R. stolonifer, C. gloeosporioides and B. cinerea, and the pathogenic bacterium S. Saintpaul. In addition, regardless of emulsifier type and emulsification process, BW–SN applied on the tomato surface exhibited low contact angles (38.5° to 48.6°), resulting in efficient wettability (−7.0 mN/m to −8.9 mN/m). These nanoemulsions may be useful to produce edible coatings to preserve fresh-produce quality and safety.Nanoemulsions are feasible delivery systems of lipophilic compounds, showing potential as edible coatings with enhanced functional properties. The aim of this work was to study the effect of emulsifier type (stearic acid (SA), Tween 80 (T80) or Tween 80/Span 60 (T80/S60)) and emulsification process  on nanoemulsion formation based on oxidized corn starch, beeswax (BW) and natural antimicrobials (lauric arginate and natamycin). The response variables were physicochemical properties, rheological behavior, wettability and antimicrobial activity of BW–starch nanoemulsions (BW–SN). The BW–SN emulsified using T80 and microfluidized showed the lowest droplet size (77.6 ± 6.2 nm), a polydispersion index of 0.4 ± 0.0 and whiteness index (WI) of 31.8 ± 0.8. This BW–SN exhibited a more negative ζ-potential: −36 ± 4 mV, and Newtonian flow behavior, indicating great stability. BW–SN antimicrobial activity was not affected by microfluidization nor the presence of T80, showing inhibition of the deteriorative fungi  The food industry is consistently looking for innovative technologies to improve quality, safety and functional properties of food products . Nanotecoc) that was 61.47 ± 0.02 °C. This curve exhibited two peaks, where the dominant crystallization peak (Tc) was at 57.15 ± 0.09 °C with a crystallization enthalpy (ΔHc) of −186.15 ± 1.34 J/g. For the endothermic process, a clear melting onset temperature (Tom) was observed at 50.64 ± 0.04 °C, showing two peaks, where the maximum melting peak (Tm) appeared at 63.09 ± 0.06 °C, with a melting enthalpy (ΔHm) of 188.85 ± 0.35 J/g. The presence of different peaks from the BW thermal behavior may be attributed to the heterogeneous composition of the BW matrix [c  and ΔHm , probably associated with different composition. The estimation of BW thermal parameters defined minimal temperature conditions for nanoemulsion formation, to avoid wax solidification.The thermal behavior of BW was evaluated through the differential scanning calorimetry (DSC) thermogram , in whicW matrix ,17. The p < 0.05) between emulsifier type and emulsification process affected the average droplet size (z-average) and PDI of the BW–SN (v/v) emulsified with T80 in 1:3 (v/v) ratio. Salvia-Trujillo et al. [v/v) ratio using the same conditions as those reported here. The slightly higher droplet sizes obtained in this work were attributed to the low crystallization temperature of BW droplets, tending to coalesce due to their heterogeneous composition.The dynamic light scattering (DLS) technique assumes that large particles scatter light more strongly than small particles , whereashe BW–SN . Using she BW–SN . Ghosh eo et al. , workingp < 0.05) by emulsifier type and emulsification processes. Emulsions containing SA subjected to ultrasound, and SA or T80/S60 treated by microfluidization, exhibited the weakest electrical charge of −3.6 ± 0.4 mV, −5.0 ± 0.2 mV and −7.0 ± 0.3 mV, respectively. In contrast, nanoemulsions produced using T80 as emulsifier, and formed by microfluidization, showed the strongest electrical charge of −35.8 ± 3.8 mV. This value is more negative than those reported by Guerra-Rosas et al. [ζ-potential is the difference in electrical charge between the dense layer of ions around the micelle particles and that of the stationary layer of fluid surrounding them . Particls et al. , rangings et al. . Anionics et al. . Finallys et al. . The higOne of the most important factors that determine the formation and properties of nanoemulsions is the solution pH through ionization of surface groups, and therefore influencing the final surface charge density . In thisp < 0.05) between emulsifier type and emulsification process on the color of the BW–SN, expressed as WI, is shown in p < 0.05) improved to 31.7 ± 0.8 using T80 under microfluidization. Salvia-Trujillo et al. [A significant interaction  was observed between the emulsifier type and emulsification process  flow curves, associated with agglomeration of starch–wax droplets, was observed for BW–SN added with SA and microfluidized (w/w) using T80 (1:0.5 oil-emulsifier (w/w) ratio) after homogenization; all samples displayed 0.81 ≤ n ≤ 0.95. In another study, Rezvani et al. [w/w) emulsified with SA (1% and 2% w/w) using two homogenization cycles, achieving n = 0.68–0.78. The remaining BW–SN presented a Newtonian behavior (n = 1), that was attributed to the high amount of continuous phase involved in BW–SN interactions and the low particle size  of the BW–SN were tested on the surface of tomato  on Ws of BW–SN  by the emulsifier type and emulsification process, in agreement with Liang et al. [R. stolonifer, 28.7 ± 1.9 mm for C. gloeosporioides, 27.9 ± 8.0 mm for B. cinerea and 13.9 ± 0.7 mm for S. Saintpaul. For BW–SN using T80 , C. gloeosporioides (27.5 ± 0.3 mm), B. cinerea (31.5 ± 1.7 mm) and S. Saintpaul (14.4 ± 0.5 mm). Similarly, Salvia-Trujillo et al. [Escherichia coli with sodium alginate nanoemulsions incorporated with lemongrass or clove oil and T80 as emulsifier under a microfluidization process. On the other hand, ultrasonically produced BW–SN did not show an inhibitory effect against any of the tested microorganisms, indicating a significant (p < 0.05) interaction with the emulsifier type-emulsification process, which prevented antimicrobial activity. According to Teixeira et al. [w/w), and natamycin (NAT) (0.04% w/w) [Arredondo-Ochoa et al.  have repg et al. . Inhibitsing T80 B, inhibi mixture C, eithera et al.  and Lu ea et al. , the antw/w), and non-crystallizable sorbitol were provided by Ingredion . BW, stearic acid (SA), Tween 80 (T80), Span 60 (S60) and morpholine were supplied by Sigma . Lauric arginate ester (LAE) was acquired from Vedeqsa-Lamirsa  while natamycin (NAT) was purchased from EcoBio .Commercial oxidized starch (OS) (carbonyl + carboxyl groups = 15.7 ± 1.2% Rhizopus stolonifer (CDBB-H-318) and Botrytis cinerea (CDBB-H-1556) were provided by the national collection of microbial strains and cell cultures , whereas Colletotrichum gloeosporioides strain was ATCC 42374. Salmonella Saintpaul S70 was supplied from the culture collection of the center for food research and development .R. stolonifer, C. gloeosporioides and B. cinerea. Tryptone soy agar (TSA) (Bioxon) was used for S. Saintpaul growth. Potato dextrose agar (PDA)  was used to grow The thermal behavior of BW was evaluated using a DSC  equipped with a refrigerated intracooling system. High-purity indium was used as standard and dry nitrogen as purge gas. Ten mg of BW was weighed into a hermetically sealed aluminum pan. The sample was equilibrated at 90 °C before being cooled to −40 °C at a rate of 10 °C/min, kept isothermally at −40 °C for 10 min, and reheated to 90 °C at 5 °C/min. Measurements were performed in triplicate and heat flow (W/g) was plotted against temperature (°C). Onset temperature and maximum peak (°C) were obtained from thermal curves of crystallization and melting, while enthalpy (J/g) was determined by integrating the area under each thermogram in the range 10–80 °C, using the Universal Analysis 2000 V 4.5 software (TA Instruments) . w/w) and non-crystallizable sorbitol (2.4% w/w) were heated at 85 °C for 20 min under magnetic stirring to complete starch gelatinization. Nanoemulsions formation was carried out following the reports of Hagenmaier and Baker [w/w) plus morpholine (0.15% w/w) as cosolvent were added with the selected emulsifier in varying ratios (w/w): BW:SA (5:1), BW:T80 (1:2.5) or BW:T80/S60 (1:2.5). The starch dispersion was kept at 85 °C along with a mixture of two antimicrobial agents (LAE (0.2% w/w) and NAT (0.04% w/w)) according to Arredondo-Ochoa et al. [2 factorial design was conducted. Main factors were the type of emulsifier, and emulsification process, at three levels: three types of emulsifiers, and three emulsification processes, whereas the response variables were physicochemical, rheological, wettability and antimicrobial properties. The obtained BW–starch nanoemulsions were stored at 4 ± 1 °C for subsequent characterization.OS suspension , by dynamic light scattering (DLS) technique working at 633 nm at 25 °C with a backscatter detector at 173°. Samples were previously diluted with ultra-pure water 1:20) to avoid multiple scattering effects. Size distribution curves as a function of intensity (%), average droplet size (z-average) (nm), and PDI were used to characterize the BW droplet dispersion in the emulsions [ to avoidThe lipid droplet surface charge  of BW–SN was evaluated by measuring their electrophoretic mobility when an electric field was applied by phase-analysis light scattering (PALS) using a Zetasizer Nano ZS . The Smoluchowski model was applied with the instrument’s software to obtain ζ–potential (mV) values, indicating potential stability of colloidal systems .The pH was obtained from direct measurements of BW–SN with a pH meter .The color of BW–SN was determined with a colorimeter , set up for illuminant D65 and 10° observer angle at room temperature, calibrated with a standard white plate. The Commission Internationale de lÉclairage (CIE) L*, a*, and b* values were determined and the whiteness index (WI) was calculated using Equation (1) :WI = 1w/v) ammonium molybdate, pH 6.5, for 1 min. The grids were observed using TEM  at an acceleration voltage of 80 kV and the images was obtained using a digital micrograph 3.1 software  [Nanoemulsions were observed by negative-staining electron microscopy as a direct measurement of droplet size and shape. The sample was adsorbed onto carbon-coated copper/palladium grids for 1 min, and then negatively stained by floating the grids face-down on a drop of 2%  .−1; whereas linear oscillatory shear flow was used to estimate viscoelastic properties  in a frequency range of 0.1–100 rad/s in the linear viscoelastic regime. Measurements were carried out in duplicate and experimental data were analyzed directly with the TA Rheology Advantage Data Analysis V.5.7.0  software [Simple shear flow and oscillatory tests of BW–SN were conducted with a controlled stress rheometer , fitted with double-concentric cylinder geometry , at 25.0 ± 0.1 °C controlled by a circulating water bath (Cole Parmer Polystat and Peltier AR-G2). Estimation of viscosity (η) as a function of shear rate software .Ws) of a solid by a liquid is determined by the balance between adhesive forces (Wa) of the liquid on the solid (Equation (2)), and cohesive forces (Wc) of the liquid (Equation (3)) expressed in mN/m, which takes into account the action of three surface forces (Equation (4)) [The wettability coefficient (ion (4)) :(2)Wa=(θ), using a drop shape analyzer DSA30 , fitted with DSA4 software. A drop (9 µL) of reference liquid , with known surface tension, was gently dispensed on horizontal tomato surface for contact time of 60 s. Finally, a Zisman plot of the surface tension of each liquid versus its corresponding cos (θ) was carried out, and extrapolation of the curve when cos (θ) = 1 corresponded to the value of The e Zisman  method be of γSV .The g method . A correg method  was intrθ) formed by each BW–SN on the tomato surface was measured according to The R. stolonifer, C. gloeosporioides and B. cinerea) and a pathogenic bacterium (S. Saintpaul S70) commonly present in fresh produce according to Arredondo-Ochoa et al. [5 CFU/mL of tested fungi in PDA plates, followed by incubation at 28 ± 1 °C for 24 h. In addition, TSA plates similarly inoculated with the pathogenic bacterium were incubated at 37 ± 1 °C for 24 h. The antimicrobial effect was determined by the inhibition zone diameter (mm) [Antimicrobial activity of BW–SN was evaluated against three deteriorative fungi  .p < 0.05) among mean values, employing the JMP statistical software, version 5.0.1 . Physicochemical, rheological, wettability, and antimicrobial properties of nanoemulsions were conducted in triplicate, and the Tukey test was used to determine significant difference (BW–SNs were successfully produced and a significant influence of emulsifier type and nanoemulsification process was evidenced from the physicochemical, rheological and antimicrobial properties. Using T80 and microfluidization, BW–SNs of low particle size were produced, suggesting homogeneous distribution of ingredients leading to stability and translucency, showing a Newtonian behavior, and efficient wettability properties. This BW–SN led to complete inhibition of deteriorative fungi and a pathogenic bacterium that are commonly present in fresh produce, making it useful as a suspension able to produce edible coatings to protect and preserve fresh food products.


