Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of  Escherichia coli DH5α is the most studied commercial strain to improve the pDNA production, although several strains have been developed for the same objective [µ); the percentage of dissolved oxygen (%DO); the increase of temperature and heating rate; and the pH control and gradients  are extrachromosomal molecules that replicate independently to the host chromosome but using their own replication machinery . pDNA isbjective ,6,7. pDNo et al. ). Organihydrates ,10. Highhydrates ,12. In ahydrates .nptII) and other antibiotic resistance genes have still been used for pDNA production research, even though the current trend is to use antibiotic-free plasmid in pDNA vaccines production due to the risk of serious hypersensitivity reactions in patients [nptII is approved by regulatory authorities to use in pDNA development [In recent years, pDNA containing kanamycin resistance gene  [µ were lower and metabolic overflow was higher than that achieved using cells without pDNA. It’s suggested that pDNA yield expected could theoretically be higher than that experimentally obtained by weakening nptII expression, which would allow more precursor metabolites and energy, also reduce power needed to be used for plasmid production [cps gene clusters expression of E. coli. These clusters encode several enzymes involved in the synthesis and translocation of exopolysaccharide and capsular polysaccharide (CPS) layers [E. coli [Antibiotic concentration in the fermentation is necessary to maintain a selection pressure and to ensure the segregation stability of the pDNA containing  (NPTII) . In celloduction ,23. Thisoduction . Some reoduction ,24. The ) layers  which sh[E. coli ,27,28.cps cluster and the HSPs, these proteins protect bacterial membrane potential against exposure to kanamycin and can rescue the cell growth [Escherichia coli DH5α has been not evaluated using temperature increase in fed-batch cultures. Thus, the objective of this work was to evaluate the synergic effect of the temperature and the kanamycin concentration on the production of pDNA, NPTII, organic acids (mainly acetate), sc-pDNA and cell morphology in batch and high cell density cultures (HCDC) of E. coli containing pVAX1-NH36 plasmid (E. coli DH5α-pVAX-NH36).Kanamycin and temperature induce the synthesis of several proteins, the ones encoded by l growth ,30,31. Sl growth . Some wol growth ,21. HoweE. coli DH5α-pVAX-NH36 (3936-bp), which contains a DNA fragment that encodes NH36 antigen of Leishmania donovani [nptII and a pUC origin (pMB1-derived) for high-copy number replication . E. coli DH5α-pVAX1-NH36 was propagated on LB agar plates with soy peptone and kanamycin (50 µg/mL). A single colony from this plate was used to inoculate a 1000 mL baffled flask with aeration system containing 200 mL of chemically defined mineral medium  plus glycerol (12.5 g/L). The shake flask was incubated in an orbital shaker  carried out at 30 °C, 250 rpm and 0.5 vvm. For cell cryo-protection, 40 mL of glycerol 80% (w/v) was added when the culture reached an optical density (OD600nm) of 8, and aliquots of 10 mL were frozen immediately on dry ice and stored at −70 °C (CDMM seed bank).The host-plasmid combination was donovani . pVAX-NHE. coli DH5α growth 2SO4, 5.92; MgSO4·7H2O, 2.3; NaCl, 2; FeCl3·6H2O, 2.85 mg/L; and 2 mL/L of trace mineral solution (TMS). The TMS contained the following composition (g/L): ZnCl2·4H2O, 2; CoCl·6H2O, 2; Na2MoO4·2H2O, 2; CuCl2·2H2O, 1.9; H3BO3, 1.6; MnSO4·H2O, 1.6; citric acid, 0.6; and CaCl2, 1. The fermentations were performed with CDMM plus glycerol and thiamine 25 g/L and 40 µg/mL, respectively.The CDMM for modified ) had the2HPO4, 5.8; KH2PO4, 7.5; antifoam 204 from Sigma-Aldrich , 0.0125; (NH4)2SO4, 2.5; MgSO4·7H2O, 2.3; NaCl, 2; FeCl3·6H2O, 22.7 mg/L; and 4 mL/L of TMS. The feeding solution had the following composition (g/L): glycerol, 650; kanamycin, 0.05; thiamine hydrochloride, 0.04; MgSO4·7H2O, 1.15; TMS, 4 mL/L.The CDMM for the initial batch culture had the following composition (g/L): Kv/v) when an increase of %DO was registered, which meant the complete consumption of glycerol with an OD600nm of 24 (DCW of 9 g/L). This inoculum was used for all experiments.An amount of 10 mL of CDMM cryo-conserved seed bank was inoculated into a 750 mL jar bioreactor containing 200 mL of CDMM plus 12.5 g/L glycerol, 50 µg/mL kanamycin and 40 µg/mL thiamine and carried out at 30 °C, 1200 rpm, 1 vvm without pH control. The culture was used as inoculum  was evaluated in batch cultures at 30 and 42 °C.4OH solution throughout the whole fermentation. Samples were taken periodically in the fermentation to determine dry cell weight (DCW) and residual glycerol. A 5 mL sample was taken when the glycerol was totally consumed to determine pDNA yields, purity as % sc-pDNA, organic acids, NPTII yields, and to visualize morphology changes by SEM.Duplicated fermentations were carried out in a 2 L bioreactor with 1 L of CDMM plus 200 mL of inoculum and performed at 1200 rpm, 1 vvm. The %DO was maintained above 30% air saturation by enriching air with pure oxygen and pH of 7.2 was controlled by automatic addition of 30% NH4OH solution. The %DO was maintained above 30% air saturation by enriching air with pure oxygen.Prior to carrying out the experiments to evaluate the effect of high kanamycin concentration on pDNA production in HCDC, a control glycerol-limited fed-batch culture was carried out in a 2 L bioreactor at 1200 rpm, 1 vvm and pH of 7.2 controlled by automatic titration with 30% NH600nm of 1.3 (0.5 gDCW/L); both pH control and fed-batch phase were started. The fed-batch culture was designed to obtain three growth phases described below at different temperatures. The feeding pump was programmed according to Equation (1)µ is the desired specific growth rate (h−1), X is the initial biomass concentration (gDCW/L), V is the initial volume of media of culture (L), Yx/s is the yield coefficient of biomass-substrate (g/g), and t is the time when the phase began (h). Si is the glycerol concentration in feeding solution (650 g/L) used during all fed-batch phaseThe glycerol-limited fed-batch culture was done as follows: when a batch phase with 25 mL of CDMM cryo-conserved seed bank plus 1 L of CDMM plus kanamycin (25 mg/L) and thiamine (40 µg/mL) at 30 °C reached an OD600nm = 21 (8 gDCW/L), F(t) was calculated using a µ = 0.27 h−1 and Yx/s = 0.35 g/g.Phase A. For cell propagation at 30 °C until the culture reached OD600nm = 21 (8 gDCW/L) was reached during fed-batch phase A, F(t) was adjusted using a µ = 0.27 h−1 and Yx/s = 0.5 g/g and the temperature increase was started with a heating rate of 0.025 °C/min until 37 °C was reached.Phase B. When an ODµ = 0.15 h−1 and Yx/s = 0.5 g/g until an OD600nm = 132 (50 gDCW/L) was reached, the heating rate was maintained at 0.025 °C/min until 42 °C was reached.Phase C. When temperature reached 37 °C in phase B, the F(t) was adjusted using a The evaluation of the effect of high kanamycin concentration on pDNA production in HCDC was performed using the same strategy of fed-batch culture described above. During fed-batch culture, the kanamycin concentration was increased to 300 mg/L when temperature increase was started. The DCW, residual glycerol, pDNA, organic acids, and NPTII yields were analyzed taking 5 mL of sample in each experiment.600nm provided that OD600 < 0.378) to determine the DCW using an appropriate sample dilution. To generate the calibration curve, samples of 10 mL were centrifuged in a Sorval centrifuge at 10,000 rpm and 4 °C for 10 min, washed with distilled water and dried in an oven overnight at 85 °C. The supernatant was used for glycerol quantification using a spectrophotometric method [A calibration curve was built previously .260nm and OD280nm using the Nano-Drop UV spectrophotometer ND-1000 . To calculate pDNA concentration, the correlation OD260nm = 1 corresponding to a pDNA concentration of 50 mg/L in the purified solution was used.The pDNA concentration in purified solution from 300 μg of DCW was measured spectrophotometrically at OD3BO3, 1 mM EDTA pH 8.0) at 50 V for 90 min and stained with ethidium bromide (5 µg/mL). Total pDNA fractions, including sc-pDNA isoform were estimated using a densitometric analysis of pDNA band intensities in the ImageJ Software version 1.47 and a known amount of a calibrated 1 kb DNA Ladder  as a standard.sc-pDNA isoform in purified solution was quantified by electrophoresis using a 0.8% agarose gel in TBE buffer , as detailed elsewhere .2HPO4, and 2 mM KH2PO4 from J.T Baker, Phillipsburg, NJ, USA). Then, the wells were blocked with 100 µL of block buffer solution (wash buffer solution plus 3% Skim Milk from Difco) at 37 °C for 1 h and washed five times again. After these washes, 100 µL anti-neomycin phosphotransferase II antibody from rabbit , which had been diluted 1:200, was added and incubated for 1 h at 37 °C, and then each well was washed five times. A total of 100 µL Goat Anti-Rabbit IgG, (H + L) HRP conjugate from Merck Millipore , which had been diluted 1:2000, was added and incubated for 1 h at 37 °C. The wells were washed and 100 µL of peroxidase substrate solution was added. To stop the reaction, 100 µL of stop solution (1N H2SO4) was added to each well, and the absorbance was read at 492 nm in a Multiskan EX from Labsystems (USA). A NPTII protein standard stock solution was obtained , dissolved in 20 mM Tris-HCl  pH 8.0 and stored at −80 °C until use. Working solutions were prepared by dilution of NPTII protein standard stock with carbonate buffer solution containing 0.3 M Na2CO3 and 0.7 M NaHCO3 from J.T Baker , to use it in ELISA method. Six calibration curve solutions  were used to calibrate ELISA method.Determination of NTPII was done by enzyme-linked immunosorbent assay (ELISA), taking 1 mL of each sample and centrifuged at 10,000 rpm for 10 min at 4 °C to obtain cellular extract. The pellets were re-suspended at same initial volume in extraction buffer . Samples were sonicated by a cycle of 50% amplitude for 45 s, then shut off for 1 min, repeating the cycle 4 times. A total of 100 µL of cell extracts were added to each well, in duplicate, of the microtiter plate and incubated for 2 h at 37 °C. Followed by three washes with 300 µL of wash buffer  solution plus 0.05% Tween 20 from GE Healthcare; PBS solution contained 137 mM NaCl, 2.7 mM KCl, 10 mM NaE. coli DH5α-pVAX1-NH36 culture, SEM was performed. For all treatments included in experimental design, 1 mL of culture broth was centrifuged for 10 min at 12,000 rpm and 4 °C. Then, the samples were washed twice with PBS buffer (pH = 7.2) and re-suspended in 1 mL of same buffer. Next, 10 µL of suspension was used to coat a glass slide (1 cm × 1 cm). A fixative was placed on the samples  in