Papiliotrema laurentii ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specific activity of α-L-rhamnosidase was 29.89 U·mg−1 through purification, and the molecular mass of the enzyme was 100 kDa, as assayed by SDS-PAGE. The characterization of the purified enzyme was determined. The optimal temperature and pH were 55 °C and 7.0, respectively. The enzyme was stable in the pH range 5.5–9.0 for 2 h over 80% and the temperature range 30–40 °C for 2 h more than 70%. The enzyme activity was inhibited by Ca2+, Fe2+, Cu2+, and Mg2+, especially Fe2+. The kinetic parameters of mK and maxV were 1.38 mM and 24.64 μmol·mg−1·min−1 using pNPR as the substrate, respectively. When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of mK and maxV were 3.28 mM and 0.01 μmol·mg−1·min−1, respectively. The conditions of enzymatic hydrolysis were optimized through single factor experiments and response surface methodology. The icariin yield increased from 61% to over 83% after optimization. The enzymatic hydrolysis method could be used for the industrialized production of icariin. At the same time, this enzyme could also cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside in naringin and neohesperidin, which could be applicable in other biotechnological processes.Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain,  As a kind of famous traditional Chinese herbal medicine, Herba Epimedii has been used for a long time in China because it could treat impotence and infertility, strengthen bone, and dispel rheumatism. The major active components of epimedium are prenyl-flavonoids including icariin, epimedin A, epimedin B, epimedin C, 2″-O-rhamnosyl icariside II, and baohuoside I . The phaE. brevicornu, and its content (15.96 mg·g−1) could even be 3–5 times that of icariin (5.36 mg·g−1) in the root of E. brevicornu [Bacteroides thetaiotaomicron has the ability to transform epimedin C into icariin, and the bioconversion of epimedin C into icariin could reach 90.5% under the optimum conditions [Escherichia coli cells expressing α-L-rhamnosidase from Aspergillus nidulans could hydrolyze epimedium extracts to prepare icariin [To increase the production yield of icariin, the biosynthesis and bioconversion of icariin are gradually being studied. Biosynthesis of icariin from glucose or phenylpropionic acid has not been realized because it needs a series of reactions and some of these enzymes are not well-characterized. So far, it has been only reported that the biosynthesis of icaritin, the precursor of icariin, from glucose has been realized, and the biosynthesis of icariin needs further research . Biotranevicornu . Therefonditions . Identic icariin .Alternaria alternata SK37.001 can make citrus juices less bitter [Aspergillus niger can improve wine aromas [α-L-Rhamnosidase (3.2.1.40) can release the terminal L-rhamnose from a series of glycosides, glycolipids, and some natural products by cleaving α-1,2, α-1,3, α-1,4, or α-1,6 glycosidic bonds . This ens bitter , and α-Le aromas . Moreovee aromas  and prepe aromas ,20.Papiliotrema laurentii ZJU-L07. Meanwhile, we aimed to purify and characterize a new intracellular α-L-rhamnosidase and to research whether α-L-rhamnosidase has potential applications in hydrolyzing some natural products. The enzyme showed great catalytic activity specifically towards the rhamnoside-α-1,2-rhamnoside glycosidic linkage at the C-3 position in epimedin C. To improve the efficiency of the bioconversion of epimedin C into icariin and increase the content of icariin in the epimedium water extract, we also conducted the optimization of the enzymatic hydrolysis condition.In recent decades, α-L-rhamnosidase-producing fungi and bacteria have been widely studied. However, the studies about the enzyme from yeasts are few. In this study, a strain producing an intracellular α-L-rhamnosidase was screened and identified as p-nitrophenyl-α-L-rhamnopyranoside (pNPR) was bought from Nantong Feiyu Biological Technology Co., Ltd. . Modified BCA Protein Assay Kit and protein markers for SDS-PAGE were produced from Sangon Biotech Engineering Co., Ltd. . HPLC-grade acetonitrile was produced from Tianjin Siyou Fine Chemical Co., Ltd. . Epimedium water extract (EWE) was provided from Jing Brand Chizhengtang Pharmaceutical Co., Ltd. . Two kinds of epimedium water extract were used in this work, one kind of epimedium water extract contained 50% of epimedin C, and another kind of epimedium water extract contained 20% of epimedin C. To make it easier to distinguish, the former was named EWE-I and the latter was named EWE-II. Other chemicals were of reagent grade.Soil samples used for the strain screening were collected from Herba Epimedii botanical garden in Huangshi, China. Epimedin C, prunin, and hesperetin-7-O-glucoside were obtained from Chengdu Desite Biotechnology Co., Ltd. . Icariin, neohesperidin, hesperidin, naringin, and isoquercitrin were purchased from Aladdin . Rutin was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. . E. brevicornu. The soil sample was dissolved in 25 mL of the sterilized liquid medium having the composition: (g·L−1): EWE-II 20. The media were cultured at 30 °C, 180 rpm. After 24 h of incubation, the fermentation solution was diluted and then seeded into the solid medium of the first screening (g·L−1): EWE-II 20, agar 20. Then, the strains were picked up and plated out on the liquid medium of the secondary screening, which contained (g·L−1): epimedin C 0.05, sucrose 30, NaNO3 3, KCl 0.5, K2HPO4·3H2O 1, MgSO4·7H2O 0.5, and FeSO4 0.01 . The target strain was determined by whether icariin was produced. The content of icariin was analyzed using a HPLC system. In order to increase enzyme activity, various intensities of gamma rays  and different concentrations of nitrosoguanidine  were applied to mutagenesis of the target strain [−1): epimedin C 0.05, NaNO3 6, peptone 6, yeast extract 6, CaCl2 0.1, K2HPO4 0.1, and FeSO4·7H2O 0.1. The strain with the highest yield of icariin was isolated.The strain, producing α-L-rhamnosidase specifically for bioconverting epimedin C into icariin, was isolated from soil samples growing t strain . After m® DNA Handbook. The ITS sequence was amplified by PCR using primers ITS1(5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4(5′-TCCTCCGCTTATTGATATGC-3′). The genotypic identification of the strain was completed by analyzing the ITS sequence using the BLAST program . The physiological and biochemical identification of the strain was finished in the Zhejiang Institute of Microbiology using Merieux VITEK2 compact automatic microbial analysis system. The neighbor-joining tree was drawn using MEGA-X.Then, the total DNA of the strain was extracted using Puregene3, 0.3% yeast extract, 0.3% peptone, 0.001% K2HPO4, 0.001% FeSO4·7H2O, and 0.001% CaCl2. After liquid cultivation, the cells were collected by centrifugation at 2328× g for 10 min, washed twice with distilled water, resuspended in phosphate buffer , with ultrasonic crushing for 10 min at a low temperature (<10 °C). Then, the cellular extracts were centrifuged at 6790× g, 4 °C for 10 min, and the supernatants were considered as crude enzyme.The strain was cultivated aerobically at 28 °C for 4 d in a medium containing 1% L-rhamnose, 1% NaNOg, 4 °C for 10 min, and the precipitate was resuspended and dialyzed in citric acid-Na2HPO4 buffer (pH 7.0). The enzyme solution was concentrated by using PEG 6000 and separated on a Sephacryl S-200 gel filtration column (Φ1.75 × 43 cm). Finally, the elution was completed with the citric acid-Na2HPO4 buffer  at a flow rate of 0.6 mL·min−1. The fractions with higher α-L-rhamnosidase activity were collected, concentrated and purified further using a DEAE SepharoseTM Fast Flow column (Φ1.6 × 20 cm) from Shanghai Chenqiao Biotechnology Co., Ltd. . The column was eluted with a NaCl linear gradient elution (0–1.0 M) in citric acid-Na2HPO4 buffer  at a flow rate of 0.6 mL·min−1. Collected fractions were screened for assaying protein concentration and α-L-rhamnosidase activity. Active fractions were combined, dialyzed in citric acid-Na2HPO4 buffer (pH 7.0), and concentrated by using PEG 6000. The molecular weight of the purified enzyme was detected by Polyacrylamide gel electrophoresis (SDS-PAGE) [α-L-Rhamnosidase