Optically pure acetoin (AC) is an important platform chemical which has been widely used to synthesize novel optically active α-hydroxyketone derivatives and liquid crystal composites.slaC and gldA encoding meso-2,3-butanediol dehydrogenase  and glycerol dehydrogenase (GDH), respectively, in S. marcescens MG1 were knocked out to block the conversion from AC to 2,3-butanediol . The resulting strain MG14 was found to produce a large amount of optically pure (3R)-AC with a little 2,3-BD, indicating that another enzyme responsible for 2,3-BD formation except meso-2,3-BDH and GDH existed in the strain MG1. Furthermore, SlaR protein, a transcriptional activator of AC cluster, was overexpressed using PC promoter in the strain MG14, leading to enhancement of the (3R)-AC yield by 29.91%. The recombinant strain with overexpression of SlaR, designated as S. marcescens MG15, was used to perform medium optimization for improving (3R)-AC production.In this study, 3R)-AC was produced by strain MG15 with the productivity of 1.11 g/l h and the conversion rate of 80.13%.Under the optimized conditions, 39.91 ± 1.35 g/l ( The current highest production of 75.2 g/l AC was achieved from Serratia marcescens H32 without considering its isomer -AC catalyzed by α-acetolactate decarboxylase (α-ALDC), while a small amount of α-acetolactate was transformed into diacetyl (DA), by the non-enzymatic oxidation decarboxylation -AC which decreased the stereoisomeric purity of (3R)-AC. Both (3R)-AC and (3S)-AC were then transformed into 2,3-BD by the meso-2,3-BDH and the GDH encoded by slaC and gldA  -AC producing strain, both the slaC and gldA were knocked out in S.marcescens MG1 using pUTKm1 as the suicide vector. The resulting mutant strain MG14 was found to produce single configuration of optically pure (3R)-AC from sucrose. Besides, three endogenous promoters were attempted to express the transcriptional regulator SlaR for the purpose of enhancing the production of (3R)-AC. Finally, the optimal culture conditions and the suitable fermentation medium component were developed for the engineered strain using statistical methods.In this study, to obtain a (Restriction enzymes were purchased from Thermo Fisher (USA). T4 DNA ligase and Primestar were purchased from TaKaRa Biotech . Taq DNA polymerase was purchased from CW Bio . Bacterial DNA kit was purchased from SBS Genetech , plasmid mini kit, cycle-pure kit, and gel extraction kit were purchased from Omega Biotech . AC and 2,3-BD were purchased from Sigma-Aldrich .slaC and gldA in Serrata marcescens MG1. In the case of slaC, the left and right flanking sequences of slaC were amplified from the genomic DNA of S.marcescens MG1 and then fused by overlap PCR. The ∆slaC fragments were gel purified and then ligated into pUTKm1. The resulting plasmid was designated pUT-slaC. For conjugation, E. coli S17-1 λpir was used as the donor strain to amplify and transfer the suicide plasmid pUT-slaC. The conjugation cells were recovered with fresh LB and plated on selection medium plates. One of the kanamycin-resistant transconjugants (single-crossover) was picked on LB plate containing 50 μg/ml kanamycin. The single-crossover was then inoculated at 30 °C for 12 h on LB plate without antibiotic; Kanamycin-sensitive colonies from a double crossover event were verified by PCR using primers slaC-F1 and slaC-R2 to obtain the slaC mutant strain MG12. The gldA from MG1 and MG12 was knocked out individually in the same way to construct the gldA mutant strain MG13 and the double-knockout strain MG14.The strains, plasmids, and primers used in this study are listed in Table slaR gene encoding the transcriptional regulator SlaR was amplified from the genomic DNA of S.marcescens MG1 using primers slaR-1 and slaR-2. The expression plasmid PAC-slaR was constructed by inserting gene slaR into pACYC184 , PA (promoter of slaA), and PR (promoter of slaR) were amplified from the genomic DNA of MG1 using primer pairs PC-1/PC-2, PA-1/PA-2, and PR-1/PR-2. The three purified PCR products were double-digested with XbaI/HindIII and ligated to the corresponding sites of pAC-slaR, respectively, to generate pAC-PC-slaR, pAC-PAB-slaR, and pAC-PR-slaR. The vectors were then introduced into S. marcescens MG14 separately by electroporation. The recombinants were selected on LB plates supplemented with 25 μg/ml chloramphenicol and designated as MG15, MG16, and MG17, respectively.The construction of recombinant plasmids is also shown in Table The engineered strains were grown at 30  C for 10 h, and then the fermentation broth was centrifuged at 8000 rpm for 10 min. After washing twice and resuspending with 50 mM phosphate buffer (pH 7.0), the cells were disrupted with an ultrasonic cell breaking apparatus . The resulting supernatant was finally obtained through centrifugation and was used for assaying enzyme activities  activities were determined by measuring NADH oxidation at 340 nm using a UV/visible spectrophotometer . Wayne . The rea2, and then the reaction was terminated by adding 0.5 ml 2 M NaOH. One unit of the integrated activity of α-ALS and α-ALDC was defined as the amount of enzyme required for the formation of 1 μM AC per milligram protein per minute at 30 °C. Total protein concentrations were determined using the Bradford method described by Marion , 20 mM sodium pyruvate, 0.01 mM flavin adenine dinucleotide (FAD), 0.5 mM thiamine pyrophosphate (TPP), and 0.5 mM MgCly Marion .2SO4 and determined using a bioanalyzer  after centrifugation. The intracellular NADH and NAD+ concentrations were determined by procedures described in the previous studies  with the flow rate at 0.6 ml/min. AC and 2,3-BD in the fermentation broth were extracted by ethyl acetate with the addition of isoamyl alcohol as internal standard and then quantified by GC (Agilent GC9860) equipped with a chiral column . The operation conditions used were as follows: N2 was used as the carrier gas at a flow rate of 1.2 ml/min; the injector temperature and the detector temperature were 215 and 245 °C, respectively; the oven temperature was maintained at 50 °C for 1.5 min, and then raised to 180 °C at a rate of 8 °C/min. A calibration curve was used to determine the concentration of the products.The cell density was monitored by determining the absorbance at 600 nm using a spectrophotometer . To determine the residual sucrose concentrations of the samples, the sucrose of the samples was hydrolyzed to glucose by 2 M HE. coli, S. marcescens MG1, and its derivatives on a rotary shaker at 200 rpm. E. coli was grown at 37 °C and S. marcescens MG1 was grown at 30 °C. If necessary, the LB medium was supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin, or 25 μg/ml chloramphenicol.Luria–Bertani (LB) medium of 50 ml in 250 ml flask was used for culturing 4)2SO4 6, KH2PO4 10, NaCl 0.5, and MgSO4 0.5 at pH 7.2. The initial fermentation medium was composed of the following (g/l): sucrose 90, yeast extract 25, sodium citrate 14, sodium acetate 4, NH4H2PO4 3, MgSO4 0.5, and MnSO4 0.05  into a 250 ml shake flask with 50 ml of fermentation medium at 30 °C on a rotary shaker at 200 rpm. The seed medium was composed of the following (g/l): glucose 10, peptone 2, yeast extract 1, (NH3R)-AC production on engineered S. marcescens were investigated. The whole fermentation process was conducted for 36 h. The optimum culture conditions were chosen for experiments of medium optimization.For culture conditions, the effects of temperature , pH , liquid volume , and inoculation size  on  design was used to select factors that significantly influenced (3R)-AC production. Table 3R)-AC production were screened from PB design for further optimization. Response surface methodology (RSM) based on central composite design (CCD) was conducted to determine the optimal levels of the variables with significant effects for improving (3R)-AC production. The design details are given in Table For medium optimization, sucrose, yeast extract, sodium citrate, sodium acetate, NHfluenced R-AC prodSerratia marcescens was regarded to be a good 2,3-BD-producing strain. As shown in the study of Rao et al. -AC concentrations of 19.79 and 6.52 g/l were obtained from strains MG 12 and MG 13, respectively, while only 2.92 g/l (3R)-AC was produced from S. marcescens MG1 (Table slaC or gldA had positive effects on (3R)-AC production. However, lower cell density, longer fermentation time, and lower comprehensive titer of AC and 2,3-BD were observed after slaC inactivation. The slaC and gldA double-knockout strain MG 14 accumulated the highest concentration of (3R)-AC without (3S)-AC. This indicated that slaC and gldA were the only two genes responsible for the production of (3S)-AC in S. marcescens MG1. Therefore, S. marcescens MG14 was chosen for further investigation for (3R)-AC production.As shown in Fig. + concentration was also observed. Table slaC and/or gldA could increase the NADH/NAD+ ratio, especially when slaC was knocked out. This was probably because the 2,3-BD pathway which participated in the regulation of NADH/NAD+ ratio  has been proved to efficiently increase acetoin production in Bacillus subtilis, similar procedure was attempted in Serratia marcescens MG1. In our previous work, PC was proved to be a moderate promoter in MG1 (data not shown). PC in MG1 might function similar to PbdhA in Bacillus subtilis. PA and PR were the only two related promoters in the AC operon. In order to find the most suitable promoter, experiments were carried out with these promoters.SlaR, as the transcriptional regulator of the AC cluster in slaR expression under the promoter of PC, PA, and PR, respectively. The resulting plasmids pACPC-slaR, pACPA-slaR, and pACPR-slaR were transformed into MG14 by electroporation. The resulting strains were named MG15, MG16, and MG17. The integrated activity of α-ALS and α-ALDC in strains MG14, MG15, MG16, and MG17 was determined at approximately 30 h in flask fermentation -AC production were determined in the engineered strains. Strain MG15 had the lowest cell density (OD600 = 6.24), but the highest (3R)-AC production of 27.74 g/l, which was 29.91% higher than that of MG14. No obvious difference was detected in cell density and sucrose consumption of MG16 compared to MG14, while the (3R)-AC production was improved by 7.07%. Lower cell growth rate and sucrose consumption rate was observed from MG17 than MG14, and the (3R)-AC concentration of 19.08 g/l produced by MG17 was the lowest. In summary, the highest (3R)-AC production was achieved in MG15, so PC was chosen as the most suitable promoter to control the expression of SlaR.As shown in Fig. 3R)-AC production of strain MG15, the fermentation conditions and medium composition were optimized. As shown in Fig. 3R)-AC production was highest at 30 °C, pH 7.0, 20 ml liquid volume, and 5% inoculation size. Then the above conditions were used for optimizing the medium composition.To further improve the -AC production from 19.78 to 32.31 g/l in the 12 experiments. After analysis of the regression coefficients, sucrose, yeast