PBS) overnight, then washed three times with PBS followed by fixation in 1% osmium tetroxide at 4 °C for 2 h.To evaluate the effect of the temperature and kanamycin concentration on the morphology of The fixed cells were washed three times with PBS. Dehydration of samples was achieved by washing with increasing concentrations of ethyl alcohol . Lastly, all samples were freeze-dried , then fixed on carbon tape and sputter-coated with gold particles for 2 min .Images were taken by Scanning Electron Microscope JSM-6510LV .pDNA/X) pDNA yields, pDNA-volumetric productivity (pDNA-VP), volumetric (NPTII) and specific yield (YNPTII/X) NPTII yields are given in The analysis of variance (ANOVA) results for volumetric (pDNA) and specific . The predicted R2 are close to the adjusted R2. This shows that the model fits the data adequately. Usually, for the predictions to be significant, F-value and t-value should be higher than the low probability p values. Values of p < 0.001 indicate that the model terms are significant at 99% confidence level.Here, ANOVA for all yields manifest that they are highly significant (model p < 0.01) and kanamycin concentration (p < 0.01) dominate in all treatments studied, having a significant influence on pDNA and NPTII production.The ANOVA results for variables imply that the linear positive effects of temperature  was obtained in the central point of the experimental design (T36-150), carried out at 36 °C with kanamycin concentration of 150 mg/L. This result was to be expected since 36 °C is within the optimum temperature growth range for E. coli. It is similar to the ones described previously [The initial glycerol concentration was 20 g/L for all batch cultures in the experimental design B. The maX/S 0. g/g was Biomass concentration increment was lower when growth temperature was at 30 or 42 °C, consequently the biomass yields on glycerol were also lower .X/S are reduced by increasing temperature in pDNA production, however those were evaluated in shake flasks in which the %DO and pH were not controlled [X/S was lower when kanamycin was added (50 and 300 mg/L). Nonetheless, at 42 °C, the YX/S value significantly increased only when the initial kanamycin concentration was 300 mg/L. All batch cultures showed a first exponential growth phase with a high µmax value, then the µmax value decreased. The exponential growth phase continued for approximately over two hours before reaching the stationary phase. The highest µmax (0.35 h−1) was obtained at 42 °C, but this high rate was reached during first 3.5 h, subsequently, it reduced to 0.095 h−1 until the stationary phase was reached. At 30 °C, the µmax decreased when the initial kanamycin concentration was increased from 0 to 300 mg/L ), pDNA value of 57.4 mg/L and YpDNA/X value of 6.4 mg/g were similar to those reported at 37 °C using the CDMM with glycerol concentration of 50 g/Land kanamycin concentration of 50 mg/L [All pDNA, Y 50 mg/L . Yields  50 mg/L ,12,38.42-300 with 300 mg/L and 42 °C, these increased more than five times compared with those obtained at 30 °C, regardless of kanamycin concentration. The pDNA replication is highly regulated at temperatures below 37 °C [E. coli is exposed to β-lactam antibiotics such as ampicillin , the DpiA protein overexpression affects the stability and replication of plasmid by activating DpiAB system in high copy number plasmids such as pVAX1 which contains A+T-rich sequences at replication origin region, and, also, this protein induces SOS response. The kanamycin as a selection marker has molecular advantages for pDNA production, since it does not increase the DpiA protein expression, therefore, it does not activate the DpiAB system [When initial kanamycin concentration was increased from 0 to 50 mg/L, no significant effect was detected on these parameters, but, in Tow 37 °C . Thus, tB system .The densitometric analysis is presented in E. coli DH5α-pVAX1-NH36 fermentation process, likewise, it is considered a contaminant in downstream purification. However, NPTII does not have any of the characteristics associated with allergenic proteins and toxicity in humans. Maximum NPTII concentration (558 mg/L) and YNPTII/X (61.9 mg/g) were obtained from the central point of experimental design (T36-150). These values were 73% and 56% lower at 30 and 42 °C, respectively. The NPTII accumulation was equal at 30 and 42 °C with initial kanamycin concentration of 0 mg/L. However, at 42 °C this accumulation increased by increasing the initial kanamycin concentration. The RpoH protein is an indicator of heat resistance in E. coli cultures at high temperature. It regulates the HSPs expression, which confers protection from protein denaturation due to heat. In addition, the exposure to kanamycin causes an increase in RpoH protein levels, which increase heat tolerance [The quantification of the NPTII synthesis is important, because it is an indicator of the metabolic burden in olerance ,45. At tThe overflow metabolism products evaluated in this work are shown in It can be observed that high concentrations of antibiotic reduced the production of overflow metabolites (mainly acetate), evidencing a synergistic effect allowing higher pDNA and NPTII. The lower amount of overflow metabolites is a positive effect, because they inhibit the production of biomass and, at higher concentrations, the plasmid replication.42-50) the maximum acetate production reached 4.62 g/L. This value decreased 40 and 74% when the initial kanamycin concentration was 0 and 300 mg/L, respectively. The exposure of E. coli to high concentrations of kanamycin evaluated in T30-300, T36-150, and T42-300 resulted in a lower production of overflow metabolism products. This phenomenon could be due to the protein misfolding (involved in the metabolic pathways of glycerol and tricarboxylic acids (TCA) [Ac/S values, obtained in the present work, were lower and can be explained by the use of glycerol instead of glucose in the fermentations [At 30 °C, the acetate concentration decreased 54% when initial kanamycin concentration was increased from 0 to 300 mg/L. On the other hand, in the fermentation carried out at 42 °C and kanamycin concentration of 50 mg/L (Tds (TCA) , which ads (TCA) . Likewisds (TCA) , which rntations ,50. Howentations .E. coli DH5α-pVAX1-NH36 cultures under stress condition by temperature and kanamycin concentration are shown in images taken by SEM  condition (B-T(30-0)) shows similar characteristics to image A (Control). However, changes were observed in the membrane integrity in all other treatments.The image A (Control) in E. coli cells are rod-shaped and are approximately 800 nm wide and 2.5 µm long in micrograph (30-50) at 30 °C and kanamycin 50 mg/L.The cells growing with kanamycin concentration of 50 mg/L at 30 and 42 °C are shown in micrograph (42-50) showed a filamentous morphology essentially when cells elongate and replicate their DNA, but do not septate and divide. These morphological characteristics are in agreement with the ones reported by Silva et al. [30-300 at 30 °C and kanamycin concentration of 300 mg/L showed sinking in the middle section and a shriveled membrane, indicating partial cytoplasm leakage. This damage can be attributed to the high concentration of antibiotic.On the other hand, cells grown at 42 °C and kanamycin 50 mg/L in micrograph a et al. . Cells iE. coli cells after treatment with antimicrobial agents has been reported previously [(36-150), the SEM images clearly show limited filamentous morphology and the production of considerable amounts of extracellular CPS. It is important to mention that endotoxins are generally referred as lipopolysaccharides and they are major contaminants in pDNA production by gram negative bacteria, like E. coli.The alteration of the surface morphology of eviously . At centE. coli to obtain biomass at 36–37 °C, since it can be observed that CPS production is associated, in this case, with the growth rate. Micrographs (42-0), in E. coli DH5α, resulting in down-regulation of cell wall biosynthetic genes [(42-300) at 42 °C with high kanamycin concentration filamentous morphology and pleomorphism was observed. This can also be observed in E. coli when ampicillin is added to growth media [(42-300) show the same filamentous morphology. Pleomorphism was also observed, but only under high temperature and kanamycin concentration. Morphological changes including filamentation, has been showed previously in detail for different culture conditions, as starvation and high hydrostatic pressure [They damage the processes of separation and purification, thus increase the process cost. Consequently, it is proposed not to start cultures of ic genes . In T on biomass, reaching 49.10 ± 1.23 gCDW/L  in HCDC with temperature increase in fed-batch culture.In this study, a synergic strategy for pDNA production was evaluated to increase pVAX1-NH36 final yields, using temperature and kanamycin concentration as stress factors. At 42 °C and 0 mg/L, results similar to those reported in the literature were obtained. However, when the combined effect at 42 °C and 300 mg/L was evaluated, an increase of 49%, 35% and 65% in volumetric yield, specific yield and productivity, were obtained. The increase in both pDNA volumetric yield and productivity is mainly due to the increase in the specific yield. The results allow us to provide evidence about the capacity of using high antibiotic concentration to reduce metabolic overflow products (mainly acetate) and NPTII synthesis related to metabolic burden. The combined use of two stress factors as temperature and high kanamycin concentration did not affect the quality of the plasmid produced (sc-pDNA ≥ 90%), being an advantage over the upstream process and its subsequent purification. Finally, based on the micrographs taken to the cultures, it is concluded that starting a culture at 36–37 °C, in the pDNA production process compromises the downstream steps because it involves the synthesis of exopolysaccharides. In addition, the same success was obtained using the high kanamycin concentration (300 mg/L) in HCDC with temperature increase in fed-batch culture. Through this strategy, the pDNA volumetric and specific yields and the volumetric productivity were approximately twice  than what were obtained in fed-batch control culture . These results suggest that a stress-based process simultaneously caused by high temperature and high kanamycin concentration can be successfully applied to increase the pDNA production by