was obtained after purification of the crude enzyme. In order to maintain enzyme activity, all the purification steps were finished at 4 °C. First, the crude enzyme was mixed with ammonium sulfate salt up to 30% saturation, and the mixture was kept for 4 h and centrifuged to remove protein impurities. The ammonium sulfate was added to the supernatant to achieve a final concentration of 70% saturation. Second, the mixture was centrifuged at 10,611× DS-PAGE) .2HPO4 buffer  were mixed and preheated for 5 min in 55 °C. Then, 50 μL of the enzyme solution was added to the mixture and incubated at 55 °C for 10 min. The reaction was stopped by adding 750 μL of 1.5 M Na2CO3. The liberated p-nitrophenyl was determined at 405 nm by a microplate reader. One unit (U) of α-L-rhamnosidase was defined as the amount of enzyme liberating 1 μmol of p-nitrophenol per min at 55 °C and pH 7.0. The protein concentration was quantified using a modified BCA Protein Assay Kit with bovine serum albumin as the standard.The activity of α-L-rhamnosidase was assayed using the method described previously . Amounts2HPO4 buffer  for 10 min [pNPR as the substrate was used to determine the optimum temperature of α-L-rhamnosidase. The reaction mixture was incubated at different temperatures (30–70 °C) in the citric acid-Nar 10 min . The higr 10 min . Then, tThe optimum pH of α-L-rhamnosidase was measured by incubating the reaction mixture at 55 °C in the pH range of 4.0–9.0 for 10 min, using pNPR as the substrate . The hig2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, Co2+, Ba2+, K+ and Al3+ were added to the reaction system to make the final concentration of 0.2 mM [To study the effect of metal ions on α-L-rhamnosidase activity, Caf 0.2 mM . The enzmaxV) and Michaelis constant (mK) were computed from Lineweaver–Burk plots.The kinetic parameters of α-L-rhamnosidase were investigated by using different concentrations of pNPR (2.0–10.0 mM) as artificial substrate and different concentrations of epimedin C (1.0–5.0 mM) as natural substrate . The max−1 of 200 μL substrates and 100 μL enzyme solution were mixed and reacted at the optimum conditions for 6 h. The reaction was stopped by ice bath and adding 900 μL methanol. Then, the mixtures werecentrifuged at 6790× g for 5 min. The supernatants were filtered using a 0.22 μm nylon membrane ahead of detecting released products via high performance liquid chromatography (HPLC).The substrate specificity of the enzyme was analyzed by using naringin, neohesperidin, epimedin C, hesperidin, and rutin as substrates. A total of 1 g·L−1. The concentration of epimedin C and icariin was determined by HPLC with incubation for 0.5, 1, 2, 4, 6, and 8 h. The effects of different purities of epimedin C on bioconversion of epimedin C to icariin were investigated according to icariin yield and epimedin C remaining. The calculation method of icariin yield and epimedin C remaining is in the Standard epimedin C, EWE-I, and EWE-II were each hydrolyzed by the crude enzyme solution at 50 °C. The concentration of epimedin C in the reaction systems was 500 mg·L−1, 1000 mg·L−1, 1500 mg·L−1), the amount of α-L-rhamnosidase , reaction temperature  and pH  were selected for single factor experiments. Secondly, the yield of icariin and epimedin C remaining were used as indexes to investigate the influence of the four single factors on the preparation of icariin by biological enzymatic method. Then, the important factors and levels on icariin yield were determined and the production of icariin was optimized by response surface methodology (RSM). The Design Expert software for Box–Behnken Design was used to design a response surface analysis test with three factors and three levels, with the yield of icariin and epimedin C remaining as the response values. The experimental design is shown in X1, X2, and X3 are the values of enzyme dosage, pH, and time, respectively, and Y1 and Y2 represent the values of the yield of icariin and epimedin C remaining, respectively. In the single factor experiments and response surface methodology, 1 mL of EWE-II and 1 mL of the crude enzyme were mixed to react, and the reaction volume was always 2 mL.To increase the content of icariin in the EWE-II, enzymatic hydrolysis conditions were optimized. Firstly, the concentration of epimedin C  species is shown by a neighbor-joining tree  [Pichia angusta X349 (33.9 U·mg−1 of protein) [P. laurentii ZJU-L07 was similar to those of α-L-rhamnosidases from Bacillus sp. GL1 (100.0 kDa) [Bifidobacterium dentium (100.0 kDa) [Thermotoga petrophila (101.7 kDa) [Aspergillus nidulans (102.0 kDa) [Penicillium griseoroseum MTCC-9224 (97.0 kDa) [The intracellular α-L-rhamnosidase from tography a and anitography b. The fiprotein)  and simiprotein) . The mol0.0 kDa) , Bifidob0.0 kDa) , Thermot1.7 kDa) , Aspergi7.0 kDa) .The catalyzed reaction rate of the enzyme is closely related to temperature, so temperature is an important factor in the study of the enzymatic properties of α-L-rhamnosidase. The optimal temperature of α-L-rhamnosidase was determined to be 55 °C a. FigurepH is also another important factor affecting enzyme activity and the catalyzed reaction rate of the enzyme. It could be seen from 2+, Fe2+, Cu2+, and Mg2+ showed inhibition on α-L-rhamnosidase. The relative activity decreased to 31.4% and 64.4% with the addition of Fe2+ and Cu2+, respectively. Ca2+ and Mg2+ had a slight inhibitory effect, reducing 6.4% and 15% of α-L-rhamnosidase activity, respectively.The effects of metal ions on α-L-rhamnosidase activity were shown in mK and maxV were 1.38 mM and 24.64 μmol·mg−1·min−1, respectively  containing lower levels of epimedin C was easy to obtain in practical production because of its cheapness and simple production process. Therefore, in order to improve the content of icariin, EWE-II was selected as the substrate to optimize the enzymatic conditions.The effects of different purities of epimedin C on the yield of icariin were investigated using standard epimedin C, EWE-I, and EWE-II as the substrates. The result was shown in −1 epimedin C was 74.99 ± 0.42%, the value was 51.81 ± 6.56% and 26.67 ± 1.02% in the reaction system loaded 1000 mg·L−1 epimedin C and 1500 mg·L−1 epimedin C, respectively. However, regardless of the concentration of epimedin C in the reaction system, the conversion rate of epimedin C was the fastest before 3 h of incubation, and the yield of icariin did not show an upward trend after 9 h of incubation, which means α-L-rhamnosidase basically stopped working. To verify the effect of enzyme activity on the yield of icariin, different amounts of α-L-rhamnosidase were incubated with EWE-II  and the residual of epimedin C was 5.05%.As the important factors affecting the enzyme-catalyzed reaction rate, the concentration of epimedin C, the amount of α-L-rhamnosidase, the reaction temperature, and the pH were studied for their effect on the yield of icariin and epimedin C remained. As shown in h EWE-II b. When th EWE-II . In the −1. The optimal temperature of epimedin C degradation was determined at 45 °C. As the four factors were optimized, it was found that the optimal yield appeared at different time points. The reaction time was also an important factor and set as a factor for further optimization.In view of the above results in Y1 (icariin yield) = 78.500 + 3.866X1 + 1.941X2 + 2.432X3 − 3.733X1× X1 + 0.171X2× X2 − 2.784X3× X3 + 1.005X1× X2 − 5.137X1× X3 + 3.628X2×X3. In the ANOVA table, F-value and p-value are applied to determine significance of the regression model for each response selected. If the model possesses a high F-value and the p-value is less than 0.05, it is considered to be significant [F-value for the quadratic model of icariin yield showed a value of 4.714 and the p-value was 0.027, which indicated that the build the quadratic model for icariin yield was significant  indicating that the built regression equation is appropriate. p-value of the model was 0.0002 (<0.05) and p-value of “Lack of Fit” is 0.055 (>0.05)  . The resB. thetaiotaomicron [A. nidulans [T. petrophila [P. laurentii ZJU-L07 could hydrolyze the rhamnoside-α-1,2-rhamnoside glycosidic bond and transform epimedin C into icariin. Moreover, α-L-rhamnosidase from P. laurentii ZJU-L07 