extract, sodium citrate, and NH4H2PO4 showed positive effects, while sodium acetate, MgSO4, and MnSO4 showed negative effects. The p values of sucrose and sodium acetate were >0.05, so they were considered significant, whereas the other factors were considered insignificant. Although sucrose and sodium acetate were identified to be two significant medium components, the optimal levels of them were still need to be determined in further experiments.Plackett–Burman design was first used to screen significant factors from the seven variables including sucrose, yeast extract, sodium citrate, sodium acetate, NH3R)-AC production (Y) and the concentrations of sucrose (X1) and sodium acetate (X2).Sucrose and sodium acetate were further optimized based on the results of PB design using CCD. The experimental design and results are shown in Table R2 value of this model was 0.9793, indicating the model could explain 97.93% of the variability in the response. The adjusted R2 was 96.45%, which indicated a high significance of the model.The 3R)-AC production. It was obvious that the response surface was convex in nature, suggesting that the optimum conditions were well defined. Based on the equation model and the response surface, the optimal concentration of sucrose and sodium acetate was 105 and 1 g/l, respectively; the corresponding maximum (3R)-AC production was predicted to be 39.27 g/l.The three-dimensional response surface Fig.  was also3R)-AC production by S. marcescens MG15 was as follows (g/l): sucrose 105, yeast extract 25, sodium citrate 15, sodium acetate 1, NH4H2PO4 0.5, MgSO4 0.2, and MnSO4 0.02. Validation experiment was carried out in triplicate test to confirm the reliability of the model equation. As shown in Fig. 3R)-AC yield of 39.91 ± 1.35 g/l was obtained under the optimized condition, which was very close to the predicted maximum value of 39.27 g/l. Besides, the conversion rate was 80.13% and the productivity was 1.11 g/l h. Therefore, this result indicated the validity of the model.From the above results, the optimum medium composition for -AC, with a little 2,3-BD. In addition, overexpression of the transcription regulator SlaR under the control of the PC promoter was successful for enhancing (3R)-AC synthesis by S. marcescens MG14. Furthermore, the suitable culture conditions were optimized using single-factor experiments and the optimal medium component was developed using PB design and RSM. Based on the suitable culture conditions and the optimized medium, the engineered S. marcescens MG16 could produce 39.91 ± 1.35 g/l (3R)-AC with a conversion rate of 80.13% and productivity of 1.11 g/l h in the flask fermentation.In this study, the


Escherichia coli (STEC) contamination. The capacity of surfactant micelles loaded with the plant-derived antimicrobial eugenol to reduce STEC on beef trimmings that were later ground and refrigerated for five days at 5 ± 1 °C was tested to determine their utility for beef safety protection. STEC-inoculated trimmings were treated with free eugenol, micelle-encapsulated eugenol, 2% lactic acid (55 °C), sterile distilled water (25 °C), or left untreated (control). Following treatment, trimmings were coarse-ground and stored aerobically at 5 ± 1 °C. Ground beef was then sampled for STEC immediately post-grinding, and again at three and five days of storage. STEC minimum inhibitory concentrations (MICs) in liquid medium for free eugenol and 1% sodium dodecyl sulfate (SDS)-loaded micelles were 0.5% and 0.125%, respectively. STEC numbers on beef trimmings treated by sterile water (6.5 log10 CFU/g), free eugenol (6.5 log10 CFU/g), micelle-loaded eugenol (6.4 log10 CFU/g), and lactic acid (6.4 log10 CFU/g) did not differ compared to untreated controls (6.6 log10 CFU/g) (p = 0.982). Conversely, STEC were significantly reduced by refrigerated storage  (p = 0.014). Antimicrobial treatments did not significantly decontaminate ground beef, indicating their low utility for beef safety protection.Beef safety may be compromised by O157 and non-O157 Shiga toxin-producing  Escherichia coli (STEC) are known causes of human foodborne disease, inducing both acute and chronic sequelae ranging from mild diarrheal disease to hemolytic uremic syndrome (HUS), renal failure and potentially death [E. coli O157:H7, six serotypes of the non-O157 STEC have been declared adulterants in fresh non-intact beef products by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) [The O157 and non-O157 Shiga toxin-producing ly death ,3. In adDA-FSIS) . EstimatDA-FSIS) .10 CFU/cm2. Others have reported greater reductions in STEC on trimmings and ground beef following application of lactic acid as a beef safety intervention. Harris et al. [10 CFU/g or 2.1 to 2.5 log10 CFU/g following application of 2% or 4% lactic acid, respectively. This group recently reported that application of acetic acid (2% or 4%) and acidified sodium chlorite (1000 ppm) produced reductions in STEC on beef trimmings ranging from 0.3 to 0.5 log10 CFU/g (p < 0.05) [Cattle have been identified as a reservoir for the STEC, potentially facilitating cross-contamination of beef products during handling of meat ,8,9. Fres et al.  reported < 0.05) . Nonethe10 CFU/g in STEC numbers [E. coli O157:H7 in ground beef patties during refrigerated storage, but did enhance reductions of the pathogen upon subsequent cooking to an internal temperature of 65 °C [In addition to exploring the use of traditional antimicrobials for reducing STEC on fresh beef products, researchers have investigated plant-derived antimicrobials (PDAs), including essential oils and their components, for beef safety protection. Application of commercial herb extract-containing products to STEC-inoculated beef trimmings, two rounds of grinding, and up to seven days’ refrigerated (4 °C) storage, produced reductions of 0.1 to 0.2 log numbers . More reof 65 °C ,17,18. TApplication of such antimicrobial interventions may be useful for beef safety protection in the manufacture of natural beef products. Hence, the objectives of this study were to: (i) quantify the capability of free and micelle-encapsulated eugenol to produce statistically significant reductions in numbers of inoculated STEC on beef trimmings prior to beef grinding when applied at concentrations not likely to produce long-term negative impacts on consumer acceptability; and, (ii) determine the capacity of antimicrobial treatments applied to beef trimmings to inhibit STEC growth and/or reduce STEC numbers on ground beef during refrigerated storage post-treatment.R), were provided by J.B. Luchansky  and preserved at −80 °C upon receipt in the culture collection of the Food Microbiology Laboratory . Isolates were revived by duplicate identical passages in sterile tryptic soy broth , followed by incubation at 35 ± 1 °C for 24 h. Following revival, isolates were individually streaked onto Petri dishes containing MacConkey Agar  supplemented with 100 µg/mL rifampicin , and incubated for 24 h at 35 ± 1 °C to verify RifR capacity, as well as organisms’ ability to utilize lactose and decompose bile salts. Well-isolated colonies were picked with sterile needles and slant cultures of each isolate were prepared on tryptic soy agar  for later use during experimental trials. Slants were layered with sterile mineral oil and stored at 5 ± 1 °C until being prepared for use via the revival procedure described above.STEC isolates belonging to serotypes O157:H7, O26:H11, and O121:H19, resistant to 100 µg/mL rifampicin (Rifw/v) stock was prepared by mixing 12.0 g SDS in 40.0 mL sterile distilled water in a sterile container. SDS has been previously reported to exert antimicrobial activity against members of the STEC at concentrations as low as 0.5% SDS [10 CFU/mL inoculum in double-strength TSB ; 100.0 µL of each isolate was added into surfactant solution-containing wells. Surfactant was added first (200.0 µL), and then 100.0 µL was extracted and loaded into the adjacent well containing 100.0 µL sterile distilled water, according to the method of Pendleton et al. [® Instruments, Inc., Winooski, VT, USA). In addition to the experimental wells described, microplate wells containing identically prepared and loaded solutions of SDS, 2× TSB, and sterile water, but no STEC, were prepared and read at OD630. This was completed in order to provide for baseline adjustment in experimental data to allow for identification of minimum inhibitory concentrations (MICs) of SDS and eugenol, using the method of Brandt et al. [Sodium dodecyl sulfate  was purchased from Sigma-Aldrich Co.; a 30%  were diluted in 95% EtOH to desired eugenol concentrations; eugenol and EtOH were purchased from Sigma-Aldrich Co. The working stock was stored at 5 ± 1 °C in a foil-wrapped container until ready for use. Following calculation of required eugenol and surfactant for micelle generation , eugenolDetermination of STEC isolate-specific MICs for free and micelle-loaded eugenol was completed according to published methods . MicroplFrozen beef trimmings (90% lean) were obtained from the Rosenthal Meat Science and Technology Center at Texas A & M University  and transported to the Food Microbiology Laboratory. Trimmings were thawed at 4 °C for 24 h prior to inoculation and use. On the day of experiment initiation, 1000 g of thawed beef trimmings were inoculated with a three-strain mixture of STEC and sealed in a sterile plastic bag. The STEC mixture was produced by blending equivalent volumes of overnight cultures of revived STEC isolates  accordinFollowing antimicrobial treatment, trimmings were coarse-ground through a 3/8 inch plate using a LEM Products electric meat grinder . In between the grinding of each batch of treated trimmings, all grinder components were disassembled and residual beef tissue removed by brush-scrubbing while soaking in chlorinated (0.6% FAC) tap water. After all beef was removed, the grinder components and scrubbing brush were soaked for an additional 10 min in chlorinated water (0.6% FAC). Components were then removed and sprayed with 70% EtOH to further sanitize prior to rinsing with water and re-assembly of the grinder. Beef was aseptically separated into 10.0 g samples, randomly assigned to an antimicrobial treatment and storage period, and subjected to assigned antimicrobial intervention treatment. STEC survival was determined immediately after antimicrobial treatment, immediately after grinding, and again at 3 and 5 days of aerobic refrigerated (5 ± 1 °C) storage in order to determine whether STEC reductions would be detected quickly or require additional holding to allow for eugenol release. Samples were placed into stomacher pouches, diluted with 90 mL sterile 0.1% peptone diluent, and stomached for 1 min at 230 rpm. Decimal dilutions were subsequently prepared in 0.1% peptone diluent and surviving STEC enumerated on TSAR following 24 h at 35 ± 1 °C.n = 3); the lowest inhibitory concentration of free or micelle-loaded eugenol for each STEC isolate across all replicates was identified as the experimental