Kluyveromyces marxianus can be considered an ideal consolidated bioprocessing (CBP). A major obstacle to ethanol production using K. marxianus is the low production of lignocellulolytic enzymes, which reduces the cellulose hydrolysis and ethanol production. Thus, further improvement of enzyme expression and secretion is essential.Taking into account its thermotolerance, high growth rate, and broad substrate spectrum, K. marxianus was optimized through mutagenesis. A T(-361)A mutation inside the promoter, a deletion of AT-rich region inside 5′UTR (UTR∆A), and a P10L substitution in the signal sequence increased the secretory expression of the feruloyl esterase Est1E by up to sixfold. T(-361)A and UTR∆A increased the mRNA expression, while the P10L substitution extended the hydrophobic core of signal sequence and promoted secretion of mature protein. P10L and T(-361)A mutations increased secretory expressions of other types of lignocellulolytic enzymes by up to threefold, including endo-1,4-β-glucanase RuCelA, endo-1,4-β-endoxylanase Xyn-CDBFV, and endo-1,4-β-mannanase MAN330. During the fed-batch fermentation of strains carrying optimized modules, the peak activities of RuCelA, Xyn-CDBFV, MAN330, and Est1E reached 24 U/mL, 25,600 U/mL, 10,200 U/mL, and 1220 U/mL, respectively. Importantly, higher yield of enzymes by optimized promoter and signal sequence were achieved in all tested carbon sources, including the major end products of lignocellulose saccharification and fermentation, with growth on xylose resulting in the highest production.To improve the expression of lignocellulolytic enzymes, the inulinase promoter and signal sequence from K. marxianus by means of various carbon resources. Activities of lignocellulolytic enzymes in fed-batch fermentation were the highest activities reported for K. marxianus so far. Our engineered modules are valuable in producing lignocellulolytic enzymes by K. marxianus and in constructing efficient CBP strains for cellulosic ethanol production.The engineered promoter and signal sequence presented increased secretory expressions of different lignocellulolytic enzymes in The online version of this article (10.1186/s13068-018-1232-7) contains supplementary material, which is available to authorized users. Kluyveromyces marxianus is a homothallic hemiascomycetous yeast species commonly isolated in dairy products, grape, and henequen [K. marxianus can be considered to be a potential alternative to Saccharomyces cerevisiae for the production of ethanol from cellulosic feedstocks [Ethanol production from the lignocellulosic biomass involves four steps that include feedstock pretreatment, fractionation, enzymatic hydrolysis (saccharification), and microbial fermentation . Kluyverhenequen . It is ahenequen , 4. Due edstocks .Cellulolytic thermophiles, Caldicellulosiruptor bescii, Thermoanaerobacterium saccharolyticum, and Geobacillus thermoglucosidasius, and filamentous fungi, such as Trichoderma sp., Aspergillus sp., Fusarium oxysporum, and Penicillium sp. to produce cellulosic ethanol via CBP [C. bescii, can directly convert cellulosic and hemicellulosic compositions to ethanol without chemical pretreatment of the feedstock [S. cerevisiae is one of the most promising microbes to develop CBP strains with cellulolytic capacity, since it exhibits both high ethanol production and tolerance [S. cerevisiae strain expressing both an endoglucanase D from Clostridium cellulovorans and a β-glucosidase from Saccharomycopsis fibuligera produced 9.15 g/L ethanol from 20 g/L β-glucan after 50 h of fermentation, achieving 80.3% of the theoretical ethanol yield [S. cerevisiae strain that integrated endoglucanase, exoglucanase, and β-glucosidase into the chromosomal ribosomal DNA and delta regions can convert 63% cellulose from pretreated corn stover into ethanol with a titer of 26 g/L [S. cerevisiae strain engineered to use hemicellulose via coexpression of the Trichoderma reesei xylanase (xyn2), the Aspergillus niger xylosidase (xlnD), the Scheffersomyces stipitis xylulose kinase (xyl3), and Bacteroides thetaiotaomicron isomerase (xylA), produced 9 g/L of ethanol after 40 days under anaerobic fermentation [Breakdown of lignocellulose into fermentable sugars requires three major cellulase enzymes, including endo-1,4-β-glucanase (EC 3.2.1.4), cellobiohydrolase EC 3.2.1.91), β-glucosidase (EC 3.2.1.21), and several hemicellulases, such as endo-1,4-β-xylanase EC 3.2.1.8), endo-1,4-β-mannanase EC 3.2.1.78), β-xylosidase (EC 3.2.1.27), and hemicellulolytic esterases , 7. Enzy8, β-xylo, endo-1,1, β-gluceedstock . Howeverolerance . An S. col yield . An induf 26 g/L . In addientation .Kluyveromyces marxianus is another CBP yeast that has attracted much attention recently with respect to its ethanol production, tolerance, and high-temperature resistance [K. marxianus strain co-displayed T. reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, produced 7.02 and 2.12 U/OD600 of endoglucanase and β-glucosidase, respectively, and converted β-glucan to ethanol with a yield of 4.24 g/L ethanol from 10 g/L β-glucan [K. marxianus, five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases, a β-glucosidase gene, and a cellodextrin transporter gene from different fungi were simultaneously expressed in K. marxianus KY3, and the resultant strain produced 0.6 g/L ethanol from 10% avicel [sistance . A genetβ-glucan . To impr% avicel .K. marxianus strains are still low. One of the major reasons is the low production of secreted enzymes by these strains [K. marxianus inulinase-encoding gene (INU1) to improve the secretary expression of lignocellulolytic enzymes. Inulinase is responsible for the degradation of inulin and that supports the growth of K. marxianus on fructans [K. marxianus strain, the latter fraction of inulinase compromises more than 60% of total secreted proteins in the supernatant [INU1 promoter and signal sequence, Bergkamp et al. obtained high β-galactosidase production (153 mg/L) and secretion efficiency (99%) in K. marxianus [INU1 promoter and secretion signal sequence could be further improved.However, compared with separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF), the efficiencies of cellulose hydrolysis and ethanol production by engineered CBP  strains . In thisfructans . A smallfructans . In a ceernatant , suggestarxianus . HoweverK. marxianus.In this study, we performed mutagenesis to increase the efficiency of the inulinase promoter and signal sequence. A mutation inside the promoter and a deletion of an A-T-rich region inside the 5′UTR increased mRNA levels and improved the secretory activity. Optimization of the signal sequence by extending the central hydrophobic region significantly improved secretion of the mature protein. The expressions of lignocellulolytic enzymes, including endo-1,4-β-mannanase, endo-1,4-β-endoxylanase, endo-1,4-β-glucanase, and feruloyl esterase, were increased by two to sixfold using optimized promoter or signal sequence. Improved expression by optimized modules can be achieved via growing in carbon sources that are the end products of lignocellulose saccharification . Our study provides useful modules for high-level secretory expression of lignocellulolytic enzymes and contributes to the optimization of CBP for cellulosic ethanol production by K. marxianus strain FIM-1∆U used in this study was derived from FIM-1 . Genomic DNA was extracted from FIM-1 by Yeast Genomic DNA Extraction Kit . The URA3 gene, including 703 bp promoter, 804 bp ORF and 488 bp terminator, was amplified from the genomic DNA of FIM-1 by means of Phanta Max Super-Fidelity DNA Polymerase  and primer pair URA3-F/URA3-R. 3′ adenine overhangs were added by Taq polymerase . The purified PCR product was ligated into the pMD18-T vector  to obtain pZP12. The URA3 ORF in pZP12 was removed by PCR mutagenesis to obtain pZP13. Briefly, pZP12 was amplified by Phanta Max Super-Fidelity DNA Polymerase with primer pair URA3∆-F/URA3∆-R. After 18 cycles of PCR, DpnI  was added into the PCR mixture to digest the methylated template. DpnI-treated product was transformed into Escherichia coli DH5α and correct transformant was named as pZP13. The URA3 deletion cassette was amplified from pZP13 by Phanta Max Super-Fidelity DNA Polymerase with primer pair URA3-F/URA3-R. 1 μg purified PCR product was transform into FIM-1 by lithium acetate method as described before [The host d before . TransfoINU1 gene, including 1136 bp promoter, 1671 bp ORF and 865 bp terminator, was amplified by Phanta Max Super-Fidelity DNA Polymerase and primer pair INU1-F/INU1-R. The same polymerase was used in the PCR reactions described below. 3′ adenine overhangs were added by Taq polymerase. Purified PCR product was ligated with pMD18-T vector to obtain pZP14. 79–1668 bp of INU1 ORF in pZP14 was removed, and multiple cloning sites (MCS) were introduced by PCR mutagenesis with primer pair MCS-F/MCS-R as described above. Resultant plasmid was called pZP15. A fragment containing the URA3 gene, pBluescript II KS(+) vector part, and autoreplicating KD element was amplified from pUKD [Est1E (Genbank: MH212232) was amplified from metagenome of yak rumen with primer pair Est1E-F/Est1E-R. Purified PCR product of Est1E was inserted between SpeI and NotI sites of pUKDN132 to obtain pZP17. M1, M2, and M3 promoters were amplified from pZP17 by means of primer pairs M1-F/MP-R, M2-F/MP-R, and M3-F/MP-R, respectively, and were inserted between SacII and SmaI sites of pZP17 to obtain pZP18–pZP20. UTR∆A mutation were introduced into pZP17 to obtain pZP22 by PCR mutagenesis using primer pairs UTR∆A-F/UTR∆A-R. A(-1104)T, A(-566)T, T(-351)A, A(-266)G, T(-233)A, or P10L mutation was introduced into pZP17 by PCR mutagenesis using primer pairs A(-1104)TF/A(-1104)TR, A(-566)T-F/A(-566)T-R, T(-351)A-F/T(-351)A-R, A(-266)G-F/A(-266)G-R, T(-233)A-F/T(-233)A-R, or P10L-F/P10L-R, and resultant plasmids were named as pZP23, pZP24, pZP25, pZP26, pZP27, or pZP28. UTR∆A or P10L mutation was introduced into pZP25, respectively, by PCR mutagenesis using primer pair UTR∆A-F/UTR∆A-R or P10L-F/P10L-R, and the resultant plasmids were named as pZP29 or pZP30, respecively. P10L mutation was introduced into pZP29 by PCR mutagenesis using primer pair P10L-F/P10L-R, and the resultant plasmid was named as pZP31. His6-tagged Est1E was amplified from pZP17 by primer pair Est1E-F/His6-R and inserted between SpeI and NotI sites of pUKDN132 or pZP28 to obtain pZP32 or pZP33. P10I, P10G, P10S, P10K, or P10D substitutions inside inulinase signal sequence of pZP17 were performed by PCR mutagenesis with primer pairs P10I-F/P10I-R, P10G-F/P10G-R, P10S-F/P10S-R, P10K-F/P10K-R, or P10D-F/P10D-R to obtain plasmid pZP34, pZP35, pZP36, pZP37, or pZP38. Liner double-strand DNA containing inulinase signal sequence from K. lactis was synthesized by Genewiz  and named as KLSS. KLSS was used as a megaprimer to replace inulinase signal sequence of K. marxianus in pZP17 by mutagenesis PCR, and the resultant plasmid was named pZP39. P10L mutation was introduced into inulinase signal sequence of pZP39 by PCR mutagenesis using primer pairs KL-P10L-F/KL-P10L-R to obtain pZP40. MAN330 [manA (Genbank: AY534912) was amplified by primer pair Man-F/Man-R, and inserted between SpeI and NotI sites of pUKDN132, pZP25, pZP22, or pZP28 to obtain pZP41, pZP42, pZP43, or pZP44. Xyn-CDBFV [xynC (Genbank: AF123252) was amplified by Xyn-F/Xyn-R, and inserted between SpeI and NotI sites of pUKDN132, pZP25, pZP22, or pZP28 to obtain pZP45, pZP46, pZP47, or pZP48. RuCel1A (Genbank: GU132859) was amplified by CelA-F/CelA-R [SpeI and NotI sites of pUKDN132, pZP25, pZP22, or pZP28 to obtain pZP49, pZP50, pZP51, or pZP52. Primers used in the plasmid construction are listed in Additional file All plasmids used in this study are listed in Additional file rom pUKD , by meanINU1 promoter and signal sequence was conducted by MEGAWHOP Cloning [INU1 promoter and signal sequence were amplified from pZP17 by error-prone PCR using the GeneMorph® II Random Mutagenesis Kit  with primer pair MutP-F/MutP-R. The PCR product was purified, and used as a megaprimer to amplify pZP17 by Phanta Max Super-Fidelity DNA Polymerase. At the end of PCR, DpnI was added into the PCR mixture to digest the methylated template. DpnI-treated product was transformed into FIM-1∆U by lithium acetate method [Random mutagenesis of the  Cloning . Brieflye method . Transfo2HPO4, 1.8 mM KH2PO4, 2.5% Triton X-10, pH 6.4). 