T lines by 0.97% and 0.87%, respectively Table . Patternns Table . HoweverTriticum aestivum. Fragments F5 and F7  and histone-lysine N-methyltransferase, respectively. The identified histone-lysine N-methyltransferase belonged to the class V-like SAM-binding methyltransferase superfamily. The F3 fragment  lines before (or after) and absence of the same band after (or before) Al treatment revealed by KpnI/MseI digests indicates a point mutation event rather than methylation change, as KpnI is insensitive towards site methylation. The direct confirmation of putative mutation could be confirmed via DNA sequencing. However, this is challenging due to the absence of the metAFLP fragment. Sequencing of the respective fragment followed by resequencing of genomic DNA of the control and stressed materials is one approach. However, if the mutation reflects a methylation change then bisulfite sequencing would be needed. Alternatively, the mutagenic action of Al stress could be confirmed from experiments with ISSR markers. Correia et al.  changes in T and NT genotypes, particularly in roots and somewhat in leaves. The results indicated the possibly mutagenic action of Al on triticale lines. This analysis is based on the specificity of the a et al.  showed tNtGPDL gene, which encoded a glycerophosphodiesterase-like protein and was linked to the response to Al, paraquat, salt, and cold stresses in tobacco, resulting in enhanced expression of the gene Table S2(DOCX 14 kb)Fig. S13+. Examples show patterns for the following primer combinations: HM-TCG/E-AGA (a), HM-TGA/E-AGG (b), HM-TGC/E-ACA (c), and HM-TCAA/E-ACC (d). H, HpaII/EcoRI digestion; M, MspI/EcoRI digestion; C, control. S15 and S30 indicate aluminum in concentrations of 15 and 30 ppm, respectively. (PDF 168 kb)Methylation patterns in roots and leaves of triticale lines (tolerant L198 and non-tolerant L444) treated with 15 and 30 ppm AlFig. S2a, b MSAP fragments exhibited homology with the coding region of peptide chain release factor subunit 1-2-like (F3), receptor-like protein kinase (F5), and histone-lysine N-methyltransferase (F7) (fragment names according to the Table