MIC [n = 3); log10-transformed plate counts were determined from sample plate counts prior to statistical analysis. Reductions in STEC on beef were determined by analysis of variance (ANOVA) and statistically differing means separated by Tukey’s Honestly Significant Differences (HSD) test at α = 0.05.MIC experiments were repeated three times in identical fashion  eugenol in 1% SDS   . Similarlsewhere ,26. Prevlsewhere ,25,27,2810 CFU/g. No treatment produced statistically significant STEC reductions on trimmings versus untreated controls (p = 0.902); the greatest numerical reduction in STEC (0.3 log10 CFU/g) as compared to the control was observed for 55 °C lactic acid-treated beef trimmings (E. coli O157:H7 numbers (0.1 to 0.2 log10 CFU/g) have been reported following the treatment of beef trimmings (80% lean) with sprays of either 2% or 4% acetic/lactic acid blends heated to 55 °C [E. coli O157:H7 following plant extract application in micro-encapsulation systems and subsequent holding.The mean number of STEC inoculated onto beef trimmings was 6.6 ± 0.4 logrimmings . Similarto 55 °C . Researcto 55 °C  likewiseE. coli O157:H7 following hot water and heated acid in this study were 0.5 and 1.1 log10 CFU/g, respectively. In a study applying 5% lactic acid via immersion to lean trimmings inoculated with O157 and non-O157 STEC, researchers reported STEC reductions varied from 0.5 to 1.4 log10 CFU/cm2, depending on whether the acid solution was applied at 25 °C or 55 °C [E. coli O157:H7 numbers of 0.5 or 0.7 log10 CFU/cm2 on inoculated trimmings immersed in 0.1% acidified sodium chlorite or 0.02% peroxyacetic acid, respectively [10 CFU/g following 20 h post-treatment refrigerated storage, whereas spray-treatment produced only a 0.5 log10 CFU/g reduction following post-treatment refrigerated storage [In contrast to these studies, other studies have reported higher STEC reductions on trimmings when immersed in solutions of traditional antimicrobials. Ellebracht et al.  submergeor 55 °C . This grectively . Finally storage . Researc storage . Neverth storage .p = 0.371), and no effect of interaction of antimicrobial treatment with storage duration (p = 0.812) (p = 0.014)  following treatment by water, free or micelle-entrapped eugenol, or 55 °C lactic acid spraying indicated no effect of antimicrobial treatment (= 0.812) . However= 0.014) . Lack of= 0.014) . GrindinE. coli O157:H7 0.6 to 0.7 log10 CFU/g following five days’ storage at 4 °C under vacuum packaging [E. coli O157:H7 numbers on minced beef following application of plant-derived antimicrobial essential oil components and holding for 3 h at 5 °C approximated 0.1 to 0.5 log10-cycles [Salmonella [Others have also reported numbers of STEC on ground beef to be reduced only to a low extent following application of plant compounds with antimicrobial activity. The application of allyl isothiocyanate (AIT) via impregnated disc to ground beef reduced ackaging . Reducti0-cycles . Nonethelmonella .10-cycle) reductions in STEC were observed as a function of storage period (p < 0.05)  to members of the O157 and non-O157 STEC in vitro was able to produce growth inhibition at MICs similar to those reported elsewhere. Nonetheless, the application of free or micelle-loaded eugenol to STEC-inoculated beef trimmings, as well as the application of 2% lactic acid (55 ± 1 °C) did not reduce STEC on trimming surfaces . Followi < 0.05) . Eugenol


Enteromorpha linza displayed potent antimicrobial activity against Prevotella intermedia and Porphyromonas gingivalis. To elucidate the active component of E. linza, isolation procedures were performed.We found that the edible green seaweed 1H nuclear magnetic resonance (NMR) spectroscopy, and 13C NMR spectroscopy. The yields of SA and GLA from dried seaweed tissue were 6.33 × 10-3% and 6.47 × 10-3%, respectively. The minimal inhibitory concentration values of SA and GLA were 39.06 μg/mL against P. intermedia and 9.76 μg/mL against P. gingivalis, respectively. SA and GLA were also active against several other oral pathogens, including Aggregatibacter actinomycetemcomitans, Candida albicans, Fusobacterium nucleatum subsp. vincenti, and Streptococcus mutans, at micromolar concentrations.The main active compound was isolated by polarity fractionation, Sephadex LH-20 gel chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The active compounds were eluted at isocratic 95% acetonitrile by RP-HPLC and identified as unsaturated fatty acids, stearidonic acid  and gamma-linolenic acid  by gas chromatography–mass spectrometry, E. linza extracts SA and GLA are useful antimicrobial agents for the prevention and/or treatment of periodontitis.These data suggest that the The online version of this article (doi:10.1186/1999-3110-54-39) contains supplementary material, which is available to authorized users. Prevotella intermedia and Porphyromonas gingivalis. Systemic or topical antibiotic therapies have been found to be useful in the treatment of periodontal disease , gingival index (GI), bleeding on probing (BOP), and activity against two bacterial strains  , Cheju Island, Korea, in April 2009. After rinsing with tap water to remove salt and miscellanea, the samples were dried for 1 day at room temperature using an electric fan. Samples were then ground to a powder using a coffee grinder for 5 min and stored at −20°C until use.The green seaweed High-performance liquid chromatography (HPLC)-grade reagents (or the highest grade available) and culture media were purchased from Sigma Aldrich, Ltd. . All solutions were made with ultrapure deionized water .E. linza powder was extracted with a 600-mL solution of methanol–water (4:1) at room temperature overnight. The extract was then filtered through Toyo No. 2 filter paper under reduced pressure. This extraction procedure was repeated three times and the extracts were combined. The methanol–water extract was concentrated to a dark-green residue (30.2 g) under reduced pressure in an evaporator. The extract was fractionated according to polarity, as described by Harborne . Gas chromatography–mass spectrometry (GC–MS) was performed on a 5975C gas chromatograph  equipped with an HP-5MS capillary column (30 m × 250 μm × 0.25 μm). The column temperature was programmed from an initial temperature of 80°C for 2 min and raised to a final temperature of 300°C at a rate of 5°C/min. The samples were converted to methyl ester derivatives by the protocol reported by Lepage and Roy  spectrometer , operating at 500 and 100 MHz for  and Roy . The inj and Roy . The strIn vitro antimicrobial activity assays were performed with the anaerobic bacteria of P. intermedia KCTC25611 and P. gingivalis KCTC381 obtained from the Korean Collection for Type Culture . These strains were cultivated on tryptic soy agar  media containing 10% sheep blood, 0.1% hemin  and vitamin K1  in a Bactron IV chamber  at 37°C, 5% CO2, 10% H2, and 85% N2.Aggregatibacter actinomycetemcomitans KCTC 3698, Candida albicans KCTC 17485, Fusobacterium nucleatum subsp. vincenti KCTC 5105, and Streptococcus mutans KCTC 3065. A. actinomycetemcomitans was cultivated on brucella medium  containing 3% horse serum  in a Bactron IV chamber. C. albicans was cultivated on RPMI 1640 medium , F. nucleatum subsp. vincenti was cultivated on Schaedler medium , and S. mutans was cultivated on BHI medium  with 3% horse serum . All microorganism incubations were performed at 37°C.Following identification of the active compounds, their antimicrobial activity was measured against several oral pathogens, including anaerobic 8 CFU/mL. Microtitre plates were prepared by dispensing the inoculum and active compound into each well. The first well of each strip contained 100 μL broth with no compound and 100 μL inoculum, and represented the negative control. The second well on each strip contained 90 μL broth, 10 μL methanol, and 100 μL inoculum, and represented the vechicle control. The final volume in each well was 200 μL. Following incubation, the MIC value was defined as the lowest concentration that yielded no bacterial cell growth  was extracted with 10 mL dichloromethane on a rotator for 1 h at 30 rpm. Following centrifugation at 2,000 × g for 5 min, 4 mL of the clean supernatant was evaporated to 5 mg/mL for RP-HPLC. Each 100-μL aliquot was separated on an Alltima C18 column (10 mm ID × 25 cm) (W. R. Grace & Co.) using the same isolation procedure. The compound levels were assessed by measuring the dimensions of the HPLC peaks using the standard curve of the pure compound.To measure the levels of antimicrobial compounds in P. intermedia and P. gingivalis. Fraction S14 was further fractionated by RP-HPLC. The active compounds were eluted at 5.1 min (S14-1), 6.3 min (S14-2), and 7.9 min (S14-3), respectively, in isocratic 95% acetonitrile by RP-HPLC. Among them, the antimicrobial activities of S14-2 and S14-3 were much stronger than those of S14-1. Thus, further isolation was conducted using S14-2 and S14-3, which were oily and yellow. The weights of S14-2 and S14-3 were 9.5 mg and 9.7 mg, and the yields were 6.33 × 10-3% and 6.47 × 10-3%, respectively, from the dried seaweed powder. The antibacterial potency of the purified compounds was determined by measuring MIC values. The MIC values of the extracts and fractionates were measured against the anaerobic bacteria of P. intermedia and P. gingivalis , heteronuclear multiple-bond correlation (HMBC), and correlation spectroscopy (COSY) spectra. Infrared analysis of the purified compounds showed absorption for OH and carbonyl function. The ta Table  and 13C-a Figure  of S14-2P. intermedia and P. gingivalis were compared, we observed no difference. The commercially available SA and GLA were then tested for antimicrobial activity against several oral pathogens, including A. actinomycetemcomitans, C. albicans, F. nucleatum subsp. vincenti, and S. mutans  and GLA S14-3) and the commercially available SA (Sigma-Aldrich 49509) and GLA (Sigma-Aldrich L2378) against  and the E. linza powder.To quantify the active compounds directly from the seaweed tissue, 0.0131 g (2.62%) of dichloromethane extract was obtained from 0.5 g of powder. The S14–2 and S14–3 peaks were integrated using the Empower™ software (Waters Co.), and the percentile dimensions were measured as 18.87% and 5.22% by HPLC. The amounts of S14–2 and S14–3 were calculated as 2.5 mg (0.50%) and 0.7 mg (0.14%) from 0.5 g of E. linza) extract displayed potent antimicrobial activity against P. intermedia and P. gingivalis  for use as a food ingredient in the United States (Lemke et al. E. linza extract are effective against gingivitis disease (Cho et al. In conclusion, we have shown for the first time that the fatty acids SA and GLA inhibit the growth of oral pathogens. Moreover, mouth rinses containing the