20 μL supernatant was mixed with 180 μL CNPF solution and incubated at 37° for 20 min. The release of 2-chloro-4-nitrophenol (CNP) was measured by the absorbance at 410 nm. One unit (U) of feruloyl esterase activity was defined as 1 nmol of CNP from the substrate per minute. Plasmids were extracted from the transformants expressing high level of Est1E by Yeast Plasmid Extraction Kit  and were transformed into E. coli. Plasmids recovered from E. coli were transformed back into FIM-1∆U by the lithium acetate method. Transformants were cultured in 50 mL YG for 72 h, and enzymatic activities in the supernatant were measured as above. Plasmids that drove high-level expression of Est1E were Sanger sequenced to identify mutations inside the inulinase promoter and signal sequence.6500 transformants containing mutant plasmids were subjected to the following screen. Transformants were inoculated into 600 μL YG medium  in 24-well microplates and cultured for 72 h. Plates were centrifuged at 3000 rpm for 5 min, and supernatant was collected. The activity of Est1E in the supernatant was measured by means of 2-chloro-4-nitrophenyl ferulate (CNPF) as substrate . CNPF wa600 of 0.2. 1 × 108 cells were collected after 6 h. Genomic DNA was prepared by Yeast Genomic DNA Extraction Kit. qPCR was performed by means of SYBR Premix Ex TaqII  in a LightCycler 480 II Real-Time PCR System . Plasmid copy numbers were determined by comparing the level of Est1E to that of endogenous LEU2. For transcriptional analysis, transformants were grown in SD liquid medium overnight. Cells were diluted into YG or YI medium  to start at an OD600 of 0.2. 1 × 108 cells were collected after 3, 6 and 9 h. Total RNA was extracted using ZR Fungal/Bacterial RNA MiniPrep  and reverse transcribed into cDNA by PrimeScript RT . qPCR was performed as above, and primers used are listed in Additional file pZP17, pZP22, pZP25, pZP28 were transformed into FIM-1∆U. Transformants were grown in SD liquid medium  overnight. Cells were diluted into YG medium to start at an OD8 cells were collected after indicated times. Cells were washed and resuspended in 400 μL lysis buffer (50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate) supplemented with protease inhibitors cocktail . Cells were mixed with 400 μL acid-washed glass beads  and processed by a bead-beater  at 6 m/s for 2 min. Lysate was centrifuged at 13,200 rpm for 20 min at 4°. 100 μL Supernatant was supplemented with 25 μL 5XSDS PAGE loading buffer (150 mM Tris–HCl (pH 7.0), 12% SDS, 6% 2-mercaptoethanol, 30% glycerol (V/V), 0.05% Coomassie Brilliant Blue G-250) and boiled. 10 μL Samples were subjected to Western blot assay [pZP32 and pZP33 were transformed into FIM-1∆U. Transformants were grown in YG medium and 3 × 10ot assay . Anti-HipZP17–pZP31 and pZP34–pZP40 were transformed into FIM-1∆U separately for expressing Est1E, pZP41–pZP44 for expressing MAN330, pZP45–pZP48 for expressing Xyn-CDBFV, and pZP49–pZP52 for expressing RuCelA. Transformants were selected on SD plates. Transformants were grown in 50 mL YG for 72 h, and supernatant was collected for enzymatic assay. The activity of Est1E was measured as described above. Activity of MAN330 was assayed by means of 0.5% locust bean gum  in 50 mM glycine–NaOH buffer (pH 9.5) at 68° . The act600 nm) and wet cell weight. Supernatant collected at indicated time points was subjected to enzymatic assays as described above.Seed culture was prepared by growing transformants in SD liquid medium for 16 h. Fed-batch fermentations were carried out in a 5 L bioreactor  equipped with controllers for pH, temperature, agitation, and dissolved oxygen. The temperature was maintained at 30°, and pH value was controlled automatically to 5.5 with the ammonium hydroxide during the fed-batch fermentation. The dissolved oxygen concentration maintained above 10% of air saturation automatically varied with the agitation speed at the fixed air rate 3 L/min. The recipe of synthetic medium used in the fermentation was based on a previous one, in which sucrose was replaced by 1% glucose [l-arabinose, mannose, galactose, or ethanol. Transformants were grown in the indicated medium for 72 h, and the supernatant was collected and subjected to enzymatic assays as described above.Medium was prepared with 2% of yeast extract and 4% (w/v) of carbon source: inulin, glucose, cellobiose, xylose, K. marxianus strains. To explore potential regions that contribute to the activity of the inulinase promoter, we used a region starting at 1136 bp 5′ of the start codon , − 735 bp (M2 promoter), and − 370 bp (M3 promoter). M1 promoter corresponded to − 1053 bp promoter, and M3 promoter to − 353 bp promoter identified by Gao et al. . The M3 INU1 promoter and signal sequence that increased Est1E activity sixfold. Subsequently, single point mutants were constructed separately to evaluate their contributions to the improvement. As shown in Fig. Using random mutagenesis and screening for increased Est1E activity, we identified a mutant MM97 with a 6-site mutation in the To investigate the mechanism underlying the improvement of secretory activity, copy numbers of plasmids were analyzed. We found the plasmid copy number was inversely correlated with expression. All higher expressing mutants has significantly reduced plasmid copy number compared to WT Fig. a. Thus, Est1E mRNA levels between cells carrying WT and those carrying P10L plasmid when cells were grown in a medium containing glucose or inulin A plasmid was doubled in glucose medium and increased by threefold in inulin medium, comparing that expressed from the wild-type plasmid. Since the T(-351)A mutation resides 183 bp upstream of the transcription start site, T(-351)A might increase the amount of mRNA by promoting transcriptional initiation [AACACAGG, complementary strand). InuR is a Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in A. niger [K. marxianus so far. Whether the T(-351)A mutation increases the affinity of the promoter to a transcriptional activator requires further study.We found no significant difference in lin Fig. b, c. Thud from T-1A plasmiA. niger . No InuRThe UTR∆A mutation caused a 22-fold increase in mRNA level in glucose medium and a 13-fold increase in inulin medium A mutation showed no distinct effect on endo-β-xylanase production  Schematic map of pUKDN132. b) MCS region of pUKDN132. Amino acids encoded by MCS region was labelled in red.Additional file 4: Figure S2. Purification and specific activity of Est1E. Transformants containing pZP28 were grown in 5L fermentor for 56 h. 200 mL culture was centrifuged at 12, 000 rpm for 30 min at 4 degree. 100 mL supernatant was mixed with 400 mL 20mM Bis-Tris buffer (pH 6.6). 200 mL sample was purified by ion exchange chromatography with Q Bestarose FF column  and eluted with 20 mM Bis-Trish Buffer with 0~1 M NaCl. Fractions containing enzymatic activity were subjected to gel filtration  in an AKTA purifier 100 FPLC system . Flow rate was controlled at 0.5 mL/min in PBS buffer . Peak of Est1E was eluted at 15 mL. The protein concentration of purified Est1E was 90 μg/mL, as measured by a BCA Protein Assay Kit . The enzymatic activity of purified Est1E was 215 U/mL and the specific activity of Est1E was 2400 U/mg. Supernatant of the culture, samples after purification by ion exchange and gel filtration were subjected to SDS-PAGE.Additional file 5: Figure S3. Purification and specific activity of RuCelA. Transformants containing pZP52 were grown in 5L fermentor for 56 h. 200 mL culture was centrifuged at 12, 000 rpm for 30 min at 4 degree. The supernatant was purified by the ion exchange chromatography and gel filtration in the same procedure as described for Est1E. Peak of RuCelA was eluted at 16.3 mL. The protein concentration of purified RuCelA was 55 μg/mL, as measured by a BCA Protein Assay Kit. The enzymatic activity of purified RuCelA was 3.6 U/mL and the specific activity of RuCelA was 65 U/mg. Supernatant of the culture, samples after purification by ion exchange and gel filtration were subjected to SDS-PAGE.Additional file 6: Figure S4. Deglycosylation of Xyn-CDBFV. Transformants containing pZP46 were grown in a 5L fermentor for 60 h. 1 mL culture was centrifuged at 12, 000 rpm for 5 min. Supernatant was collected and subjected to deglycosylation by Endo H  according to manufacturer’s manual. Sample before or after Endo H treatment was mixed with 5XSDS PAGE loading buffer and boiled. Samples were subjected to SDS-PAGE.Additional file 7: Figure S5. Purification and specific activity of MAN330. Transformants containing pZP42 were grown in a 5L fermentor for 56 h. Culture was centrifuged at 12, 000 rpm for 30 min at 4 degree. 100 mL supernatant was mixed with 65 g (NH4)2SO4 and centrifuged at 12,000 rpm for 30 min at 4 degree. Supernatant was discarded. Precipitate was dissolved in 10 mL H2O and subjected to gel filtration  in an AKTA purifier 100 FPLC system. Flow rate was controlled at 0.5 mL/min in PBS buffer (pH 7.4). Peak of MAN330 was eluted at 12 mL and fractions containing the peak were pooled. The protein concentration of purified MAN330 was 1mg/mL, as measured by a BCA Protein Assay Kit. The enzymatic activity of purified MAN330 was 1590 U/mL and the specific activity of MAN330 was 1590 U/mg. Precipitate dissolved in H2O and fractions eluted at 12 mL in gel filtration were subjected to SDS-PAGE.Additional file 8: Table S3. OD600 of cells growing in different carbon sources.