could also hydrolyze the glucoside-α-1,2-rhamnoside glycosidic bond and transform neohesperidin and naringin into hesperetin-7-O-glucoside and prunin, respectively. Therefore, α-L-rhamnosidase from P. laurentii ZJU-L07 could also be used for debittering citrus juices [Saccharomyces cerevisiae, Hanshula anomala, Debaryomyces ploymorphus [C. albidus [P. angusta X349 [S. cerevisiae, H. anomala, and D. ploymorphus show low levels of α-L-rhamnosidase activities, which are not good sources of α-L-rhamnosidase production. α-L-rhamnosidase from C. albidus [P. angusta X349 [As an important biotechnology enzyme, α-L-rhamnosidase has been paid attention because it could hydrolyze various natural compounds. With the pharmacological effects of icariin being paid more and more attention, the preparation of icariin by α-L-rhamnosidase has also been gradually researched. Different sources of α-L-rhamnosidase have different abilities to hydrolyze different glycosidic bonds . It was aomicron , A. nidunidulans , and T. trophila  could cos juices  and the s juices . In prevymorphus , C. albi albidus , and P. sta X349 . S. cere albidus  could trsta X349  could hyA. nidulan [B. thetaiotaomicron [C. albidus (60 °C) [T. petrophila DSM 13995 (90 °C) [P. angusta X349 (40 °C) [A. flavus MTCC-9606 (50 °C) [A. aculeatus JMUdb058 (50 °C) [B. amyloliquefaciens 11568 (45 °C) [A. alternata SK37.001. The optimal temperature of α-L-rhamnosidase from A. alternata SK37.001 was 60 °C, and it was relatively stable at a temperature between 30 and 50 °C [F. moniliforme MTCC-2088 was 10.5 [Aspergillus awamori MTCC-2879 [Penicillium griseoroseum MTCC-9224 [mK means the affinity of enzyme and substrate, and the larger the value of mK means the smaller the affinity between enzyme and substrate. Metal ions also affected enzyme activity. Among them, Fe2+ and Cu2+ had a strong inhibitory effect on enzymes, which might destroy the active group of the enzyme [A. alternata SK37.001 [P. citrinum MTCC-8897 [B. thetaiotaomicron [2+ had extremely high levels of rhamnosidase inhibition. The mK of the α-L-rhamnosidase studied with pNPR as the substrate was lower than that of α-L-rhamnosidase from some other microorganisms, which indicated that the affinity of the enzyme for pNPR was superior to that of other-sourced enzymes [mK of α-L-rhamnosidase with epimedin C as the substrate was higher than that of α-L-rhamnosidase from A. nidulans and T. petrophila [The efficiency of enzyme conversion of natural products is not only related to the specificity of the enzyme, but also related to the activity of the enzyme. Enzymatic properties show the effects of temperature, pH, and metal ions on enzyme activity. In this study, the optimum temperature of α-L-rhamnosidase was 55 °C a, which  nidulan  and B. taomicron , lower t (60 °C)  and T. p (90 °C) , and hig 40 °C) , A. flav (50 °C) , A. acul (50 °C) , and B.  (45 °C) . Howevernd 50 °C . Howevernd 50 °C . The optnd 50 °C . The optnd 50 °C ,38, and  °C , A. e enzyme . This waSK37.001 , P. citr enzymes ,31. Km otrophila ,23, whicIn all the experiments of enzymatic hydrolysis of epimedin C, epimedin C in the reaction system was not completely converted to icariin. The crude enzyme not only contained α-L-rhamnosidase, but also other enzymes. α-L-Rhamnosidase and β-D-glucosidase usually coexist in microorganisms. Naringinase is an enzyme complex that possesses the activities of both α-L-rhamnosidase and β-D-glucosidase, which cleave terminal α-L-rhamnose and β-D-glucose from flavonoids and other products . β-D-GluP. laurentii ZJU-L07. α-L-Rhamnosidase from P. laurentii ZJU-L07 has a highly efficient enzymatic hydrolysis effect on epimedin C. When the concentration of epimedin C in the reaction systems was 500 mg/L, 87% and 85% of epimedin C in standard epimedin C and EWE-I were converted into icariin, respectively. After optimizing the reaction conditions, more than 83% of epimedin C in EWE-II was converted into icariin. The efficient and highly specific α-L-rhamnosidase provides a valuable protocol for industrialized production of icariin in the future. Nevertheless, there are some difficulties in large-scale preparation because this enzyme is an intracellular enzyme. Therefore, in future studies, an α-L-rhamnosidase gene from P. laurentii ZJU-L07 could be cloned and expressed in P. pastoris [In summary, icariin as a chemical marker for the quality control of Herba Epimedii in Chinese Pharmacopeia , has extpastoris . Alterna


Vaccinium vitis-idaea L.) are well established; however, their proanthocyanidins are still heavily underutilized. Optimizing their delivery systems is key to enabling their wider applications. The present study investigates the phytochemical and ‘wound-healing’ properties of proanthocyanidin-rich fraction(s) (PRF) from lingonberry leaves as well as the development of optimal dermal film as a proanthocyanidin delivery system. The obtained PRF was subjected to HPLC-PDA and DMAC analyses to confirm the qualitative and quantitative profiles of different polymerization-degree proanthocyanidins. A ‘wound healing’ in vitro assay was performed to assess the ability of PRF to modulate the wound environment for better healing. Low concentrations of lingonberry proanthocyanidins were found to accelerate ‘wound‘ closures, while high levels inhibited human fibroblast migration. Fifteen dermal films containing PRF were prepared and evaluated based on their polymer  compositions, and physical, mechanical, and biopharmaceutical properties using an experimental design. The composition containing 0.30 g of MC, 0.05 g of HEC, and 3.0 g of PEG 400 was selected as a promising formulation for PRF delivery and a potentially effective functional wound dressing material, supporting the need for further investigations.The compositions and health-beneficial properties of lingonberry leaves ( Vaccinium vitis-idaea L.), a small evergreen shrub of the family Ericaceae, is among the best natural sources of bioactive compounds with beneficial health effects [Lingonberry , selected the optimal compositions, and determined the physical and mechanical properties of methyl-cellulose-hydroxyethyl cellulose films containing PRF.A representative HPLC-PDA chromatogram of PRF is presented in The additional cytotoxicity experiment by the MTT assay was performed on human foreskin fibroblast (HF) to evaluate the PRF effect on HF viability in the range of concentrations from 3.9 to 125 µg/mL . It was The PRF showed a concentration- and time-dependent effect on HF migration by the ‘wound healing’ assay . The strp ≥ 0.05) from MH0.400-P2.0 (No. 6) and MH0.400-P2.0 (No. 10) films with thicknesses of 285 ± 13 µm and 304 ± 6 µm, respectively. The MH0.425-P2.5 film (No. 9) had the highest thickness (522 ± 33 µm). This film differed statistically significantly (p < 0.05) from other experimental MC-HEC films. It appeared that, as the amount of PEG 400 in the film increased, its thickness increased. This was confirmed by a statistically significant (p < 0.05) moderate correlation (r = 0.624) between the amount of PEG 400 and film thickness.Quality parameters of experimental methylcellulose-hydroxyethyl cellulose (MC-HEC) films are given in p ≥ 0.05) among:(a)Experimental MC-HEC films  contained 2.5–3.0 g of PEG 400, and the moisture content in the film ranged from 3.9 to 5.1%;(b)Experimental MC-HEC films  contained 2.0 g of PEG 400 and MH0.375-P2.5 film (No. 8) contained 2.5 PEG 400; the moisture content of the films ranged from 5.5 to 7.1%.The moisture content in the experimental MC-HEC films ranged from 3.9 to 7.1% . The lowp < 0.01) and very strong correlation (r = −0.918) between the amount of PEG 400 and the moisture content.It was found that as the amount of PEG 400 in the film increased, the moisture content in it decreased . This wap ≥ 0.05) from MH0.450-P2.0 (No. 12) film, containing 0.35 g of methylcellulose, 0.10 g of hydroxyethyl cellulose, and 2.0 g of PEG 400, with a stickiness of 0.179 ± 0.017 N.The stickiness of the experimental MC-HEC films ranged from 0.143 to 0.419 N . The MH0p ≥ 0.05) from MH0.350-P3.0 (No. 14) and MH0.400-P3.0 (No. 5) films with a stickiness of 0.412 ± 0.027 N and 0.386 ± 0.024 N, respectively. It was found that by increasing the amount of PEG 400 and