The mink circovirus (MiCV), a newly discovered pathogen, is associated with diarrhea in farmed minks. The prevalence and economic importance of this virus remain poorly understood, and a quantitative method for diagnosis of MiCV infection has not been established. This research aims to develop a highly specific, sensitive, and quantitative assay for MiCV. A Real-Time quantitative polymerase chain reaction (qPCR) assay was developed to detect different isolates of the MiCV in mink samples. The qPCR system is highly sensitive with a detection limit of as low as 10 viral DNA copies. The specificity of this qPCR assay was supported by the absence of cross-reaction with other pathogens. The coefficients of variation were low for both inter-assay and intra-assay variabilities. In addition, the results also expressed the distribution of MiCV in infectious mink tissues with high levels of virus in the skeletal muscle and heart. The heart occupied a higher proportion than other tissues, which can be considered the primary source of test material. This qPCR method could be a useful tool for epidemiological studies and disease management. This method for MiCV is highly specific, sensitive, repeatable, quantitative, and can rapidly determine viral load levels in different tissues samples. Circovirus belonging to the Circoviridae family. MiCV is a small non-enveloped icosahedral virus with a circular single-stranded DNA genome of 1,753 nucleotides  as reported initially as a novel pathogen in diarrheal minks in Dalian, China  techniques have proven to diagnose important viral livestock diseases and have become established scientific tools in veterinary virology and disease control  and used as the positive control for the qPCR and conventional PCR assay. Negative controls to test the specificity of qPCR included AMDV and DogCV isolated by our laboratory from Heilongjiang Province, respectively (GenBank accession No. KY680280 and No. MF797786). The porcine circovirus 1 (PCV1) and PCV2 were obtained from Dr. Yanwu Wei, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences . The primers used were cap2-F: 5′-CGTATTGTCCAGGTTTGTATGAAG-3′, and cap2-L: 5′-TCACAGGCATTCCCGCTAC-3′.DNA samples were extracted from liver, lung, spleen, heart, duodenum, jejunum, ileum, colon, brain, kidney, skeletal muscle, marrow, and mesenteric lymph nodes. Theses tissue samples were collected from mink samples. Generally, 0.1 gram of each tissue specimens was weighed, added into 500 μL of phosphate buffered saline, all the samples were homogenized, freeze thawed three times, then centrifugated. Total genomic DNA was extracted from 200 μL supernatant of tissue homogenates using genomic DNA Kit , according to the manufacturer's protocol. After elution in 100 μL elution buffer, DNA was stored at −80°C before use.E. coli DH5α cells  using the pMD18-T vector . Recombinant plasmids were purified by the plasmid purification kit  and validated by sequencing company . The target plasmid pMDT-Cap with the original concentration was identified by using a Nanodrop 2000 spectrophotometer . The serial tenfold dilutions of the standard plasmid templates were used for the quantitative analysis and the copy number of plasmid was computed referred to the previous study , 0.4 μL 50 × ROX Reference Dye, 0.8 μL of each primer (10 μM), and 12 μL of nuclease-free water. The qPCR reaction was conducted in 8-tube strip using a ABI Prism 7500 thermocycler  under the following conditions: 95°C for 15 min, then 40 cycles of denaturation for 10 s at 95°C, annealing for 35 s at 55°C and elongation for 45 s at 72°C. A melting curve analysis was performed at the end of the amplification. The dilutions were tested in triplicate and used as quantification standards to construct the standard curve. by plotting the plasmid copy number logarithm against the measured cycle threshold (Ct) values. The standard curve, correlation coefficient of the standard curve, and efficiencies were calculated automatically by the LightCycler software. The amount of DNA quantified for each sample was expressed as number of copies/reaction.Serial 10-fold dilutions containing copies of 3.5 × 10Ten viruses that may cause potential cross-reactions in qPCR assay for MiCV were used to evaluate the specificity in this study. The MiCV HEB15 strain was used as the positive control and sterile water was used as the negative control.8–101 copies/μL) were used as templates. The conventional PCR reaction was performed using the protocol described above with the primer pair McapF and McapR.To compare the sensitivity of qPCR with conventional PCR, 10-serial pMDT-Cap plasmid dilutions  was determined following the formula: CV =  × 100.The repeatability was determined using four different DNA concentrations of viral agent tested. Concentrations of the DNA standard 10A total of 401 clinical samples including 52 serum sample, 319 different tissues of minks and 30 healthy mink fecal samples were collected from 5 provinces, including Heilongjiang, Jilin, Shandong, Hebei, and Liaoning in China in 2014–2017. These samples were examined in terms of presence of MiCV infection in qPCR and were assayed in duplicate. Furthermore, these samples were also detected using conventional PCR simultaneously.Ct-value determined from the standard curve. All the samples were tested in triplicates.There are 8 artificial infection minks, 22 naturally infected minks and the 3 healthy control minks were collected to characterize the tissue distribution of MiCV in minks. The tissues, including liver, lung, spleen, heart, duodenum, jejunum, ileum, colon, brain, kidney, skeletal muscle, marrow, and mesenteric lymph nodes, were detected by qPCR to evaluate viral load. The viral load was quantified by qPCR using 2 μL of DNA per reaction. The DNA copy number of each sample was converted to copy number per gram by using the calculated 8 copies/μL to 3.5 × 101 copies/μL. Ct-values were plotted against the known copy numbers of the standard controls. Thus, a correlation coefficient (R2) of 0.974, a slope of −3.155, an efficiency of 107.447% and the Y intercept 39.202 were obtained in this study and the following formula: Y = −3.155X+39.202  was achieved.As shown in Figure 1 copies, while the conventional PCR, 1 × 106 copies  within runs (intra-assay variability) and between runs (inter-assay variability) that ranged from 0.73 to 1.69% and 0.97 to 2.18%, respectively , followed by skeletal muscle (4.75 × 108 copies/g), brain (1.36 × 108 copies/g), duodenum (1.36 × 108 copies/g), kidney (6.16 × 107 copies/g), jejunum (2.44 × 107 copies/g), lung (1.82 × 107 copies/g), mesenteric lymph nodes (1.54 × 107 copies/g), colon (1.32 × 107 copies/g), spleen (1.28 × 107 copies/g), liver (1.24 × 107 copies/g), and lastly, marrow (9.27 × 106 copies/g), and ileum (8.80 × 106 copies/g).To investigate MiCV distribution in infected minks, 8 artificially infected minks, 22 naturally infected minks, and 3 control minks were used and determined by the qPCR. All of the 8 artificially infected minks were MiCV positive, and MiCV can be detected in all these 13 tissues. The viral loads of 8 artificially infected minks are shown in Table 9–4.95 × 106 copies/g), skeletal muscle (4.89 × 109–9.49 × 105 copies/g), brain (1.75 × 109–3.50 × 106 copies/g), kidney (2.11 × 109–1.53 × 106 copies/g), duodenum (8.23 × 108–2.33 × 106 copies/g), jejunum (3.75 × 108–7.23 × 105 copies/g), lung (1.23 × 108–5.16 × 106 copies/g), liver (1.64 × 108–5.80 × 105 copies/g), colon (7.89 × 107–2.38 × 105 copies/g), mesenteric lymph nodes (6.64 × 107–3.05 × 105 copies/g), spleen (3.16 × 107–3.48 × 105 copies/g), marrow (2.03 × 107–2.09 × 104 copies/g), and ileum (3.18 × 107–2.51 × 104 copies/g).Moreover, the number of MiCV DNA copies in different tissues of 22 naturally infected minks was shown below: heart (3.35 × 108 copies/g), heart (5.02 × 108 copies/g), kidney (2.94 × 108 copies/g), brain (2.45 × 108 copies/g), and duodenum (1.31 × 108 copies/g) samples contained the highest viral loads, whereas jejunum (5.11 × 107 copies/g), lung (4.68 × 107 copies/g), colon (2.68 × 107 copies/g), mesenteric lymph nodes (2.59 × 107 copies/g), liver (1.88 × 107 copies/g), marrow (1.44 × 107 copies/g), spleen (1.04 × 107 copies/g), and ileum (9.58 × 106 copies/g) had the lowest viral loads. Meanwhile, no MiCV was detected in the 3 healthy control minks. In general, the viral load between the naturally and experimentally infected minks in the different tissues were similar.In a descending order, the mean viral loads per tissue were as follows: skeletal muscle , in heart 28/30 (93.33%), in duodenum 26/30 (86.67%), in jejunum 24/30 (80.00%), in skeletal muscle 24/30 (80.00%), in colon 22/30 (73.33%), in kidney 22/30 (73.33%), in ileum 18/30 (60.00%), in marrow 16/30 (53.33%), and in mesenteric lymph nodes 16/30 (53.33%). Furthermore, all the infected minks had detectable virus in the liver, spleen, and brain.MiCV is a newly novel pathogen in diarrheal minks in China , in which it indicated that this method is highly efficient, and may prove a suitable approach for MiCV diagnosis.According to previous study, when 5-fold higher sensitivity than that of conventional PCR , skeletal muscle (4.75 × 108 copies/g), brain (1.36 × 108 copies/g), then duodenum (1.36 × 108 copies/g). However, the viral DNA loads in lymphoid tissues are not very high. The heart has a higher mean viral loads in both naturally infected minks and experimentally infected minks. We also found that the heart can be tested positive for MiCV easily, due to its relatively high testing rate in infected minks. However, other tissues can't satisfy these two requirements simultaneous. Based on these reasons, the heart can be considered the first choice of tissue for detecting MiCV in infected minks and might be a key index for MiCV detection.The qPCR method was able to quantify the level of viral DNA in different tissues of the MiCV infected minks. In the present study, viral DNA loads of MiCV infected minks were found to be widely distributed in various tissues of artificial infection and naturally infection samples. The naturally infected minks mean viral loads of skeletal muscle, heart, kidney and brain was present at higher levels, 5.31 × 10Furthermore, the pathogenic role of MiCV is still unclear, though the virus distribution in tissues may be related with the pathogenicity. Hence, the result of the viral loads in different tissue samples of MiCV-infected minks may be conducive to MiCV pathogenicity-related research in the future.The qPCR assays for PCV2 and DogCV detection have been previously reported . Samples were collected only from animals for laboratory analyses, avoiding unnecessary pain and suffering of the animals. The owners gave their written consent for sample collection, and the locations where we sampled are not privately owned or protected in any way. The studies did not involve endangered or protected species.JG and HC: Conceived and designed the experiments; XC, JG, YS, SW, and LZ: Performed the experiments; JG, XC, and LZ: Analyzed the data; LZ, SG, CW, and YY: Contributed reagents, materials, and analysis tools; JG and XC: Wrote the paper. All authors read and approved the final manuscript.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.