Food raw materials can contain the mycotoxin ochratoxin A (OTA). Thermal processing of these materials may result in decreased OTA levels but also in the formation of the thermal isomerization product 2′R-ochratoxin A (2′R-OTA). So far, only 2′R-OTA levels reported from 15 coffee samples in 2008 are known, which is little when compared to the importance of coffee as a food and trading good. Herein, we present results from a set of model experiments studying the effect of temperatures between 120 °C and 270 °C on the isomerization of OTA to 2′R-OTA. It is shown that isomerization of OTA starts at temperatures as low as 120 °C. At 210 °C and above, the formation of 25% 2′R-OTA is observed in less than one minute. Furthermore, 51 coffee samples from France, Germany, and Guatemala were analyzed by HPLC-MS/MS for the presence of OTA and 2′R-OTA. OTA was quantified in 96% of the samples, while 2′R-OTA was quantifiable in 35% of the samples. The highest OTA and 2′R-OTA levels of 28.4 µg/kg and 3.9 µg/kg, respectively, were detected in coffee from Guatemala. The OTA:2′R-OTA ratio in the samples ranged between 2.5:1 and 10:1 and was on average 5.5:1. Besides coffee, 2′R-OTA was also for the first time detected in a bread sample and malt coffee powder. Aspergillus and Penicillium as well as in food products derived from these commodities [The mycotoxin ochratoxin A OTA,  can be fmodities . Considemodities .Most food items undergo food processing, such as milling, baking, roasting, frying, or fermentation. All of these steps can have an impact on the mycotoxin burden of the product, for instance by physical removal or chemical modification ,3. To whDepending on the commodity, OTA undergoes different types of processing, such as baking, extrusion cooking, roasting, and fermentation. The reduction of OTA levels during these processes has been found to range between no impact and almost a 100% decrease ,10,11,12OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic, and immunotoxic in various species and was classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans (group 2B) in 1993 ,24. The Little is known on the kinetics of the isomerization of OTA to 2′R-OTA, and the minimum temperature required for the thermal conversion of OTA to 2′R-OTA has not been elucidated . DependiHerein, we report the analysis of 51 coffee samples from Germany, France, and Guatemala to extend the database on 2′R-OTA levels in food. Furthermore, the kinetics of the isomerization of OTA to 2′R-OTA were studied in a model system covering temperatures between 120 and 270 °C. As isomerization of OTA to 2′R-OTA has already been observed at 120 °C, further thermally treated food samples were analyzed on the occurrence of OTA and 2′R-OTA.The isomerization of OTA to 2′R-OTA as well as the formation of other degradation products were studied in model heating experiments. To that end, OTA was heated at temperatures of 120, 150, 180, 210, 240, and 260 °C without solvent for 1 to 30 min, respectively. The observed degradation curves of OTA are shown in The published dataset on 2′R-OTA contamination in food and the OTA:2′R-OTA ratio is limited to data from the analysis of 15 coffee samples in 2008 . TherefoIn two decaffeinated coffee samples (The isomer 2′R-OTA was quantifiable in 18 of the 51 coffee samples (35%) with a mean concentration of 0.27 µg/kg. The highest 2′R-OTA level of 3.9 µg/kg was determined in the coffee sample from Guatemala containing 28 µg/kg OTA . However, despite the low OTA contamination, six food samples were found to be positive for 2′R-OTA. In particular, coffee surrogates, such as instant malt coffee powder and malt coffee powder, were all found to be positive for this compound. With an OTA concentration of 0.62 ± 0.04 µg/kg and a 2′R-OTA concentration of 0.22 ± 0.02 µg/kg, comparable racemization rates as for roasted coffee were observed. Additionally, one bread sample (pumpernickel) contained detectable amounts of 2′R-OTA. Pumpernickel is a long-term heated bread. To produce this type of bread, a loaf of rye is baked for 16–24 h at an oven temperature of approximately 110 °C.The thermal instability of the mycotoxin OTA has been reported for several food processing technologies. However only for roasted coffee have the degradation products 2′R-OTA and DC-OTA been described and quantified so far, and in a limited number of samples. In this study, it was shown in model heating experiments that a slow racemization of OTA to 2′R-OTA occurs at temperatures as low as 120 °C. Above these temperatures, a fast racemization of OTA was observed, with 25% 2′R-OTA being formed after 30 min at 150 °C, after 5 min at 180 °C, and after 1 min or less at 210 °C and above. Longer heating periods at temperatures of 240 °C and above resulted in a fast degradation of OTA and 2′R-OTA, which can be explained by other reactions, such as pyrolysis or polymerization. Compared with previously reported data, these results confirm the importance of 2′R-OTA as the main OTA degradation product. A slow conversion of OTA to 2′R-OTA occurring at temperatures as low as 120 °C has not yet been reported .Thus, food samples, processed at temperatures far lower than those applied for coffee roasting, might be additional sources of 2′R-OTA exposure. However, to date, 2′R-OTA has only been detected in blood samples from 34 coffee drinkers in concentrations between 0.021 and 0.411 ng/mL (mean: 0.21 ± 0.066 ng/mL) compared to 0.071–0.383 ng/mL (mean: 0.11 ± 0.093 ng/mL) for OTA. An average OTA:2′R-OTA ratio of 2:1 was determined and in some cases, the concentration of 2′R-OTA even exceeded that of OTA. No 2′R-OTA was detected in the set of 14 samples from non-coffee drinkers, suggesting that 2′R-OTA is predominantly present in roasted coffee. Nevertheless, the number of non-coffee drinkers participating in that study was low, and no correlation between the 2′R-OTA levels and overall coffee consumption was observed, making further sources of 2′R-OTA plausible ,33.Screening of a set of 51 coffee samples confirmed the role of roasted coffee as the key source of 2′R-OTA exposure, with 2′R-OTA levels of up to 3.9 µg/kg in highly contaminated coffee from Guatemala and up to 0.52 µg/kg in European coffee samples. No systematic differences between espresso coffee and other coffee with respect to the 2′R-OTA levels could be observed. For decaffeinated coffee, moderate OTA levels were detected, but no 2′R-OTA in concentrations above the LOQ. Although no information on the OTA content before roasting was available, the reported reductive effect of decaffeination on OTA levels seems to be limited . These rTo confirm the results from the model experiments, other thermally processed food samples, known to contribute to OTA exposure, were analyzed for the presence of 2′R-OTA, the most abundant thermal degradation product of OTA. Besides coffee, quantities of 2′R-OTA were detected in malt coffee as well as in traditionally baked rye bread (Pumpernickel). The latter is of special importance as it confirms the results from the model experiments, showing that OTA can be converted to 2′R-OTA at low temperatures between 100 and 120 °C. However, it has to be considered that this type of bread has a minimum baking time of 16 h, which makes a comparison with other bread and bakery products rather difficult. Other analyzed food samples contained no detectable amounts of 2′R-OTA but were also low for OTA.The most relevant sources of OTA exposure are cereals, followed by coffee, cocoa, meat, and wine. For the cereal products biscuits and muesli, baking times are usually short and temperatures inside the product are mostly at 100 °C or below . For roa2HCO3 were in pro analysi (p.a.) quality from Merck KGaA . Potato dextrose agar (PDA), potato dextrose broth, and KH2PO4 were from Carl Roth , Xylene in p.a. quality was obtained from Honeywell , NaHCO3 in p.a. quality was obtained from Grüssing , and KCl in p.a. quality was obtained from VWR . Purified water of ASTM type 1 quality was prepared with a Purelab Flex 2 system from Veolia Water Technologies .Methanol (MeOH), acetonitrile (ACN), and toluene were obtained in gradient grade from Fisher Scientific . NaCl, formic acid, hexane, and NaAspergillus westerdijkiae BFE 1115, provided by the Max Rubner Institute , which were activated for 2 days in liquid potato dextrose broth at room temperature and then incubated for two weeks at 27 °C on autoclaved durum wheat adjusted to a water content of 62.5% (w/w) and supplemented with 2.5% sodium chloride (w/w). OTA was extracted from the durum wheat cultures using tBME containing 0.5% formic acid, purified by liquid–liquid extraction with water at different pH levels and silica column chromatography with the solvent system toluene/tBME/formic acid (8/1.5/0.5 (v/v/v)). Finally, residual impurities were removed by crystallization from xylene/hexane (7/3 (v/v)). The purity of OTA was >99% as determined by HPLC-UV (220 nm) and NMR. 2′R-OTA, DC-OTA, OTα-amide, d5-OTA, and d5-2′R-OTA were prepared in-house as described elsewhere [OTA was isolated from cultures of lsewhere ,22.v/v/v)) and analyzed by HPLC-FLD for OTA and 2′R-OTA.For the model heating experiments, a stock solution with a concentration of 274 µg/mL in acetonitrile was prepared. Aliquots of the solution  were transferred into 1.5 mL vials and the solvent was evaporated to dryness under a stream of nitrogen at 40 °C. The dried thin film of OTA was heated for 1–30 min at temperatures of 120, 150, 180, 210, 240, and 260 °C, respectively. Subsequently, the samples were dissolved in 1 mL methanol/water/formic acid . The coffee samples originating from Guatemala were collected from small local markets and produced by local coffee roasters. Cocoa beans and nibs were provided as a gift from August Storck KG, Berlin, Germany. Other food samples reported in 3-solution  and 50 µL of a solution containing 100 ng/mL d5-OTA and 50 ng/mL d5-2′R-OTA in methanol were added. The mixture was extracted for three minutes using an Ultra-Turrax T25 mixer  at a rotation speed of 9500 min−1. The obtained suspension was filtered through a 150 mm 3 HW folded filter  and 5.00 mL of the filtrate were diluted with 45 mL phosphate-buffered saline (PBS) pH 7.4  before purification using an OchraTest WB  immunoaffinity column (IAC). After loading the IAC with the sample, the column was washed with 10 mL PBS and 10 mL water. OTA, 2′R-OTA, and DC-OTA were eluted with 2 mL methanol according to the protocol of the IAC manufacturer. The eluate was evaporated to dryness under a stream of nitrogen at 40 °C and reconstituted in 250 µL methanol/water/formic acid .Food samples were analyzed as described in literature with slight modifications ,38. To 5For the determination of the recovery rate, a coffee sample containing only traces of OTA and 2′R-OTA was fortified with three different concentrations of OTA  and 2′R-OTA  before analysis. To that end, aliquots of a standard solution of OTA and 2′R-OTA in acetonitrile were added to the homogenized sample and the solvent was allowed to evaporate for 2 h before extraction. The recovery rates were determined in duplicate and were 104.4 ± 4.0%, 102.9 ± 4.5%, and 103.9 ± 3.8% for 0.5, 2, and 8 µg/kg OTA and 106.0 ± 0.0%, 109.9 ± 11.2%, and 99.4 ± 3.8% for 0.4, 0.7, and 1.4 µg/kg 2′R-OTA, respectively.5-OTA (1 ng/mL) and d5-2′R-OTA (0.5 ng/mL) in a concentration range from 0.1 to 10 ng/mL was used for quantification. The limit of detection (LOD) and the limit of quantification (LOQ) were determined by the signal-to-noise ratios 3 and 9, respectively. For OTA and 2′R-OTA, the LOQ was 0.1 ng/mL and the LOD was 0.03. For the HPLC-FLD experiments, a five-point calibration of OTA, 2′R-OTA (concentration range from 0.10 to 10.0 ng/mL), DC-OTA, and OTα-amide (concentration range from 0.05 to 2.00 ng/mL) was used for quantification. Samples exceeding the calibration range were diluted with methanol/water/formic acid  by an appropriate factor and reanalyzed. For all samples, calibration curves with correlation coefficient r2 > 0.95 were calculated.For HPLC-MS/MS experiments, an external seven-point calibration of OTA and 2′R-OTA with internal standards dm/z 404.2→239.1  and qualifier m/z 404.2→221.0 ; d5-OTA and d5-2′R-OTA: quantifier m/z 409.2→239.1 , and quantifier m/z 409.2→221.0 .An Agilent 1100 series HPLC  was coupled with an API 4000 QTRAP mass spectrometer  operated in electrospray ionization (ESI) positive mode with an ionization voltage of 5500 V. Data acquisition and quantification was carried out with the Analyst 1.6.2 software (Sciex). Chromatographic separation was achieved on a Nucleodur C18 Isis  column with a 5 x 2 mm guard column of the same material  using a binary gradient at a flow rate of 0.3 mL/min. Methanol containing 0.1% formic acid was used as solvent A and water containing 0.1% formic acid as solvent B. The following linear gradient was used: 0 min 60% A, 1 min 60% A, 10 min 100% A, 10 min 100% A. The injection volume was 50 µL. The mass spectrometer was operated in selected reaction monitoring mode (SRM) with Gas 1 (nebulizer) set to 35 psi and Gas 2 (drying gas) set 350 °C and 45 psi. Nitrogen served as the Curtain gas (20 psi). The following transition reactions were monitored for 100 ms each , collision energy voltage (CE), and collision cell exit potential (CXP) are given in brackets): OTA and 2′R-OTA: quantifier Samples of the degradation experiments were analyzed with HPLC Germany coupled to a fluorescence detector . Separation was achieved at 40 °C on a ReproSil-Pur C18-AQ  column  under isocratic conditions using a solvent mixture of methanol/water/formic acid  at a flow rate of 0.7 mL/min. The fluorescence detector was operated at an excitation wavelength of 330 nm and emission wavelength of 460 nm. The injection volume was 5 µL.