decreasing the amount of methylcellulose in the film, its stickiness increased (p < 0.01) strong correlation (r = 0.851) was found between the amount of PEG 400 and film stickiness. Results showed that as the moisture content of the film decreased, its stickiness increased. This was confirmed by a statistically significant (p < 0.01) strong correlation (r = −0.751) between the moisture content and film stickiness.The MH0.400-P3.0 film (No. 3), containing 0.30 g of methylcellulose, 0.10 g of hydroxyethyl cellulose, and 3.0 g of PEG 400, had the highest stickiness (0.419 ± 0.012 N). This film did not differ statistically significantly (ncreased . HoweverR2 = 0.9657–0.9942), which shows a linear relationship between the total released amount of proanthocyanidins (%) and the duration of the process (h).The release kinetics of proanthocyanidins from experimental MC-HEC films are shown in Experimental MC-HEC films  containing 2.0 g of PEG 400 released 40.8–49.3% (flux 188.7–233.0 µg/cm²) proanthocyanidins after 4 h A. Of theExperimental MC-HEC films  containing 2.5 g of PEG 400 released 38.7–58.2% (flux 206.3–294.8 µg/cm²) proanthocyanidins after 4 h B. Of theExperimental MC-HEC films  containing 3.0 g of PEG 400 released 39.8–56.5% (flux 211.4–293.7 µg/cm²) proanthocyanidins after 4 h C. Of thep < 0.01) moderate inverse correlation (r = −0.643) between the amount of hydroxyethyl cellulose in the films and the released amount of proanthocyanidins after 4 h.Statistical analysis showed a statistically significant , only statistically significant linear models of moisture, stickiness, and release after 0.25 h were obtained . These qThese selected compositions of the polymeric films corresponded to composition No. 14 of the experimental design; the practical values for the appropriate properties are given in The interest in utilizing proanthocyanidins in the pharmaceutical industry has considerably increased over time with the discovery of their beneficial functions . One posLingonberry fruits are highly valued in the food and nutraceuticals industry, while leaves, despite the considerable richness of proanthocyanidins and availability during all seasons, are still hardly used . Due to The literature pertaining to the biological activities of proanthocyanidins strongly suggests possible application in wound healing ,37. WounA PRF fraction at 20 µg/mL and lower concentrations did not significantly reduce the viability of HF, indicating that proanthocyanidins do not decrease fibroblast proliferation and functions and do not delay epithelization. Obtained concentration- and time-dependent inter-relationships on HF migration by ‘wound healing’ assays correlate well with previous findings ,41, wherThin films are receiving attention for drug delivery . PharmacPanchal et al.  pointed In general, our selected composition of the polymeric film with the highest ratio between the polymer mixture and PEG 400 (1:8.6), when the polymer mixture was predominated by MC, provided acceptable moisture, stickiness, and 56.5% practical release of proanthocyanidins after 4 h and, therefore, could be regarded as a promising tool for PRF delivery. This and previous reports ,61,62 inVaccinium vitis-idaea L.) leaves using column chromatography as described previously [The proanthocyanidin-rich fraction was obtained from the lingonberry , ethanol from Vilniaus degtine . Ultrapure water was obtained by a Milli-Q water purification system from Millipore .Standards used in the phytochemical analysis: procyanidins A1, A2, A4, B1, B2, C1, (+)-catechin, and (−)-epicatechin from Sigma-Aldrich; procyanidin B3 from Extrasynthese .The following reagents were used: 4-dimethylaminocinnamaldehyde (DMAC), 3--2,5-diphenyltetrazolium bromide (MTT) from Sigma-Aldrich, and phosphate-buffered saline (PBS) from Gibco .Methylcellulose (viscosity 15 cPs) was purchased from Alfa Aesar . Hydroxyethyl cellulose (average M = ~90 kDa) was purchased from Sigma-Aldrich. Polyethylene glycol 400 (PEG 400) was purchased from Carl Roth .2 falcons at 37 °C in a humidified atmosphere containing 5% CO2, and were used until the passage of 10.The human foreskin fibroblasts (HF) CRL-4001 were originally obtained from the American Type Culture Collection  and kindly provided by Prof. Helder Santos . HF were cultured in Dulbecco’s Modified Eagle’s GlutaMAX medium (Gibco), supplemented with 1% of antibiotics ), and 10% fetal bovine serum (Gibco). Cell cultures were grown 25 cmScreening of individual proanthocyanidins and other tannins in the obtained fraction from lingonberry leaves was performed with HPLC-PDA  system using ACE Super C18  column  and gradient elution consisting of 0.1% trifluoroacetic acid (eluent A) and acetonitrile (eluent B) . ElutionThe total proanthocyanidin content in the PRF (before incorporating) and polymeric films (during in vitro release test) was evaluated using a DMAC assay with slight modifications . Three m4 cells/well and incubated for 48 h at 37 °C in a humidified atmosphere containing 5% CO2. Then the scratch was made using a 100 µL pipette tip. The cells were washed once with PBS, and the fresh medium containing different concentrations of PRF was added. The final extract concentrations were: 20, 10, and 5 µg/mL. For the selection of these PRF concentrations, the effect on HF cells was performed using a standard MTT assay, as described elsewhere [2.The PRF effect on human fibroblast migration was assessed using the ‘wound healing’ assay, as described elsewhere . After tlsewhere . The wel‘Wounds’ were captured at intervals of 0, 5, and 20 h from scratch under phase-contrast microscopy at a 4× magnification, in two different areas in each well. The ’wound’ area was analyzed using ImageJ program, version 1.530 .α = 1) as an experimental design ) was applied to model the composition of the film [The surface response center composition model  of the moisture: Y1 = −0.973X1 − 5.360X2 − 2.131X3 + 11.208;Linear model (p = <0.0001) of the stickiness: Y2 = −1.803X1 + 0.110X2 + 0.171X3 + 0.454;Linear model (p = 0.0034) of the release of biologically active compounds after 15 min: Y3 = −5.718X1 − 19.902X2 − 0.044X3 + 5.445;Linear model  in the polymeric films, Y2—the stickiness (N) of the polymeric films, Y3—the release (%) of biologically active compounds from the polymeric films after 15 min, X1—the amount (g) of methylcellulose, X2—the amount (g) of hydroxyethyl cellulose, and X3—the amount (g) of polyethylene glycol 400.Here: YMaximum stickiness (5+);Maximum release after 15 min (4+);Minimum moisture (3+).In order to obtain the optimal composition of the polymeric films, the following criteria were chosen and arranged in descending order of importance (indicating the number of pluses):2) [2) was weighed and further analyzed.An appropriate amount  of methy2) ,70. This2) was measured (n = 3) using a digital micrometer  and expressed in µm.The thickness of the experimental films (1.77 cm2) was measured (n = 3) using an electronic moisture analyzer  and expressed in %.The moisture content of the experimental films (1.77 cm2) was measured at several random locations (n = 4) using a texture analyzer  with a mucoadhesion rig (A/MUC) and expressed in N. Conditions for the texture analysis:(1)The pre-test speed was 1 mm/s.(2)The test speed was 0.5 mm/s.(3)The post-test speed was 1 mm/s.(4)The applied force was 1 N.(5)The return distance was 10 mm.(6)The contact time was 10 s.The stickiness of the experimental films  were placed in Eppendorf® centrifuge tubes (2 mL) and filled with 2 mL of purified water. The tubes were stored at 32 °C . Samples were taken after 0.25, 0.5, 0.75, 1, 2, 3, and 4 h by adding the same volume of fresh acceptor medium. Release samples were analyzed spectrophotometrically (2).The experimental films  in terms of quality parameters, such as thickness, moisture, stickiness, and ability to release proanthocyanidins. The above results provide firm evidence that topical application of lingonberry proanthocyanidin in the form of an MC-HEC film represents a feasible approach to support dermal wound healing. Future research could continue to elucidate the exact mechanisms of lingonberry proanthocyanidins in wound healing and assess the suitability of the developed polymeric films.