This study evaluated the treatment results of lymph node (LN) oligo-recurrence in esophageal cancer patients treated with salvage radiotherapy (RT) in a multi-institutional retrospective study.Eligibility criteria for this retrospective analysis were: the primary lesion of esophageal cancer was controlled; from one to five LN recurrences; total RT dose ≥45 Gy to exclude palliative RT; without recurrence other than LN; and salvage RT for LN recurrence was given between January 2000 and April 2015. The median follow-up time for the 93 living patients was 29.6 months.p = 0.000055), disease-free interval (DFI) ≥12 months (p = 0.0013), LN max diameter ≤22 mm (p = 0.0052), and Karnofsky performance status ≥80% (p = 0.030) were associated with a significantly better prognosis. On multivariate analysis, significant differences were seen for combined chemotherapy (p = 0.000018), DFI (p = 0.0027), and LN max diameter (p = 0.018).Two hundred thirty-seven patients were matched in five hospitals. The 3-year overall survival (OS) was 37%, local control was 45%, progression-free survival was 24%, and esophageal cancer-specific survival was 42%. On univariate analysis for OS, combined chemotherapy  and small size (≤22 mm). Lymph node (LN) recurrence from esophageal cancer after surgery is one of the main types of treatment failure –3. Accoret al. in 2006 [On the other hand, radiotherapy (RT), chemoradiation therapy (CRT), or lymphadenectomy have been used in treating LR. These treatments have had a beneficial symptomatic effect for a significant proportion of these patients, and it is possible to obtain long-term survival in some patients –12. Canc in 2006 –15. LocaFor LN oligo-recurrence after primary definitive CRT, salvage surgery is first considered, and, when surgery is not indicated, salvage CRT or stereotactic radiotherapy is performed. Some researchers , 17 haveThe purpose of this retrospective study was to assess the efficacy of salvage RT or CRT for inoperable LN recurrence after primary curative therapy for esophageal cancer.The eligibility criteria for this retrospective analysis were as follows: a) the primary lesion of esophageal cancer was controlled; b) from 1–5 LN recurrences; c) total RT dose of ≥45 Gy in order to exclude palliative RT; d) without recurrence other than LN; and e) salvage RT or CRT for LN recurrence was given between January 2000 and April 2015. The eligibility criteria did not include with or without combined CTx, the kind of initial curative therapy, the disease-free interval (DFI), recurrent LN location, LN max diameter, age, Karnofsky performance status (KPS), and the histopathological type of primary tumor.The DFI was defined as the interval between initial therapy for the primary lesion and the date of identification of LN recurrence. This was the endoscopic submucosal dissection date, the operation date independent of perioperative CTx, or the starting date of initial CRT.The reason why the 11 patients with stage IV disease received curative treatment (surgery in eight patients and CRT in three) was that these patients had only supraclavicular, para-aortic, or hilar LN metastasis, and cure was considered possible. These stages were classified based on the UICC/AJCC TNM system version 7.p value on univariate analysis for overall survival (OS) was calculated by the log-rank test. The 95% confidence interval (CI) was calculated using Greenwood’s formula. The significance level was set at 5%. The events were defined as any death for OS, local recurrence within the radiation field for local control, any death and any relapse for relapse-free survival, and death from esophageal cancer for esophageal cancer-specific survival. Multivariate analysis for OS was performed with a Cox proportional hazards model, and the variables were selected by the stepwise method using the Bayesian Information Criterion (BIC). The Bonferroni correction was used for multiple comparisons; in other words, the significance level was set as 5% divided by the number of variables in the multivariate analysis.Survival curves were prepared using the Kaplan-Meier method, and the A total of 237 patients who matched the study definition of oligo-recurrence were treated by CRT or RT alone in five Japanese hospitals. The median age was 66 years . The male-to-female ratio was 207:30. The ratio of KPS ≥90% to <90% was 154:83. Primary histopathology was squamous cell carcinoma (SCC) in 231 patients, adenocarcinoma or adeno-squamous cell carcinoma in three patients, and others in three patients. Clinical stages I, II, III, and IV at the initial curative therapy were seen in 34, 89, 103, and 11 patients, respectively. The primary tumor location was cervical in 10 patients (whose primary therapy was surgery in 9 patients and CRT in one patient), upper thoracic in 22 patients, middle thoracic in 140 patients, and lower thoracic plus gastric-esophageal junction in 65 patients. The primary therapy was endoscopic submucosal dissection in five patients, radical surgery in 219 patients , and definitive CRT in 13 patients. The median DFI was 11.9 months . The number of LN recurrences was one in 161 patients (68%), two in 31 patients, three in 29 patients, four in seven patients, and five in five patients. The median maximum LN diameter (MLD), which was defined as the greatest transverse diameter in the axial plane, was 22 mm . The location of recurrent LN metastasis was loco-regional only in 144 patients (60.8%) and distant regions only, such as neck, supraclavicular, abdominal para-aortic, or hilar LNs, in 66 patients (27.8%); 27 patients (11.4%) had both loco-regional and distant region involvement. The median total radiation dose was 60 Gy . Median overall treatment time was 43 days . Radiation therapy was involved-field in 170 patients, extended-field including prophylactic irradiation in 62 patients, and stereotactic body radiotherapy of 50 Gy in 10 fractions in five patients. Systemic CTx was administered concurrently to 199 patients (84%), but two patients received it sequentially following RT. The regimen included an FP regimen (5-fluorouracil and cisplatin) in 65 patients, nedaplatin plus 5-fluorouracil in 102 patients, S1 alone in 17 patients, docetaxel alone in nine patients, a DCF regimen  in two patients, and others in four patients.The first failure site after this definitive salvage RT was lung in 38 patients, liver in 19 patients, bone in 10 patients, other distant organs in 11 patients, distant LN in 47 patients, and locoregional LN in 61 patients (31 patients within field and 30 patients outside field). When taken together, in-field recurrence of salvage RT occurred in 31 patients (16.7%), and out-of-field recurrence was seen in 155 patients (83.3%).The median follow-up time for the 93 living patients was 29.6 months . The overall follow-up was 15.0 months , and 12 patients were lost to follow-up. The 3-year OS was 36.7% (95% CI: 29.8–43.6%), local control was 45.1% (95% CI: 37.3–52.6%), relapse-free survival was 24.1% (95% CI: 18.6–30.1%), and esophageal cancer-specific survival was 41.5% (95% CI: 34.1–48.7%). The MST was 21.6 months (95% CI: 18.0–28.5 months).p = 0.000055, log-rank test)   . Age >66 vs. ≥66 years (p = 0.76), KPS >90% vs. ≤90% (p = 0.11), number of LN recurrences mono vs. multiple (p = 0.44), LN location locoregional vs. distant (p = 0.41), serum SCC antibody value >2 mg/mL vs. ≤2 mg/mL (p = 0.42), and histopathological type SCC vs. others (p = 0.55) were not significant prognostic factors.The 3-year OS was 39.7% 95% CI: 32.1–47.3%) with CRT and 20.8% (95% CI: 8.3–37.0%) with RT alone (.1–47.3% p = 0.000018), DFI (HR = 0.60 and p = 0.0027), and LN max diameter (HR = 0.65 and p = 0.012) were significant.On multivariate analysis by the BIC stepwise method Table , multiplThe number of patients with all of (a) treated with CRT, (b) DFI of not less than 12 months, and (c) LN max diameter of not more than 22 mm was 56 (24%). The 2-y OS and 3-y OS of these patients were 64.9% (95% CI: 50.1–76.3%) and 55.2% (95% CI: 40.0–68.0%), respectively, and the MST was 43.4 months (95% CI: 26.5-NA months).Eleven patients (4.6%) experienced acute or late non-hematologic adverse events of grade 3 or greater according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.0, including: grade 4 cardiac tamponade at 4.6 months after the completion of RT; grade 5 drug-induced interstitial pneumonia at 0.3 months; grade 4 hyperglycemia during treatment; grade 5 pleural effusion at 13.5 months; grade 3 anastomotic stenosis at 1.4 months; grade 5 mediastinal-bronchial fistula at 2.6 months; grade 5 esophageal bleeding at 3.3 months; grade 4 esophagobronchial fistula during treatment; grade 5 gastric ulcer bleeding at 0.2 months; grade 4 fistula of a gastric tube at 15.4 months; and grade 4 gastric tube to bronchial fistula at 1.0 months.2/day) and 5-fluorouracil . With a median observation period of 72 months, the 3-y OS was 38.4%, with an MST of 21.0 months. Three-year relapse-free survival was 29.3%, and the 3-y irradiated-field control rate was 71.5%. Although the present results should be interpreted with caution because of the short observation period, the MST of 21.6 months and the 3-y OS of 36.7% can be looked upon as favorable and encouraging.According to previous studies of RT or CRT for patients with postoperative LR of esophageal cancer, the median MST of 13 studies was 13.6 months , and the median 2-y OS was 25%  , 20–25. Several prognostic factors have been reported. For example, high RT dose, younger age, non-anastomotic recurrence, good performance status, single LN recurrence, and single recurrent region predicted better outcomes , 24–26. et al. [In the study of Jingu et al. , 24, patThe present study has several limitations associated with its retrospective design. This study could not demonstrate a survival benefit of salvage RT or CRT compared to other treatment modalities such as CTx-alone. According to the previous reports of life-prolonging chemotherapy alone for esophageal M1 patients, the median survival time was 10.0–15.5 months , 27, 28.Depypere et al.  and NakaIn the present study, 60 Gy was given to 157 patients (78.9%) and 50/50.4 Gy was given to 21 patients (10.6%) of 199 patients treated with CRT. In the primary treatment, 50–50.4 Gy of RT is standard for patients treated with definitive CRT , althougAccording to the above-cited study by Nakamura et al. , the 3-yOligo-recurrence involving LNs following treatment for esophageal cancer does not appear to be a terminal-stage event, unlike other distant metastases. Moreover, cure may be possible by chemoradiation therapy with a long DFI (≥12 months) and a small size (≤22 mm).