Gitelman syndrome (GS) is an inherited autosomal recessive renal tubular disorder characterized by low levels of potassium and magnesium in the blood, decreased excretion of calcium in the urine, and elevated blood pH. GS is caused by an inactivating mutation in the SLC12A3 gene, which is located on the long arm of chromosome 16 (16q13) and encodes a thiazide-sensitive sodium chloride cotransporter (NCCT).A 45-year-old man with Graves’ disease complicated by paroxysmal limb paralysis had a diagnosis of thyrotoxic periodic paralysis for 12 years. However, his serum potassium level remained low despite sufficiently large doses of potassium supplementation. Finally, gene analysis revealed a homozygous mutation in the SLC12A3 gene. After his thyroid function gradually returned to normal, his serum potassium level remained low, but his paroxysmal limb paralysis resolved.GS combined with hyperthyroidism can manifest as frequent episodes of periodic paralysis; to date, this comorbidity has been reported only in eastern Asian populations. This case prompted us to more seriously consider the possibility of GS associated with thyroid dysfunction. Gitelman syndrome (GS) is an inherited autosomal recessive renal tubular disorder characterized by low levels of potassium and magnesium in the blood, decreased excretion of calcium in the urine, and elevated blood pH. GS is caused by an inactivating mutation in the SLC12A3 gene, which is located on the long arm of chromosome 16 (16q13) and encodes a thiazide-sensitive sodium chloride cotransporter (NCCT). Graves’ disease (GD) is a common cause of hyperthyroidism. In our department, we diagnosed a patient with GD and GS.+, 1.4 mmol/l; Na+, 138 mmol/l, Cl−, 97 mmol/l; Ca2+, 2.61 mmol/l; free triiodothyronine (FT3) 6.96 pmol/l (1.86–6.44); free thyroxine (FT4) 38.96 mIU/l (11.45–22.14); thyroid-stimulating hormone (TSH) < 0.01 mIU/l (0.4–4.5); thyroglobulin antibody (TgAb) 16.61 IU/ml (0–150); and thyrotropin receptor antibody (TRAb) 22.36 mIU/l (0–5). Thyroid ultrasound demonstrated diffuse thyromegaly with a rich blood supply. The patient was diagnosed with GD and hypokalemic periodic paralysis and was treated with propylthiouracil (PTU) and potassium chloride. However, 2 days later, despite improvement of his weakness, his temperature increased to 41 °C, and he experienced cough and expectoration. Computed tomography (CT) imaging of his lungs revealed pneumonia. He was subsequently treated with cefazolin and transferred to our hospital 2 days later.The patient was a 45-year-old male with a 12-year history of paroxysmal weakness of the limbs. He was diagnosed with hypokalemic periodic paralysis in 2005 and hyperthyroidism in 2008. He had taken antithyroid drugs on an irregular basis since 2008 but had not undergone proper biochemical examination. Whenever he felt that his weakness was becoming severe, he would self-prescribe potassium chloride. In June 2017, the extent of his lower limb weakness increased such that he could no longer walk. He took potassium chloride without improvement. Subsequently, he was admitted to another hospital. His temperature was 36.7 °C, and his pulse was 96 beats/min. The muscle strength in his lower limbs was grade II  K+ 2.110 mmol/l, Na+ 131.6 mmol/l, Cl− 91.1 mmol/l, Ca2+ 1.850 mmol/l, and Mg2+ 0.540 mmol/l; [thyroid function and thyroid antibodies] triiodothyronine (T3) 1.40 mmol/l (1.34–2.75), thyroxine (T4) > 300 nmol/l (78.38–157.40), FT3 5.32 pmol/l (3.60–6.00), FT4 51.23 pmol/l (7.86–14.41), TSH 0.01 mIU/l (0.34–5.65), thyroid peroxidase antibody (TPOAb) 36.33 IU/ml (0–30), TRAb 9.011 IU/ml (0–30), TgAb 6.04% (< 30%), and thyroid microsomal antibody (TMAB) 6.48% (< 20%); and creatine kinase (CK) 1398 U/l (38–174) and CK-MB 29 U/l (0.0–25.0). The patient’s liver and kidney functions were normal. We treated him with cefazolin, propranolol, PTU and potassium chloride. The patient’s vital signs and strength normalized after 3 days, and his leukocyte count had decreased to 5.97 × 109/l, his neutrophils had decreased to 3.59 × 109/l, and his CK had decreased to 40 U/l. However, his serum potassium level remained low despite 24 g/d of potassium supplementation. Additionally, the patient had hypomagnesemia and metabolic alkalosis : [blood count] leukocytes 13.10 × 10http://bio-bwa.sourceforge.net/), and ANNOVAR (Date: 2015-06-17) was used to annotate the mutation sites based on dbSNP, Clinvar, ExAC, and 1000 genomes, among others. We found a homozygous mutation in the SLC12A3 gene (Exon12 1562-1564delTCA) with an amino acid change of 522delIle, which was first reported as a compound heterozygous mutation.by Vargas-Poussou [Based on these results, we suspected that the patient did not have thyrotoxic periodic paralysis (TPP) but rather GS. Therefore, we sent a blood sample to Beijing Huada Company for sequencing. The Next Generation Sequencing (NGS) was used. The sequencing protocol was based on the Roche Nimblegen SeqCap EZ Choice XL Library for exon trapping. A total of 25 genes Table  known to Date: 205-06-17 w131 therapy. His serum potassium level remained low despite a sufficiently large daily dose of potassium, but no paroxysmal paralysis occurred after discharge.In addition to antithyroid drugs (methimazole 30 mg/d), we gave the patient potassium chloride (3 g/d), potassium citrate (6 g/d), and magnesium potassium aspartate (1.788 g/d). At the follow-up visit, we found that the patient often forgot to take his medicine. The results for thyroid function and electrolyte levels before and after treatment are listed in Table More than 400 SLC12A3 variations have been identified to date. The prevalence of GS among Japanese populations is 10.3/10000 . The preNevertheless, we still lack sufficient data to demonstrate whether AITD is more likely to occur in patients with GS. Patients with GS may undergo more extensive testing, including thyroid functional analysis, compared to healthy individuals, which may facilitate the identification of additional abnormalities. Although sufficient data are available regarding the induction of hypokalemia and hypomagnesemia by hyperthyroidism, research on the long-term effects of hypokalemia and hypomagnesemia on the thyroid is lacking. Iodine and magnesium metabolism have been found to be closely linked . One stuAutoimmune thyroid diseases (AITDs) are complex genetic diseases. The genes contributing to AITD can be divided into two categories: immunomodulatory genes, including the human leukocyte antigen (HLA), cytotoxic T lymphocyte-related antigen 4 (CTLA-4), protein tyrosine phosphatase, nonreceptor type 22 (PTPN22), CD40, CD25, and Fc receptor-like 3 (FCRL3) genes, and thyroid-specific genes, including the thyroid-stimulating hormone receptor (TSHR) gene and the thyroglobulin (Tg) gene. However, no studies have indicated that a correlation exists between these genes and the SLC12A3 gene.In conclusion, GS combined with hyperthyroidism (or other AITDs) can cause hypokalemic periodic paralysis. Our patient was misdiagnosed with hypokalemic TPP for a long time, indicating that the possibility of GS should be considered in clinical cases with hyperthyroidism and persistent hypokalemia.