The as-prepared 2MgO/BC-450-0.5 had phosphorus adsorption capacities of 171.54 mg g−1 at 25 °C and could remove 100% of phosphorus from livestock wastewater containing 39.51 mg L−1 phosphorus. The kinetic and isotherms studied show that the pseudo-second-order model (R2 = 0.999) and Langmuir models (R2 = 0.982) could describe the adoption process well. The thermodynamic analysis indicated that the adsorption of phosphorus on the MgO-modified biochars adsorbent was spontaneous and endothermic. The effect of pH, FTIR spectra and XPS spectra studies indicated that the phosphorus adsorption includes a protonation process, electrostatic attraction and precipitation process. This study provides a new strategy for biochar modification via a facile mechanochemical method.Adsorption is an efficient technology for removing phosphorus from wastewater to control eutrophication. In this work, MgO-modified biochars were synthesized by a solvent-free ball milling method and used to remove phosphorus. The MgO-modified biochars had specific surface areas 20.50–212.65 m However, excessive discharge of phosphorus from agricultural, industrial and domestic sources into water will cause eutrophication, damage the ecological environment and even threaten human health. According to the Integrated Wastewater Discharge Standard (GB 8978-1996) established in China, phosphate discharge (in terms of P) is divided into the primary standard (≤0.5 mg L−1) and the secondary standard (≤1.0 mg L−1). In addition, the US Environmental Protection Agency (EPA) recommends that the total phosphorus concentration in surface water should not exceed 0.1 mg L−1, which is considered safe for aquatic life [Phosphorus is an indispensable nutrient element in the growth process of aquatic plants ,2, exist2O3 [At present, the methods for phosphorus removal mainly include biological methods, chemical methods and physical methods. Among them, biological methods such as assimilation  are ofte2O3 , biochar2O3  and so o2O3 .2O3 [43− from water [−1. Compared with traditional impregnation, which involves introducing metal oxides onto the surface of biochar, the emerging ball milling method is a more facile strategy due to its solvent-free modification approach [Generally, pristine biochar prepared by direct pyrolysis of biomass has poor adsorption performance for phosphorus due to its negatively charged surface, the low specific surface area and limited adsorption sites ,15. Ther2O3  on the som water ,20,21. Fom water  used theapproach ,24.3O4− or CuO-modified biochar, have been synthesized using the ball milling method for the removal of tetracycline [Ball milling has emerged as a favorable method for the synthesis of nanomaterials and is becoming recognized as an environmentally friendly and low-cost method for enhancing the physicochemical properties of metal oxides/biochar nanomaterials ,26,27. Tacycline , methyleacycline  and reacacycline . The resacycline ,33, thus2PO4, AR, 99.5%), potassium nitrate , potassium bicarbonate  and potassium sulfate  were purchased from Shanghai Macklin Biochemical Co., Ltd. . Potassium chloride  were supplied by Tianjin JiangTian Chemical Technology Co., Ltd. . Magnesium acetate tetrahydrate (Mg(CH3COO)2·4H2O, AR, 99%) was provided by Yuanye Biological Technology Co., Ltd . The livestock wastewater used in this study was taken from a cattle farm in the Dali Experimental Station. Before use, the wastewater was pretreated to remove suspended solids through a 0.2 μm filter membrane. Ultrapure water was produced by the Direct-Q3 UV system.Rice straw was obtained from Xiangtan in Hunan province (China). Potassium phosphate monobasic (KH3COO)2·4H2O (Mg precursor) and rice straw powder  were loaded into a ball milling tank with 27.3 g of ZrO2 balls (diameter 6.0 mm). Then, the tank was placed in a planetary ball mill . The rotation speed of the planetary disk was set as 350 rpm. After ball milling for a certain period of time (0.25–10 h), the resulting ball mixing sample was placed in a tube furnace  and heated to the designed temperature (250–850 °C) with a heating rate of 2 °C min−1 under N2 atmosphere and maintained for 2 h. After pyrolysis treatment, the resulting biochar was ground to powder and denoted as xMgO/BC-y-z . The biochar named BC was prepared by direct pyrolysis of pure rice straw at 450 °C after ball milling for 0.5 h.Rice straw was washed, dried, ground and finally sieved through a 60 meshes sieve (0.3 mm). In a typical run, Mg. The specific surface area (SBET) and pore size (DBJH) of adsorbents were calculated by the BET method and the BJH method. The total pore volume  was determined from the amount of adsorbed N2 at a relative pressure (P/P0) of 0.99. The Mg loadings of the adsorbents were determined with inductively coupled plasma optical emission spectroscopy , while the contents of C, H, O and N were measured by an elemental analyzer . Thermo-gravimetric analysis (TGA) was performed by DSC/DTA-TG analyzer  in N2 stream with a heating rate of 10 °C min−1 from 25 °C to 900 °C. The zeta potential was measured in solutions of different pH using a zeta potential analyzer . Fourier transformed infrared spectroscopy  was performed to determine the functional group changes.The morphology structure of the adsorbents was investigated by scanning electron microscopy (SEM). Surface element analysis was also conducted simultaneously with SEM at the same surface locations, using energy-dispersive X-ray spectroscopy . X-ray diffraction  was employed to determine the crystallographic structure of adsorbents in the range of 5° to 90°, operating at 40 kV and 40 mA. X-ray photoelectron spectroscopy  was used to determine the elemental composition and chemical state of adsorbent surfaces with mono Al Kα radiation. N−1, pH = 5.2) in a shaker at 250 rpm for 24 h. All adsorption experiments were run in triplicate, and the average values were reported with a relative deviation below 5%. The adsorption capacity qe (mg g−1) of phosphorus on the adsorbent was calculated at equilibrium by the following Equation (1):C0 and Ce (mg L−1) are the initial and equilibrium concentrations of phosphorus in the solution, respectively; V (L) is the volume of phosphorus solution; m (g) is the weight of the adsorbent.In order to select the adsorbent with the best adsorption performance, a series of preparation conditions were screened, including Mg precursor-to-rice straw mass ratio , pyrolysis temperature  and ball milling time . The adsorption experiment was conducted in the 50 mL polyethylene tube loaded with 20 mg of adsorbents and 40 mL of phosphorus solution (100 mg L−1 phosphorus solution at 25 °C. Adsorption isotherms for phosphorus on the adsorbents were determined with different concentrations ranging from 0 to 200 mg L−1 (pH 4.6–6.8) at 25 °C for 24 h. Adsorption thermodynamics for phosphorus on 2MgO/BC-450-0.5 were conducted with different reaction temperatures (298–318 K) by adding 20 mg of adsorbents to 40 mL of 100 mg L−1 phosphorus solution for 24 h. The details of this process are presented in the Adsorption kinetics for phosphorus on different adsorbents were determined at different time intervals  by adding 20 mg of adsorbents to 40 mL of 100 mg L−1 phosphorus solution and adsorbing for 24 h at 25 °C. The effect of adsorbent