Saccharomyces cerevisiae is a host for the industrial production of S-adenosyl-l-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, 13C-metabolic flux analysis (13C-MFA) was conducted to investigate the metabolic regulation responsible for the high SAM production from ethanol.13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose.The comparison between the metabolic flux distributions of central carbon metabolism showed that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that of glucose. Estimates of the ATP balance from the These results suggest that the enhanced ATP regeneration due to ethanol assimilation was critical for the high SAM accumulation.The online version of this article (10.1186/s12934-018-0935-6) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae has been used in several industrial processes such as for the production of S-adenosyl-l-methionine (SAM)  ethanol was the best carbon source for the precise measurements of metabolic fluxes. Using the optimized experimental design, the intracellular metabolic flux distribution of central carbon metabolism was successfully determined for the high SAM-producing S. cerevisiae strain (Kyokai no. 6) in the ethanol limited-chemostat culture. The results showed that the metabolic flux levels through the glyoxylate shunt and the later TCA cycle were upregulated during growth on ethanol, and the resultant activation of the oxidative phosphorylation should contribute the high SAM accumulation in the ethanol-assimilating conditions.In the present study, S. cerevisiae strain used for Japanese sake brewing, Kyokai no. 6 (NBRC2346), was purchased from the National Biological Resource Center . This S. cerevisiae strain was cultivated in an aerobic carbon-limited chemostat culture with a working volume of 100 mL in a 250-mL fermentor . The pH was maintained at 5.5 by the automatic addition of 1.0 N NaOH. The temperature was maintained at 30 °C. The stirring speed was 1000 rpm. The aeration rate was 200 mL/min. The dilution rate was 0.06 h−1. The synthetic medium used for cultivation contained 3.8 g/L ethanol, 5.0 g/L (NH4)2SO4, 0.50 g/L K2HPO4, and 3.4 g/L of yeast nitrogen base without amino acids and ammonium sulfate . Vitamins and minerals were added to the medium, with the final composition as follows: 2.0 g/L KH2PO4, 1.5 g/L MgSO4, 2.5 mg/L ZnSO4, 2.4 mg/L MnSO4, 0.27 mg/L CuSO4, 0.20 g/L CaCl2, 4.0 mg/L FeCl3, 0.40 mg/L NaMoO4∙2H2O, 1.0 mg/L H3BO3, 0.20 mg/L KI, 0.20 g/L NaCl, 34 µg/L biotin, 1.6 mg/L Ca-pantothenate, 13 mg/L inositol, 7.8 mg/L thiamine-HCl, 2.3 mg/L pyridoxine-HCl, 0.40 mg/L para-aminobenzoic acid, 0.40 mg/L riboflavin, 0.80 mg/L niacin, and 4.0 µg/L folic acid. For 13C-MFA, the carbon source in the medium was replaced with [2-13C]ethanol. Samples taken from the reactor were centrifuged at 18,800×g for 5 min at 4 °C. The supernatant was used for analysis of extracellular metabolites. The cell pellet was used for 13C-labeling analysis by gas chromatography-mass spectrometry (GC–MS).The 4 extraction. CDW was estimated using OD600 = 1 corresponding to 0.21 gCDW/L. Glucose and ethanol concentrations were measured enzymatically by using an analyzer . An enzymatic kit was used for the quantification of glycerol  in the supernatant. The concentrations of acetate, pyruvate, succinate, fumarate, malate, citrate, and lactate in the supernatant were measured by HPLC .The analysis of SAM, cell dry weight (CDW), glucose, ethanol, glycerol and organic acids such as acetate were performed as described by Hayakawa et al. . SAM conl-methionine, and SAM concentrations were measured using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) according to previously described methods ethanol was predetermined. The simulated 13C-enrichments were calculated for the 21 fragments of the following amino acids: (M−57)+ fragments for Ala, Asp, Glu, Gly, Phe, and Thr; (M−85)+ fragments for Ala, Asp, Glu, Gly, Ile, Leu, Pro, Thr, and Val; (M−159)+ fragments for Glu, Ile, Leu, Pro, and Val; and (M302)+ fragment for Asp; and then the Gaussian noise at 1% levels was added to produce hypothetical measurements for the 13C-enrichment data. A metabolic flux distribution was estimated by minimizing the residual between the hypothetical measurement and simulated 13C-enrichments of amino acid fragments. The 95% confidence intervals were determined using the grid search method , , and Si are the range of the 95% confidence intervals, the range of 95% confidence intervals in 13C-labeled ethanol composition of 10:0:0:0 (100% non-labeled ethanol), and precision score for the ith flux. The maximum net flux was 500, which was normalized to an ethanol consumption rate of 100. The metabolic network shown in the following was used.A computer simulation was conducted for the design of the escribed . The intescribed . Data foh method , 11. PreS. cerevisiae for the 13C-MFA was based on previously published models, including glycolysis, the pentose phosphate (PP) pathway, the anaplerotic pathways, the TCA cycle, gluconeogenesis, and the transport reactions between the cytosol and mitochondria -N-methyl-trifluoroacetamide (MTBSTFA), and the mixture was incubated at 95 °C for 1 h. Next, 1 μL of the sample was injected into the GC–MS system , according to a previously described method [13C-MFA [13C-enrichments of the amino acid fragments at an isotopic steady state [χ(∞)] can be calculated from the following equation:Cell pellets were hydrolyzed in 6 N HCl at 105 °C for 18 h. After removal of the debris by filtration, the hydrolysate was dried and dissolved in acetonitrile. For GC–MS, the hydrolysate was mixed with an equal volume of d method , 18. The, [2-13C], and [U-13C]ethanol, and 95% confidence intervals of the estimated flux levels were investigated by a computer simulation of 13C-MFA experiment.As mentioned in the background, S. cerevisiae describing the stoichiometry equations and carbon atom transitions of each metabolic reaction was constructed from previously published models with modifications  by adding Gaussian noise at 1% levels. The metabolic flux distribution and 95% confidence intervals were determined using the artificial mass spectra datasets by the 13C-MFA procedure  with mixtures of non-labeled, [1-13C], [2-13C], [U-13C]ethanol at 10:0:0:0, 5:5:0:0, and 0:0:10:0 were estimated to be 2.5–45.1, 19.0–42.3, and 27.4–41.6, from which SIDH were estimated to be 1.0, 0.55, and 0.33, respectively  and wider (poorer precision) 95% confidence intervals, respectively. This result indicates that the range of the 95% confidence interval depended greatly on the composition of 13C-labeled ethanol. The mixture ratio at 0:0:10:0 (100% [2-13C]ethanol) showed the smallest sum of the precision score Si in the TCA cycle and PP pathway among all 13C-labeling conditions  flux  between experimental and simulated 13C-enrichment data for the best-fitted metabolic flux distributions that passed the χ2-test (α = 0.05) were small (69.5) ethanol was the best carbon source for the precise estimation of metabolic fluxes through the TCA cycle and PP pathway  at the entry point of the TCA cycle disagreed with a previous transcriptome analysis because the expression levels of PDA1, PDB1, PDX1, and LPD1 genes in the ethanol-assimilating conditions increased from that of glucose  was clearly greater than the ATP consumption flux for SAM production (3.5 µmol/gCDW/h)  compared to glucose (0.13–0.43 mmol/gCDW/h)  Fig. . These rS. cerevisiae, cytosolic acetyl-CoA was synthesized from acetate and CoA by acetyl-CoA synthase with consuming ATP ethanol was the best carbon source for the precise estimation of metabolic fluxes through the TCA cycle and PP pathway. The 13C-MFA result revealed that the metabolic flux distribution was totally rewired in the ethanol culture from that of glucose. Estimates of the ATP balance from the 13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose. These results suggest that the excess ATP regeneration via the activation of oxidative phosphorylation was a mechanism responsible for the SAM overproduction under the ethanol-assimilating conditions, which also provided general knowledge for the development of S. cerevisiae cell factories for the cytosolic acetyl-CoA-derived products.In this study, the design of the Additional file 1. Additional tables and figures.


Fusarium oxysporum f. sp. melongenae, an eggplant pathogen. The final assembly consists of 1,631 scaffolds with 53,986,354 bp  and 16,485 predicted genes.Here, we present the genome sequence of an isolate (14004) of  Fusarium species are some of the most important phytopathogenic and toxigenic fungi, causing various diseases on nearly every economically important plant species. Aside from their economic importance, species of Fusarium also serve as key model organisms for biological research . A total of 16,485 protein-coding genes were predicted from the assembly, with the highest N50  using AUGUSTUS version 3.2.2 , and 500 mg of mycelium was used for DNA extraction with the Qiagen DNeasy plant minikit ; then, 1 μg of genomic DNA was sent for sequencing at Génome Québec , specifying 100-bp paired-end reads with a 300-bp insert. Over 43 million paired-end reads totaling 10.7 Gb were received. The genome was assembled using the programs Velvet version 1.2.10 (ion 2.04 , with odMPIL00000000. The version described in this paper is version MPIL01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no.