The crystal structure was characterized using X-ray diffraction. It was found that a single perovskite phase was achieved in Pr-PMN-PT thin films annealed at 650 °C for 3 min. The dielectric constant (εr) value was 2400 in 2.5% Pr-PMN-PT thin films at room temperature, 110% higher than that of pure PMN-PT samples. Through 2.5% Pr3+ doping, remanent polarization (Pr) and coercive field (Ec) values increased from 11.5 μC/cm2 and 35 kV/cm to 17.3 μC/cm2 and 63.5 kV/cm, respectively, in PMN-PT thin films. The leakage current densities of pure and 2.5% Pr-PMN-PT thin films were on the order of 1.24 × 10−4 A/cm2 and 5.8 × 10−5 A/cm2, respectively, at 100 kV/cm. A high pyroelectric coefficient (py) with a value of 167 μC/m2K was obtained in 2.5% Pr-PMN-PT thin films on Si substrate, which makes this material suitable for application in infrared detectors.The [001]-oriented Pr Recentlon (PLD)  indicati3+, La3+, and so forth have been used to improve the up-conversion photoluminescence in bulk piezoelectric materials [d33 = 1500 pC/N) was obtained in Sm-doped PMN-PT polycrystalline [3 (PZT) thin films, the dielectric and piezoelectric properties were improved by La doping [3+ doped PMN-PT thin films were prepared by the sol–gel method at various annealed temperatures and times. The phase structure of the samples was analyzed by X-ray diffraction. Then, the surface morphology of Pr-PMN-PT thin films was measured by scanning electron microscopy, atomic force microscopy, and piezoresponse force microscopy. Finally, dielectric, pyroelectric, and ferroelectric properties of Pr-PMN-PT thin films were investigated.Rare earth elements such as Eraterials ,29. Recestalline  based ona doping . Until n2 diffusion barrier layer was first fabricated by oxidizing the Ti metal layer as follows: Ti films of 60 nm thickness were deposited on SiO2/Si (100) substrate by DC magnetron sputtering in an Ar gas environment at room temperature. The sputtering power was 175 W. The base vacuum of Ti sputtering was higher than 8 × 10−4 Pa. The ratio of Ar:O2 was 90:10. After deposition, the Ti layer was annealed in a rapid thermal processing (RTP) furnace at 700 °C for 1 h. Then, a Pt bottom electrode of 80 nm thickness was deposited on the TiO2 diffusion barrier layer. The sputtering parameters were the same as the Ti metal layer deposition. The sheet resistance was about 2.85 Ω/sq, which was measured by the four probe method.Before sol–gel processing, a TiO3+ doped Pb(M1/3Nb2/3)O3-0.30PbTiO3 (xPr-(1 − x)(PMN-0.30PT), x = 1%, 2%, 2.5%, and 3%) precursor solutions were prepared by the sol–gel method. The lead acetate trihydrate (Pb(CH3COO)2·3H2O), magnesium acetate tetrahydrate (Mg(CH3COO)2·4H2O), niobium ethoxide (Nb(OCH3CH2)5), tetrabutyl titanate (Ti(OC4H9)4), and praseodymium acetyl acetone (C15H21PrO6·xH2O) were used as starting materials to synthesize the Pr-PMN-PT sol–gel solution. An additional 20 mol% of lead acetate trihydrate was added to equalize the Pb volatilization and 5 mol% more magnesium acetate tetrahydrate was mixed to boost the production of perovskite phase. Next, 2-methoxyethanol (C5H8O2) and glacial acetic acid (CH3COOH) were added as solvent and catalyst respectively. Acetyl acetone (C5H8O2) was added as a stabilizer in the Pb-Pr-Mg-Nb-Ti solution. The concentration of the Pr-PMN-PT solution was attenuated to 0.4 mol/L. Then, the Pr-PMN-PT thin films were deposited by spin coating on Pt/TiO2/SiO2/Si substrates at 2000 rpm for 9 s before the spin coating was subsequently sped up to 4000 rpm for 20 s. To evaporate the solvent in solution, the sample was dried at 220 °C for 3 min after each deposition and then pyrolyzed at 450 °C for 5 min. Finally, Pr-PMN-PT films were annealed at 600, 650, and 700 °C for 1–5 min in air by RTP at ramp rates +20 °C/s and −5 °C/s respectively. In order to get 500 nm thick film, this entire process was repeated 10 times. For further electric measurement, Au top electrodes were deposited on Pr-PMN-PT samples using magnetron sputtering through a shadow mask with a 1 mm diameter.After that, Prpy) was calculated by the equation as below:I is the pyroelectric current, A is the surface area of the samples, and dT/dt is the rate of temperature cooling.The crystalline structure of Pr-PMN-PT thin films was analyzed by X-ray diffraction (XRD)  with Cu-Kα radiation in the 2theta range of 20°–60° and a scan step length of 0.02 at room temperature. The surface morphologies of the samples were characterized by a field emission scanning electron microscope (FE-SEM) , atomic force microscope (AFM) , and piezoresponse force microscopy (PFM) . For electric measurement, the samples were all of a pure perovskite phase structure due to the high electric performance. The dielectric properties were measured using an impedance analyzer  with a precision LCR meter connected to a heating stage . The P-E hysteresis loops and leakage current were examined by a standard ferroelectric system . The pyroelectric coefficient , (110), (111), (002), (220), and (112) reflections respectively. The pyrochlore phase peak (222) at 2θ = 29.4° was clear for samples annealed at 600 °C and 700 °C and was identified as Pb2Nb2O7 by the comparison to JCPDS No. 40-828, which showed the other characteristic diffraction peak at 2θ = 34.1°. Compared to the typical PMN-PT solid solution, the single perovskite structure was obtained at 650 °C, which showed two strong peaks at 2θ = 22.2° and 45.3°, indicating a mainly preferred crystal growth along the (001)c direction. 2/SiO2/Si substrates annealed at 650 °C for 1 min, 3 min, and 5 min respectively. It was found that 2.5% Pr-PMN-PT thin films were mostly in the perovskite phase with a little pyrochlore phase when annealed for 1 min, which was further crystallized to a single perovskite phase structure after one more minute annealing. However, a peak of the pyrochlore phase was found again in the sample annealed for 5 min, which can be ascribed to the greater lead loss caused by the longer time annealing [Avoiding the formation of a pyrochlore phase is a key factor for the synthesis of PMN-PT thin films as the addition of this phase will reduce the piezoelectric and ferroelectric properties of the resulting thin films. In order to restrict the formation of a pyrochlore phase, the annealing conditions for thin film preparation were first investigated. nnealing .αhkl, which can be calculated by the following formula [Ihkl is the relative intensity of the corresponding diffraction peaks (hkl). As shown in α001 values are 86.6% and 73.6% in Pr-PMN-PT thin films annealed at 650 °C for 2 and 3 min respectively, presenting the highly (001)-oriented textured structure. The preferred orientation was related to many factors, such as substrate materials [The degree of (001) texture in 2.5% Pr-PMN-PT thin film was evaluated by preferred orientation parameter  formula :(2)αhklaterials , growth aterials , annealiaterials , and so 2 films are around 450 nm, 80 nm, and 80 nm in thickness respectively, which are in agreement with the values measured by a surface profilometer. 2 scan area at the surface of 2.5% Pr-PMN-PT thin films is shown in Rrms) is about 2.59 nm with a scan area of 2 × 2 μm2, indicating low interference of electrode contact in electronic measurement. A schematic of the device for further electric investigation is shown in εr, py, and pr in 2.5% Pr-PMN-PT thin films were on the order of 2400, 167 μC/m2K, and 17.3 μC/cm2, showing 110%, 123%, and 50% higher factors respectively than those in pure PMN-PT thin films. Compared with PMN-0.30PT grown by a chemical solution deposition (CSD) method [εr in PMN-0.30PT thin films on Si substrate was lower than that of the sample synthesized on perovskite structure single crystal substrates. A similar tendency of pyroelectric property was found in PMN-0.30PT thin film grown on various substrates. The pr value in 2.5% Pr-PMN-PT thin films was still higher than that of PMN-0.32PT thin films deposited on Ba0.5Sr0.5RuO3/NdScO3 and La0.5Sr0.5CoO3/LaAlO3 crystals using pulsed laser deposition. In contrast, the py value showed an opposite trend, which is attributed to the lattice misfit. Compared with PVDF and normal ferroelectric material, the relaxor-PT thin films showed higher dielectric and pyroelectric properties. In contrast, the response time of the PMN-0.30PT pyroelectric sensor was longer than that of PVDF [3 [The dielectric, pyroelectric, and ferroelectric properties in 2.5% Pr-PMN-PT thin films at room temperature are summarized in ) method , the εr  of PVDF  and LiTa PVDF [3 .3+ doping effect in PMN-PT thin films, the elements distribution was measured, as shown in In order to evaluate the Pr3+ ratios of 0, 1.5%, and 2.5%. The addition of Pr3+ in PMN-PT thin films increased the value of remanent polarization (Pr) and coercive field (Ec) from 11.5 μC/cm2 and 35 kV/cm to 17.3 μC/cm2 and 63.5 kV/cm respectively. The leakage current densities of pure and 2.5% Pr-PMN-PT thin films were approximately 1.24 × 10−4 A/cm2 and 5.8 × 10−5 A/cm2 respectively at 100 kV/cm, as shown in Ec in Pr-PMN-PT thin films, indicating that the domain structure was easily reoriented owing to the presence of lead vacancies near the grain boundaries [3+ ion distribution shown in 3+ doped compositions compared to the undoped counterpart, as shown in undaries ,45, corrεr) and dielectric loss (tanδ) of Pr-PMN-PT thin films in the frequency range of 20 Hz to 200 kHz at room temperature. We see that the εr value decreased slightly with an increasing frequency, which is induced by the polarization decrease due to charge accumulation at low frequency. Meanwhile, the dielectric loss showed a very quick increase in the range 104–2 × 105 Hz, indicating conduction through the electrode surface and grain boundary capacitance interference in poled samples at high frequency. εr curve presents a broad peak centered at around 100 °C, which corresponds to the diffuse ferroelectric to paraelectric phase transition [ansition . The valpy) measured at room temperature for the obtained PMN-PT thin films as a function of Pr3+ doping content. The py in the thin films with 2.5% dopant presented the maximum value on the order of 167 μC/m2K, which is almost a two-fold increase over that of pure PMN-PT thin films, offering a new way for enhancing the pyroelectric performance in relaxor-PT thin films for use in infrared detectors. The temperature dependence of py values is illustrated in 3+ doped PMN-PT ferroelectric thin films with a single perovskite structure were prepared by a sol–gel processing and investigated by X-ray diffraction. The microstructure of Pr-PMN-PT samples was characterized by scanning electron microscopy, atomic force microscopy, and piezoresponse force microscopy. The average grain size was about 100 nm. It was found that Pr3+ ions were located at the edge of grain boundaries measured by energy dispersive X-ray spectroscopy. For 2.5% Pr3+ doping, the dielectric constant, pyroelectric coefficient, and remenant polarization all reached a maximum. Importantly, the dielectric constant in PMN-PT thin films increased from 1140 to 2400 through 2.5% Pr3+ doping. Such enhancement was ascribed to the domain wall motion induced by the Pr3+ ion distribution around the grain boundary. Finally, this rare earth dopant presents a useful method for developing high performance relaxor-PT thin films for use in energy transducers and photodetectors.The pure and Pr