dosage (0.25–1.25 g L−1) was studied with the initial phosphorus concentrations of 200 mg L−1 at pH = 5 and the rest of the conditions were kept constant. The effect of co-existing ions (0.01 M) was evaluated, including Cl−, NO3−, SO42− and HCO3−. The removal rate R (%) of phosphorus was calculated following Equation (2):A series of adsorption experiments were carried out to evaluate the effects of environmental parameters on the performance of MgO-modified biochars for phosphorus removal. The effects of solution pH (1–11) on phosphorus removal were investigated by adding 20 mg of adsorbents to 40 mL of 100 mg L−1) for 12 h at 250 rpm and 25 °C, and the removal rate (R) was calculated as above. The phosphorus-adsorbed sample was treated with 1 mol L−1 NaOH solution for 8 h for the desorption of phosphorus from 2MgO/BC-450-0.5. The regenerated 2MgO/BC-450-0.5 was again used for phosphorus adsorption under the same conditions.The reusability of 2MgO/BC-450-0.5 was tested by 5 adsorption–desorption cycle experiments. The adsorbent (80 mg) was treated with 40 mL phosphorus solution  with 40 mL of wastewater and shaking for 12 h as described above. After the mixtures were filtered, the phosphorus removal rate was calculated as above.2 adsorption–desorption experiment analysis. The isotherms results show that all MgO-modified biochars exhibited typical type-IV curves, which is the characteristic of dominated mesopores (BET (12.31 m2 g−1) and Vtotal (0.017 cm3 g−1). The surface area (SBET) and Vtotal were increased for MgO-modified biochars  and Vtotal were slightly reduced for 3MgO/BC-450-0.5, which was prepared with a higher Mg precursor-to-rice straw mass ratio. The pores in 3MgO/BC-450-0.5 were blocked by the inserted MgO particles, leading to a decrease in SBET compared with that of 2MgO/BC-450-0.5 (BJH) decreased after mixing ball-milling of biochar and Mg precursor 2·4H2O and hemicellulose. At the third stage (380–460 °C), the weight loss of only 5.09% was ascribed to the formation of MgO and degradation of cellulose and lignin. Then, four MgO-modified biochars were prepared at pyrolysis temperatures of 250, 450, 650 and 850 °C, respectively. The result of adsorption experiment showed that phosphorus adsorption capacity was rapidly increased from 17.08 to 168.85 mg g−1 when pyrolysis temperature increased from 250 to 450 °C . The morphologies (−1), suggesting that the ball milling treatment is essential for improving the adsorption performance of the adsorbents. Based on the above studies, 2MgO/BC-450-0.5 prepared at the optimum conditions had the best phosphorus adsorption; they were then employed to study the adsorption behavior in detail.Considering that ball milling time may affect the morphology and structure of the adsorbent ,44, fourhologies  of MgO-mhologies . Ball mi+ surface and negatively charged phosphate ions [R2 = 0.999) and pseudo-first-order kinetic model (R2 = 0.995). It was indicated that the adsorption behavior of phosphorus on 2MgO/BC-450-0.5 was mainly chemisorption predominated.The adsorption kinetics experiments of BC and 2MgO/BC-450-0.5 were studied for a maximum of 60 h a. The adate ions . The sloate ions . To undeR2 = 0.762), the Langmuir model (R2 = 0.982) fitted the adsorption data better, suggesting that phosphorus adsorption on the surface of 2MgO/BC-450-0.5 was a monolayer adsorption process [qmax on 2MgO/BC-450-0.5 based on the Langmuir model were 171.54 mg g−1, which was almost 27-times that of BC. The qmax of MgO-biochars for phosphorus adsorption surpassed most of MgO-modified materials and are comparable with some CaO-modified materials (qmax = 171.54 mg g−1), the mole ratio of MgO to phosphorus was 2:1, considering that only the surficial atoms of MgO particles were available for the adsorbate.The adsorption isotherms of biochars were fitted with Freundlich and Langmuir models b. Compar process . The obtaterials . On the qmax was increased from 171.54 to 212.19 mg g−1. According to the literature [G0, ∆S0 and ∆H0 were calculated. It could be observed from G0 were negative and declined with the rise in temperature, revealing that phosphorus adsorption on 2MgO/BC-450-0.5 was spontaneous. The value of ∆S0 was positive, illustrating the high randomness during the adsorption process. Furthermore, the value of ∆H0 was positive, indicating an endothermic adsorption process. Consequently, a higher reaction temperature was conducive to phosphorus adsorption on 2MgO/BC-450-0.5.The adsorption thermodynamics of phosphorus on 2MgO/BC-450-0.5 were conducted as shown in terature , the val−1 with initial solution pH varying from 1 to 3. Then, the adsorption capacity reached the maximum capacity (160.70 mg g−1) at pH 5. However, with the initial pH value rising from 5 to 11, the adsorption capacity decreased from 160.70 to 53.37 mg g−1. A rise in the final pH value was observed due to the release of OH- from the reaction of MgO and water (MgO + H2O→MgOH+ + OH−) [3PO4, H2PO4− and HPO42− at pH ≤ 2.12, 2.12–7.21 and 7.21–12.67, respectively [PZC value of 2MgO/BC-450-0.5 was determined to be 5.19  . Accordiectively ,56. The  be 5.19 b, sugges−1 2, MgHPO4 and MgO, respectively [XPS was carried out to analyze the composition changes in surface materials of 2MgO/BC-450-0.5. In ectively . After aectively ,65.+, which further reacted with adsorbed phosphate ions to produce Mg(H2PO4)2 and MgHPO4 crystals [2O→Mg − OH+ + OH−Mg − O + HProtonation process:+ + H2PO4−→Mg − OH+—H2PO4−Mg − OH+ + HPO42−→Mg − OH+—HPO42−Mg − OHElectrostatic attraction:2+ + H2PO4−→Mg(H2PO4)2Mg2+ + HPO42−→MgHPO4MgPrecipitation process:The process of phosphorus adsorption on 2MgO/BC-450-0.5 was proposed based on the above results. When 2MgO/BC-450-0.5 was loaded in phosphorus solution, MgO nanoparticles on the surface of 2MgO/BC-450-0.5 were rapidly protonated under acidic conditions (pH = 5.2), resulting in the formation of MgOHcrystals . The ads−1 and 2MgO/BC-450-0.5 dosage of 2 g L−1. As shown in The recycling adsorption experiment was conducted for 12 h with initial phosphorus concentration of 10 mg L−1 of 2MgO/BC-450-0.5 was loaded in the livestock wastewater containing 39.51 mg L−1 phosphorus. After treatment for 12 h, phosphorus removal efficiency of 63%, 85% and 100% were obtained with 0.25, 0.5 and 0.75 g L−1 of absorbent (In order to evaluate the performance in the treatment of actual wastewater, 0.25–0.75 g Lbsorbent , indicatThe ball milling method has proved to be highly efficient for biochar modification to enhance phosphorus adsorption performance. Mg precursor-to-rice straw mass ratio, pyrolysis temperature and ball milling time had a significant impact on the adsorption performance of final adsorbents. Typically, 2MgO/BC-450-0.5 prepared in optimum conditions showed the greatest phosphorus adsorption capacity and had good recycling stability. The kinetic behavior and adsorption isotherm of phosphate on 2MgO/BC-450-0.5 was fitted well by pseudo-second-order and Langmuir models, respectively. Adsorption of phosphorus on 2MgO/BC-450-0.5 was spontaneous and endothermic, mainly involving the protonation process, electrostatic attraction and precipitation process. This study demonstrated that the ball milling method is a potential strategy to prepare metal oxides/biochar composites in wastewater treatment.