Inhibition of return (IOR) is considered as a “blindness mechanism” that emotional stimuli have no impact on it. Most previous studies suggested that IOR was not modulated by emotional cues. However, one key question they ignored was that only supraliminal presentation of emotional stimuli was used in their experiments. The present experiment is aimed at exploring the possible interaction between the IOR effect and subliminal emotional process. We manipulated three different kinds of valence strength of negative stimuli  which were presented under the subliminal perception level and an event-related potentials (ERPs) recording was adopted. The results showed that, compared to MN and HN, the IOR effect triggered by peripheral cues was more significant for LN with aspects of behavioral and electrophysiological data . This indicated that IOR can be modulated by emotionally relevant stimuli. The automatic processing that was triggered by subliminally negative stimuli of peripheral cues had an influence on the shifting of spatial attention that was triggered by IOR. These two mechanisms may occur in the perceptual stage simultaneously. The limited processing capacity of the visual system makes attention select the most valuable information when our visual environment is rich. Moreover, the rapid and efficient attention to stimuli relating to the development or survival of individuals may have a crucial influence on flexible and adaptive behavior, especially for the threatening information. Inhibition of return (IOR) is widely considered as a “foraging facilitation” which contributes to adaptive behavior . Posner As compared to neutral stimuli, biologically relevant stimuli like threatening events are more sensitive to human behavior . If negaFirst, different groups of individuals are an important factor. Second, will emotional process and IOR occur in the same stage ? This question may be ignored in most previous literature with non-clinical participants. In a typical cue-target paradigm, IOR is tested during a spatially visual space and it can affect the early visual process of sensory perception . AlthougIn summary, one definite conclusion can be drawn on that individual differences  can modulate the IOR effect size. The growth of evidence suggests that a reduced IOR effect is always observed for anxious, schizophrenic, or depressive individuals for their over-attention of negative stimuli , 2014. A“This study was approved by the Human Ethics Committee of Nantong University. A written informed consent was obtained from all the participants before their participation”.According to participants’ scores in Hamilton Anxiety Scale , 22 participants  were selected from 35 volunteers to take part in this experiment and gave a reward after participation. All participants had a normal or corrected-to-normal vision, right-handed, without mental illness, color blindness, and hadn’t participated in a similar experiment before.1 for formal experiment and 6 photographs for a practice block. To ensure the validity of these three kinds of negative pictures, we tested their emotion valence and arousal. We found a significant main effect of emotion valence , pairwise comparisons found a significant difference between HN and MN, HN and LN, MN and LN, p < 0.001. There was no significant main effect of emotion arousal , pairwise comparisons found no significant difference between HN and MN, HN and LN, MN and LN, p > 0.05. Pictures converted to grayscale and reduced their visibility down to 10% compared with 100% luminance of original pictures  that, when present time of cue was 15 ms, there was no significant difference between accuracy and probability, p > 0.1, indicating up to subliminal perception.Thirty-six photographs were selected from International Affective Picture System (IAPS) . They wepictures . We condE-prime software was used for the presentation of stimuli, data collection on a Dell PC. Each trial started with two rectangle boxes and a fixation. Each box was 2.34°  × 3.36°  and fixation was 0.5°  × 0.5° . Negative stimuli were presented at cue location and a hollow circle was used as the target. Participants sat in a dimly lit and electromagnetically shielded room, with their heads located 70 cm away from the computer screen.The present experiment consisted of a 2 (cueing: cued vs. uncued) × 3  within-subject design. When cue and target appeared at the same location named cued trials, cue and target appeared at different locations named uncued trials. In the course of the experiment, participants performed a localization task as accurately and quickly as possible. Reaction times, accuracy, and EEG data were recorded simultaneously.Figure 1. Central fixation and two rectangle boxes with a variable duration between 750 and 850 ms were presented at the beginning of each trial. Emotional cues flickered in one of two potential boxes randomly with 50% probability for 15 ms. After 300 ms delay, the fixation cue for 200 ms was presented in the middle box  and 60 trials with uncued condition . There was the equal probability  for cue and target appeared at left or right, for each one of 30 pictures . The operation of the practice and experimental block was identical. Before the experiment, participants were told to try their best to reduce their actions of the swallow, frown, and blink, to keep their attention on the fixation cross, to keep the body from moving and in the blink of a black blank. Three minutes were provided after every block for a rest.We interviewed participants whether they could see the pictures after their participation. All participants answered “No,” indicating that they couldn’t be aware of these pictures explicitly.Continuous electroencephalogram (EEG) was recorded by a 64 Ag-AgCl electrode elastic cap placed according to the international 10-20 system and the Neuroscan ERP workstation (scan4.5). M1 and GND served as the reference and ground electrode on the left mastoid and medial frontal aspect separately. Vertical electrooculogram (EOG) and Horizontal EOG placed above and below the participant’s left eye and outer canthi of both eyes. The sampling rate and band pass were 1000 Hz/channel and 0.05∼100 Hz. EEG data and behavioral data were recorded simultaneously. Every participant washed their hair in the laboratory first and we started to record until the impedances were stable below 5 KΩ with the conductive paste on the scalp.After recording, scan software was used for off-line analysis. We merged behavioral data into EEG data and translated the average of M1 and M2 into a new reference. Ocular artifacts like blinks and vertical eye movements were rejected and not included in the construction of ERPs. EEG epoch was -100 ms prior to target onset and 500 ms posterior to target onset containing the rejection of incorrect or no responses trials. After baseline correction, trials with excessive artifacts (±80 μV standard) were rejected for further analysis . Finally, mean waveforms under all conditions performed a band-pass filter containing a high-pass filter of 0.1 Hz and a low-pass filter of 30 Hz (24 dB/octave) . Data ofp of all main effects and interactions were corrected by Greenhouse–Geisser.According to the overall average map and research literature, the parieto-occipital P1, N1, early posterior Nd  and Nd ERP components were analyzed. These ERP components were divided by the time windows in which they occurred: P1, 100∼150 ms; N1, 150∼200 ms; early posterior Nd, 100–200 ms; Nd, 200–300 ms. Based on the previous literature , Po7/Po8The analysis of RTs was based on the trials with the correct response and was executed within 200–1000 ms first. Then, trials with RTs faster or slower than three standard deviations below or above participants’ mean RTs were excluded. Totally, 4.15% of trials were excluded from statistic analyses of mean RTs and correct rate for each condition.On correct rate, main effect and interaction were not significant.Figure 2), 2  × 2  repeated measures analysis of variance (ANOVA) revealed a significant main effect of cueing, F = 33.03, p < 0.001, η2 = 0.61, indicating faster RTs on uncued trials (334 ± 11 ms) than cued trials (351 ± 12 ms). The main effect of cue type was not significant, F = 0.62, p > 0.05. However, there was a significant interaction between cueing and cue type, F = 5.94, p < 0.01, η2 = 0.22. Planned contrast revealed a significant main effect of cue type at uncued location, F = 3.76, p < 0.05, η2 = 0.27. LSD post hoc test showed that LN (330 ± 11 ms) was faster than MN (336 ± 11 ms) and HN (336 ± 11 ms), p = 0.048 and p = 0.023 separately. Meanwhile, we compared the IOR effect size (cued-uncued) of HN, MN, and LN. We found the main effect of cue type, F = 5.94, p < 0.01, η2 = 0.22, indicating significantly larger magnitudes of IOR for LN (22 ms) than MN (15 ms) and HN (14 ms).On mean RTs : for contralateral electrodes, there was a significant marginally interaction of cueing × cue type, F = 2.98, p = 0.064, η2 = 0.14. Planned contrast revealed a significant main effect of cueing at LN condition, F = 5.99, p < 0.05, η2 = 0.25, indicating larger amplitudes of uncued trials (0.54 ± 0.29 μV) than cued trials (0.05 ± 0.3 μV); there were no main effects of cueing at MN sand HN. For ipsilateral electrodes, main effects and interactions were non-significant .An ANOVA analysis  at the time window 0–100 ms showed that , cue type , and laterality .F = 5.19, p < 0.05, η2 = 0.22. There was a significant interaction of cueing × cue type, F = 4.16, p < 0.05, η2 = 0.19. Planned contrast revealed a significant main effect of cueing at LN condition, F = 10.62, p < 0.01, η2 = 0.37, indicating larger amplitudes of uncued trials (2.09 ± 0.51 μV) than cued trials (1.41 ± 0.44 μV); there were no main effects of cueing at MN and HN. Other main effect and interactions were non-significant .P1: The main effect of cue type was laterality, F = 8.58, p < 0.01, η2 = 0.32. Planned contrast revealed a significant main effect of cueing at LN condition, F = 14.23, p < 0.01, η2 = 0.44, indicating smaller amplitudes of uncued trials (0.22 ± 0.37 μV) than cued trials (-0.72 ± 0.39 μV); there were no main effects of cueing at MN and HN. Other main effect and interactions were non-significant .N1: There was a significant interaction of cueing × cue type, F = 7.56, p < 0.01, η2 = 0.3. Planned contrast revealed a significant main effect of cueing at LN condition, F = 17.58, p = 0.001, η2 = 0.49, indicating larger amplitudes of uncued trials (1.15 ± 0.4 μV) than cued trials (0.4 ± 0.36 μV); there were no main effects of cueing at MN and HN. There was a significant interaction of cueing × laterality, F = 6.95, p < 0.05, η2 = 0.28. Other main effect and interactions were non-significant .Early posterior Nd: There was a significant interaction of cueing × cue type, F = 12.06, p < 0.01, η2 = 0.4. There was a significant interaction of cueing × cue type, F = 4.87, p < 0.05, η2 = 0.21. Planned contrast revealed a significant main effect of cueing at LN condition, F = 11.34, p < 0.01, η2 = 0.39, indicating larger amplitudes of uncued trials (4 ± 0.59 μV) than cued trials (3.16 ± 0.62 μV); there were no main effects of cueing at MN and HN. Other main effect and interactions were non-significant .Mean amplitudes of the Nd components was assessed in a three-way, repeated-measures ANOVA with factors for peripheral cueing , central cueing , and electrodes . The main effect of electrode site was significant, F = 8.04, p < 0.01, η2 = 0.31, the amplitudes of LN (1.93 ± 0.47 μV) were more positive than MN (1.22 ± 0.45 μV) (p = 0.001) and HN (1.53 ± 0.45 μV) (p = 0.038) separately; N1: F = 7.51, p < 0.01, η2 = 0.31, the amplitudes of MN (-0.5 ± 0.36 μV) (p = 0.001) and HN (-0.22 ± 0.34 μV) (p = 0.052) were more negative than LN (0.22 ± 0.35 μV) separately; Nd: F = 4.87, p < 0.05, η2 = 0.21, the amplitudes of LN (4 ± 0.59 μV) were more positive than MN (3.35 ± 0.6 μV) (p = 0.009) and HN (3.48 ± 0.59 μV) (p = 0.017) separately].Another statistics of planned contrast of EEG data showed that: For P1, N1, Nd, there were no main effects of cue type on cued location but significant main effects of cue type on uncued locations [P1: In order to further explore the relation between subliminal emotional process and the IOR effect, we used a location task to examine these two mechanisms from behavioral and electrophysiological aspects. Consistent with our hypothesis, we found that IOR was modulated by three different kinds of valence strength of negative stimuli. There were two main results: First, the IOR effect was modulated by the subliminally emotional process. Second, this modulation occurred in the P1, early posterior Nd and Nd components. This indicates that IOR and subliminally emotional process occurred in perceptual stage simultaneously.Previous studies used biologically emotional cues usually found no conspicuous difference of the IOR effect . One keyAn analysis was operated to clarify potential difference at the time window 0–100 ms. The result showed the difference between cue type and cueing effect only at the contralateral electrodes. Especially, we found a reduction 0–100 ms component at the contralateral electrodes. This might indicate the early perceptual inhibition. The current experiment focused on ERP components of perceptual processing (P1 and N1), early posterior Nd and post-perceptual processing (Nd), wherein the oculomotor was suppressed. We all found some difference in these components. The P1 reduced amplitude of cued location than uncued location was usually associated with the IOR effect . For N1,In summary, the current experiment found a significant interaction between IOR and subliminal emotional processing, suggesting that those two mechanisms may occur in the same stages simultaneously. As compared to MN and HN, IOR was more significant for LN in behavioral and electrophysiological aspects.FP and XW designed and coordinated the study; XW, LZ, and YO carried out experiments and data process; XW drafted the manuscript; FP reviewed the manuscript. All authors gave the final approval for publication.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.