Context: Phytochemical study and biological potential of Evax pygmaea (L.) Brot. (Asteraceae) are reported for the first time.Objective: To identify the secondary metabolites of Evax pygmaea and to determine its antioxidant, antibacterial and cytotoxic activities.Materials and methods: Dried aerial parts (1 kg) were macerated in 70% MeOH (5 L) during 72 h. The concentrated hydromethanolic extract was subjected to extractions with chloroform (3 × 300 mL), ethyl acetate (3 × 300 mL) and n-butanol (3 × 300 mL), successively. VLC of combined ethyl acetate (EAEP) and n-butanol (BEP) fractions was followed by column purifications. Antioxidant activity was investigated using DPPH, CUPRAC, and metal chelating, β-carotene/linoleic acid and ABTS assays. Agar method was used in the antibacterial study. Cytotoxic activity was determined by Brine shrimp lethality test in DMSO and ethanol, at varying concentrations  and  successively.Results: Quercetin (1), isorhamnetin 3-O-β-d-xyloside (2), isorhamnetin 3-O-β-d-glucoside (3), quercetin 3-O-β-d-glucoside (4), quercetin 7-O-β-D-glucoside (5), patuletin 3-O-β-d-glucoside (6) were isolated from for the first time from Evax genus. The EAEP was the most active in ABTS (IC50: <3.125 μg/mL) assay whereas the BEEP exhibited the highest activity in the β-carotene/linoleic acid assay (IC50: <3.125 μg/mL). The EAEP and BEP exhibited good antibacterial activity (MIC: 40–80 µg/mL). The plant did not show any toxicity (LD50>80 µg/mL).Discussion and conclusions: Six flavonoids were isolated for the first time from Evax pygmaea which exhibited good antioxidant and antibacterial activities. Medicinal plants have been widely used for therapeutic purposes since ancient times. The beneficial effects of fruits and vegetables are generally attributed to the presence of phenolic compounds such as phenolic acids, flavonoids and tannins, nitrogen compounds such as alkaloids and amines, as well as vitamins, terpenoids and other metabolites, which have a high antioxidant activity  is the richest vascular plant family in the world, with 1600–1700 genera and 24,000–30,000 species  (Asteraceae) is mainly distributed in the Mediterranean area, it is represented by eight species, five of them are found in Algeria (Quezel and Santa E. pygmaea (L.) Brot. (syn. Filago pygmaea L.), growing wild in Tunisia  which is known as an antimicrobial.We report here, for the first time, the phytochemical study, the antioxidant activity, by the use of five methods, and the antibacterial and cytotoxic activities of 254 , column chromatography (CC) was performed on a polyamide SC6 and silica gel 60 spots were observed under UV light at 254 and 365 nm or visualized by heating after spraying with H2SO4 50%. Polyamide SC6 was purchased from Fluka .NMR spectra were recorded on a Bruker AVANCE DRX 500 NMR spectrometer . Thin layer chromatography (TLC) was carried out on precoated silica gel 60 F•), ascorbic acid, 2,2′-azinobis(3-ethyl-benzothiazoline-6-suphonic acid) diammonium salt (ABTS•+), β-carotene-linoleic acid were obtained from Sigma Chemical Co. . All other chemicals and solvents used were of analytical grade.Both antioxidant and cytotoxic activity measurements were carried out on a 96-well microplate reader, butylatedhydroxylanisole (BHA), butylatedhydroxyltoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPHE. pygmaea were collected on May 2014 at Constantine (North Eastern Algerian) and identified by Professor Gérard De Bélair, Faculty of Sciences University of Annaba, Algeria. A voucher specimen (LOST.Ep.05.14) was deposited at the Herbarium of the Laboratory of Therapeutic Substances (LOST), University des frères Mentouri-Constantine.The aerial parts of Evax pygmaea, were macerated at room temperature with MeOH–H2O  for 24 h, three times. After filtration and concentration, the residue was dissolved in water. The resulting solution was extracted successively with CHCl3, EtOAc and n-butanol. Concentration in vacuo led to the following fractions: CHCl3 (2 g), EtOAc (4.7 g) and n-butanol (10 g). Ethyl acetate (EAEP) and n-butanol (BEP) fractions were combined because they are similar. This mixture (10 g) was column chromatographed on polyamide SC6 with a gradient of toluene–MeOH with increasing polarity; six main fractions  were collected. Fraction F5 (154 mg), obtained from toluene 88% was further purified on a silica gel CC eluted with an isocratic system  and yielded a yellow precipitate which was identified as compound 2 (15 mg), the fractions F3 (100 mg), F7 (400 mg), F8 (289 mg), F10 (230 mg) and F11 (320 mg), obtained from toluene 92, 85, 80, 75 and 70%, respectively, yielded the compounds 1, 3, 4, 5 and 6 successively as yellow precipitates .Air-dried and powdered aerial parts (1.5 kg) of The structures of these compounds were established by chemical and spectral analysis UV, NMR and acid hydrolysis as well as by comparing their spectroscopic data with those reported in the literature.f with authentic samples. Sugars were identified in the aqueous residue by comparison with authentic samples on silica gel TLC impregnated with 0.2 M NaH2PO4, solvent Me2CO–H2O (9:1), revealed with aniline malonate. The optical rotation of each purified sugar was measured and compared with authentic samples.The pure compounds were treated with 4 M HCl at 100 °C for 1 h. The hydrolysates were extracted with EtOAc and the aglycones were identified by their UV spectra in methanol and by comparison of their R• in methanol was prepared and 4 mL of this solution was added to 1 mL of sample solutions in methanol at different concentrations. Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. BHT and BHA were used as antioxidant standards for comparison of the activity. The scavenging capability of DPPH radical was calculated using the following equation. The results were given as IC50 (µg/mL) corresponding the concentration of 50% inhibition.Acontrol and Asample are the absorbances of the reference and sample obtained from the UV–visible spectrophotometer, respectively.The free radical-scavenging activity was determined spectrophotometrically by the DPPH assay Blois . In its 2+ complexation assay measured spectrophotometrically  (50% inhibition):The metal chelating activity by the ferrene–Fe•+ scavenging activity was determined according to the method of Re et al. The ABTSe et al. . The ABT50).The bleaching activity of the fractions was evaluated by the β-carotene-linoleic acid test system Miller . β-CarotR) of β-carotene was calculated according to the following equation:The bleaching rate (a is the absorbance at time zero and b is the absorbance at time t (120 min). The antioxidant activity was calculated in terms of percent inhibition relative to the control, using following equation:where ln is the natural log, 2·2H2O), 1 mL of ammonium acetate buffer at pH 7.0 and 1 mL of neocaproin solution (0.0075 M) were mixed to 0.5 mL of plant extract or standard of different concentrations solution. The final volume of the mixture was adjusted to 4.1 mL by adding 0.6 mL of distilled water. The resulting mixture was incubated for 1 h at room temperature, and then the absorbance of the solution was measured at 450 nm by the use of a spectrophotometer against blank and BHT and BHA as standards.The cupric reducing capacity of the fractions was determined by the CUPRAC method  corresponding the concentration indicating 0.50 absorbance intensity.The results were given as The total polyphenolics was determined by the Folin–Ciocalteu method, with slight modifications. Mixture of extract (0.5 mL) and 5 mL of Folin–Ciocalteu reagent (1 mol) was neutralized with 4 mL saturated sodium carbonate (75 g/L), and kept at room temperature for 2 h. The absorbance was measured at 760 nm by the use of a spectrophotometer. The total polyphenolics were expressed as gallic acid equivalents (GAE) . Control larvae were placed in DMSO and ethanol, at varying concentrations  and  successively. After 24 h, the tubes were examined against a lighted background and the average number of larvae that survived in each tube was determined.Cytotoxicity of the BEP, EAEP and CEP fractions of 50) was determined using the microplate reader.The concentration at 50% mortality of the larvae      (Sikorska & Matławska O-β-d-glucoside (6) : 257, 375; +NaOH: 271, 425; +AlCl3: 270, 452; +AlCl3/HCl: 267, 426; NaOAc: 274, 394. +H3BO3: 269, 365. 1H NMR : 7.74 , 7.65 , 6.92 , 6.41 , 6.21 .C21H20O11, yellow powder, UV : 253, 345; +NaOH: 271, 335, 415, +AlCl3: 268, 401; +AlCl3/HCl: 268, 402; NaOAc: 275, 395. +H3BO3: 270, 346. 1H NMR : 7.86 , 7.57 , 6.93 , 6.44 , 6.19 , 5.37 , 2.70–3.70 (sugar protons), 3.84 .C22H22O12, yellow powder, UV : 254, 355; +NaOH: 271, 330, 413, +AlCl3: 268, 403; +AlCl3/HCl: 269, 400; NaOAc: 274, 396. +H3BO3: 269, 365. 1H NMR : 7.95 , 7.61 , 6.93 , 6.43 , 6.23 , 5.44 , 3.20–3.90 (sugar protons), 3.76 .C21H20O12, yellow powder, UV : 256, 357; +NaOH: 273, 328, 409, +AlCl3: 269, 435; +AlCl3/HCl: 269, 403; NaOAc: 273, 392. +H3BO3: 264, 380. 1H NMR : 7.74 , 7.61 , 6.92 , 6.41 , 6.23 , 5.27 , 3.20–3.79 (sugar protons).C21H20O12, yellow powder, UV : 256, 372; +NaOH: 271, 425; +AlCl3: 271, 461; +AlCl3/HCl: 267, 429; NaOAc: 259, 406. +H3BO3: 261, 390. 1H NMR : 7.79 , 7.69 , 6.91 , 6.78 , 6.49 , 5.08 , 3.40–3.75 (sugar protons).C22H22O13, yellow powder, UV : 263, 360; +NaOH: 269, 408; +AlCl3: 276, 456; +AlCl3/HCl: 273, 395; NaOAc: 261, 366. +H3BO3: 271, 381. 1H NMR : 7.80 , 7.69 , 6.90 , 6.69 , 5.08 , 3.10-4.10 (sugar protons), 3.71 .CEvax pygmaea were evaluated for their antioxidant activity. The results showed that the EAEP was the most active in DPPH (IC50: 6.91 ± 0.09 µg/mL), ABTS (IC50: <3.125 μg/mL) and CUPRAC (A0.50: 6.26 ± 0.04 μg/mL); this can be due to its polyphenol content which is higher than that of BEP  , however, the BEP exhibited the best activity in the β-carotene/linoleic acid decolourization assay (IC5 μg/mL) .Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 43300 and Pseudomonas aeruginosa ATCC 27853, with 40 µg/mL MIC’s value (Klebsiella pneumoniae (MIC 40 µg/mL).The EAEP and BEP fractions displayed the highest activity against ’s value . However50 values were all greater than 80 μg/mL (Cytotoxicity analysis, determined by Brine shrimp lethality test, revealed that this plant was not toxic as their LD80 μg/mL .n-butanol fractions of E. pygmaea. These fractions showed a good antioxidant activity with the five used methods namely, β-carotene-linoleic acid, DPPH• and ABTS•+ scavenging, CUPRAC and metal chelating assays. This result could be in part related to the isolated flavonoids from which some possess a quercetin squeleton. The fractions showed a good antibacterial activity against tested microorganisms which confirms its use as an antimicrobial. The brine shrimp lethality test revealed that this plant was not toxic. The results of this study indicated that E. pygmaea could be an important dietary source of phenolic compounds with high antioxidant capacity.Six flavonoids have been isolated, for the first time, from the ethyl acetate and