Decapping enzymes remove the 5′ cap of eukaryotic mRNA, leading to accelerated RNA decay. They are critical in regulating RNA homeostasis and play essential roles in many cellular and life processes. They are encoded in many organisms and viruses, including vaccinia virus, which was used as the vaccine to eradicate smallpox. Vaccinia virus encodes two decapping enzymes, D9 and D10, that are necessary for efficient viral replication and pathogenesis. However, the underlying molecular mechanisms regulating vaccinia decapping enzymes’ functions are still largely elusive. Here, we demonstrated that vaccinia D10 almost exclusively colocalized with mitochondria. As mitochondria are highly mobile cellular organelles, colocalization of D10 with mitochondria can concentrate D10 locally and mobilize it to efficiently decap mRNAs. Mitochondria were barely observed in “viral factories,” where viral transcripts are produced, suggesting that mitochondrial colocalization provides a spatial mechanism to preferentially decap cellular mRNAs over viral mRNAs. We identified three amino acids at the N terminus of D10 that are required for D10’s mitochondrial colocalization. Loss of mitochondrial colocalization significantly impaired viral replication, reduced D10’s ability to remove the RNA 5′ cap during infection, and diminished D10’s gene expression shutoff and mRNA translation promotion abilities. Again, 10.1128/mbio.00300-22.1FIG S1FIG S1, TIF file, 2.9 MB.D10 colocalizes with mitochondria in HeLa cells during infection. HeLa cells were infected with vD10-3xFlag, WT-VACV, or mock infected. Confocal microscopy was used to visualize D10 , mitochondria , and DNA  at 6, 9, and 16 hpi. Download Copyright © 2022 Cao et al.2022Cao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00300-22.2FIG S2FIG S2, TIF file, 1.2 MB.D10 colocalizes with mitochondria in A549DKO cells during infection. A549DKO cells were infected with vD10-3xFlag or WT-VACV. Confocal microscopy was employed to visualize D10 , mitochondria , and DNA  at 16 hpi. Download Copyright © 2022 Cao et al.2022Cao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We first generated and tested plasmids expressing D10 C-terminal (D10-ΔC57) and D10 N-terminal (D10-ΔN50) truncation mutants, respectively . While D10.1128/mbio.00300-22.3FIG S3FIG S3, TIF file, 1.1 MB.D10 mitochondrial colocalization signal is located at the N terminus of D10. A549DKO cells were transfected with a plasmid expressing indicated codon-optimized D10 truncation mutants with C-terminal 3×Flag. Confocal microscopy was employed to visualize D10 or its mutants using α-Flag antibody (green). Download Copyright © 2022 Cao et al.2022Cao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We then constructed two recombinant VACVs, vD10-I9/12/13T and vD10-Δ9-13, in which the D10 amino acids from 9 to 13 (ISQII) were mutated to TSQTT or deleted, respectively, yet both contained a 3×Flag tag at the C terminus. Interestingly, in both cases, the mutated D10 diffused in the infected A549DKO or HeLa cells but did not colocalize with mitochondria, using Tom20 or MitoTracker to locate mitochondria  and S5. 10.1128/mbio.00300-22.4FIG S4FIG S4, TIF file, 1.7 MB.D10 mutants with deletion of amino acids 9 to 13 or mutation expressed from recombinant VACV do not colocalize with mitochondria during infection, A549DKO cells were infected with indicated recombinant VACVs (MOI = 3) encoding D10 mutants with a C-terminal 3×Flag tag. Confocal microscopy was used to visualize D10 , mitochondria , and DNA  at 16 hpi. Download Copyright © 2022 Cao et al.2022Cao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00300-22.5FIG S5FIG S5, TIF file, 2.7 MB.D10 mutants with deletion of amino acids 9 to 13 or mutation expressed from recombinant VACV do not colocalize with mitochondria during infection. HeLa cells were infected with indicated recombinant VACVs (MOI = 3) encoding D10 mutants with a C-terminal 3×Flag tag. Confocal microscopy was used to visualize D10 , mitochondria , and DNA  at 16 hpi. Download Copyright © 2022 Cao et al.2022Cao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, we examined the impact of D10 mitochondrial colocalization on VACV replication by comparing the replication of vD10-Δ9-13 and vD10-I9/12/13T to vD10-3xFlag, a control VACV encoding wild-type (WT) D10 with a 3×Flag tag at its C terminus. Two viruses with D10 knocked out (vΔD10) and the D10 Nudix motif inactivated (vD10mu) , respect10.1128/mbio.00300-22.6FIG S6P > 0.05; *, P ≤ 0.05. Significance was compared to vD10-3xFlag. Download FIG S6, TIF file, 0.2 MB.BHK-21 cells were infected with indicated viruses at an MOI of 3 (A) or 0.001 (B). Viral titers were determined using a plaque assay at indicated times postinfection. All the viruses used encode D9. Error bars represent the standard deviation of at least three replicates. ns, Copyright © 2022 Cao et al.2022Cao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the VACV encodes two decapping enzymes, D9 and D10, that have overlapping yet distinct functions , 25, 35.Overall, we conclude that D10 mitochondrial colocalization is required for efficient VACV replication in both PKR-and RNase L-dependent and independent manners.We next determined the impact of D10 mitochondria colocalization on viral plaque size in BS-C-1 cells. The results indicated that vΔD9-D10-I9/12/13T and vΔD9-D10Δ9-13 have significantly smaller plaques than vD10-3xFlag, as well as vΔD9  and B. TOur results  demonstrIt has been shown that inactivation of D9 and D10 decapping activities stimulate PKR activation, followed by eIF2α phosphorylation , leading7G-capped RNA in the presence or absence of D9 expression during VACV infection  RNA with a 5′ poly(A) leader and m7G cap and renilla luciferase (RLuc) RNA with a 5′ untranslated region (UTR) containing Kozak sequence and m7G capped were cotransfected in cells with expression of wild-type D10 or its mutants. Notably, all those containing mitochondrial colocalization sequences promoted 5′-poly(A) leader-mediated translation, while those without mitochondrial colocalization sequences significantly reduced the translation enhancement  leader, a feature of all poxvirus mRNAs expressed after viral DNA replication . The enhnfection . We emplnfection , 43. We ancement  to C. Apancement , 45. Thepped RNA  to F. Inter mRNA . This maIn this study, we identified and characterized the first mitochondria-colocalized decapping enzyme, D10, encoded by a poxvirus. We pinpointed three hydrophobic isoleucine residues at the N terminus of D10 that are essential for D10’s mitochondrial colocalization. The mitochondrial association is required for D10’s unusual function to promote 5′ poly(A) leader-mediated mRNA translation, including cap-independent translation enhancement. The mitochondrial colocalization is also necessary for D10's optimal function to efficiently remove 5′ caps of RNAs in cells and shut off gene expression. Consequently, mitochondrial colocalization is required for efficient VACV replication. Interestingly, one group of mRNAs that are preferentially targeted by D10 for degradation are those involved in oxidative phosphorylation, which was reported by Ly et al. during the revision of this manuscript . As mRNAThere are several possible yet nonexclusive mechanisms by which mitochondrial colocalization is required for the optimal effect of D10 to shut off gene expression . First, How D10’s mitochondrial association facilitates mRNA translation, especially cap-independent translation enhancement, is thought-provoking but needs extensive investigation of D10’s molecular functions in the presence and absence of VACV infection. As inactivation of its decapping activity also renders it to lose its ability to promote translation , the mitIn addition to being required for optimal gene expression shutoff and translation promotion, D10's mitochondrial colocalization may provide additional mechanisms to promote VACV replication in VACV-infected cells. Notably, it provides a spatial mechanism for D10 to more readily decap cellular mRNAs . Viral f54–D10 mitochondria association may affect VACV replication in addition to regulating protein synthesis in VACV infection. While in A549 control cells, VACV protein synthesis rate and level decreased ; a lineaIn summary, our study identified a spatial mechanism for a poxvirus-encoded decapping enzyme to regulate mRNA metabolism and translation, resulting in an important role in viral replication. This study also provides a new strategy a decapping enzyme uses to regulate its function by riding mitochondria. As decapping enzymes are a group of diverse proteins with important physiological functions, it is of great interest to further dissect the molecular mechanisms.2.A549 control cells and A549DKO cells (kind gifts from Bernard Moss) , human fVACV Western Reserve (WR) strain ATCC VR-1354) is used in this study. Other recombinant VACVs used in this study were derived from the VACV WR strain. vD9muD10mu, vD10mu, vΔD10, and vΔD9 were kindly provided by Bernard Moss and described elsewhere , 32. vD14 is usedVACV and its derived recombinant viruses were grown in HeLa or A549DKO cells and purified on a 36% sucrose cushion. The viruses (except for vD9muD10mu) were titrated using a plaque assay as described elsewhere . The vD9Virus infection was carried out with DMEM or EMEM containing 2.5% FBS. Virus was sonicated and diluted according to the desired MOI. Medium containing viruses was added to the cultured cells and incubated at 37°C for 1 h and replaced with fresh medium. For plaque assay, virus-containing samples were 10-fold serial diluted and added on top of BS-C-1 cells in 12-well plates. After 1 h of incubation at 37°C, the medium was replaced with fresh medium containing 0.5% methylcellulose (Fisher Scientific). Plaques were visualized by staining the infected cells in 12-well plates with 20% ethanol containing 0.1% crystal violet for 5 min.To compare plaque sizes, the diameters of 25 representative plaques of each virus were picked and measured with ImageJ software.Mouse α-Flag monoclonal antibody (used for Western blotting) was purchased from Sigma-Aldrich . Rabbit α-Flag polyclonal antibody  was purchased from Thermo Fisher Scientific . Mouse α-Tom20 antibody  and mouse α-GAPDH  antibody  were purchased from Santa Cruz Biotechnology. Rabbit α-VACV and rabbit α-A7 antibodies were kindly provided by Bernard Moss . Mouse αPlasmids encoding D10 mutants are illustrated in A puromycin labeling-based method to detect newly synthesized proteins was used as previously described . BrieflyWestern blotting was performed as described previously . BrieflyMock, VACV-infected, or plasmid-transfected cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature. The cell membrane was penetrated with 1× phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 10 min following being blocked with 1× PBS containing 2% BSA for 1 h. Primary antibodies were diluted in PBS (with 2% BSA) and incubated with cells for 1 h at room temperature. After three times of washing with 1× PBS, cells were incubated with secondary Alexa Fluor (488 nm for green and 594 nm for red)-conjugated IgG diluted in 1× PBS (with 2% BSA) at room temperature for 1 h. After three times of washing with 1× PBS, cells were stained with DAPI for 5 min and washed with 1× PBS two more times. Coverslips were mounted using 40% glycerol. Zeiss 880 or Zeiss 700 confocal microscopy was used to visualize the cells.in vitro was carried out as previously described using HiScribe T7 Quick high-yield RNA synthesis kit  . The RNAs were purified using a PureLink RNA minikit  and transfected into cells using Lipofectamine 2000  according to the manufacturer's instructions. Six hours posttransfection, cell lysates were collected, and luciferase activities were measured using a dual-luciferase reporter assay system  and GloMax Navigator microplate luminometer with dual injectors (Promega) as per manufacturer protocol.Synthesis of RNA . E2050) , 42, 43.Gaussia luciferase activities were measured using a luminometer using the Pierce Gaussia luciferase flash assay kit .The t test was performed to evaluate statistical differences from at least three replicates. We used the following convention for symbols to indicate statistical significance: ns, P > 0.05; *, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; ***, 0.0001 < P ≤ 0.001; and ****, P ≤ 0.0001.The Student's


More attractively, a NSUC-0.4-assembled symmetrical supercapacitor delivers an energy output of 7.4 Wh kg−1 at 100 W kg−1 in 6 M KOH as well as a capacity retention of 92.4% after 10,000 cycles, indicating its practical application prospect. Our findings open up new prospects for the design and electrochemical application of N, S-doped ultramicroporous carbons.N, S-doped ultramicroporous carbons (NSUC-x) with a high nitrogen/sulfur content and a narrow pore-size distribution of around 0.55 nm were firstly prepared using L-cysteine as a nitrogen and sulfur source. The phase, graphitization degree, morphology, specific surface area, pore structure and surface condition of NSUC-x are investigated to analyze the key role in electrochemical performance. Such an ultramicroporous structure and N, S doping not merely provide a high-specific surface area and a suitable pore size, but also induce a good wettability for the fast transport and adsorption of electrolyte ions. Due to the above strategies, the typical NSUC-0.4 exhibits a high gravimetric capacitance of 339 F g With the fast economic development and the excessive consumption of fossil fuels, the energy dilemma and environmental pollution have made it essential to develop renewable and clean energy devices for a sustainable society ,2,3. As + (3.3 Å), Na+ (3.6 Å), OH− (3.0 Å) and SO42− (3.8 Å), the >0.5 nm micropores are usually considered to be electrochemically available in aqueous electrolytes [−1 at 1 mV s−1 in 6 M KOH [3, H2S, melamine, etc.) [−1 at 0.5 A g−1 [−1 at 0.5 A g−1 in a 6 M KOH aqueous electrolyte [Carbon materials, including activated carbon, carbon nanotubes and carbon aerogels, are mainly applied as EDLC electrodes due to their high specific surface area and designable pore structure ,11,12,13trolytes ,17,18. B 6 M KOH . They pr 6 M KOH ,21,22. Ie, etc.) ,24,25. Ue, etc.) . For exa.5 A g−1 . Via thectrolyte . −1 at a current density of 0.5 A g−1 (three-electrode system), as well as 7.4 Wh kg−1 at 100 W kg−1 (two-electrode system) in 6 M KOH with excellent cycling stability. These findings highlight that carbon materials with ultramicroporous structure and heteroatoms doping can be widely applied as high-performance supercapacitor electrodes.In this paper, an N, S co-doped ultramicroporous carbon is synthesized by using L-cysteine as a nitrogen and sulfur source and exhibits a high specific capacitance of 339 F g2 with a heating rate of 1 °C min−1. The resultant carbons were named nitrogen and sulfur co-doped ultramicroporous carbons (NSUC-x), in which x represents the mass of L-cysteine (g).All chemicals were used as received without any purification. In a typical synthesis, 0.126 g of phloroglucinol, 0.1 g of terephthalaldehyde, 0.2 mL ammonia solution and appropriate L-cysteine (0.3–0.5 g) were added to a 56 mL ethanol solution (28.6 vol.%) with stirring at 70 °C for 24 h. Subsequently, the mixed solution was transferred into a Teflon-lined autoclave and placed in an oven at 100 °C for 24 h to obtain the polymerized precursor. After washing in distilled water and alcohol, the polymer was dried in an oven at 60 °C for 48 h and carbonized at 600 °C for 4 h under pure NX-ray diffraction (XRD) patterns for characterizing the phase constitutions were tested on Bruker D8 Advance diffractometer (Germany) with the 2θ range of 10–80°. Raman spectra were recorded on a Renishaw Invia-spectrometer (UK) using a laser beam of 532.3 nm as the excitation source. After degassing at 200 °C for 2 h, the nitrogen sorption of all samples was measured at −196 °C by Micromeritics ASAP 2460 . The Brunauer–Emmett–Teller (BET) model was applied to calculate the specific surface area, and the Density Functional Theory (DFT) model was used to estimate the pore parameters . SurfaceC, F g−1) were calculated according to the Formulas (1) and (2), respectively:A CHI660D electrochemical workstation  was used to test all the electrochemical measurements. In a typical three-electrode system conducted in 6 M KOH electrolyte, Hg/HgO electrode and a Pt foil electrode were used as reference and counter electrodes, respectively. The working electrodes were fabricated by mixing 80 wt.% carbon materials, 10 wt.% polytetrafluoroethylene (PTFE) and 10 wt.% graphite, pressing on nickel foam and dried at 60 °C for 24 h. A voltage range from −1 to 0 V was applied in cyclic voltammetry (CV) and galvanostatic charge–discharge (GCD) tests, whose specific capacitance , the energy densities  and power densities  of the device were obtained according to Formulas (3)–(5):Moreover, a coin cell (NEWARE CR2032) was assembled with two identical NSUC-0.4 electrodes, 6 M KOH electrolyte, a piece of filter paper separator to analyze the two-electrode symmetric system. The capacitance of a single electrode (m (g) is the mass of all active materials, ΔV (V) is the operation potential window, Δt (s) is the discharging current time, r (V s−1) is the scan rate and I (A) is the response current (Formula (1)) and the discharging current (Formulas (2) and (3)).In Formulas (1)–(5), x in the range from 10 to 80° exhibit two broad diffraction peaks around 23 and 44°, which are ascribed to the (002) and (001) lattice facets of amorphous carbon [−1 correspond to I band (caused by the impurities near carbon atoms), D band (derived from the defects/disorders in sp3 structure), D′ band (ascribed to the defects from stacked graphene layers) and G band (attributed to the vibration of graphitic sp2-type carbon), respectively [D/IG(I) reflects the indicator of concrete graphitization degree. Apparently, the relative D/IGI in NSUC-0.4 is the lowest value (0.81) among all samples and implies the highest graphitization degree, which has a positive feedback on electronic conductivity. In s carbon . The gras carbon ,32. Suchectively ,34. The 2 physisorption isotherms of NSUC-x presented in P/0P < 0.1) and the flat plateau (0.1 ≤ P/0P ≤ 1.0) are ascribed to abundant micropores. Moreover, the pore size distribution curves of NSUC-x depicted in 2 g−1) is achieved by NSUC-0.4, which also has the largest microporous specific surface area and total pore volume. The addition of L-cysteine has an important influence on the specific surface area of NSUC-x. When L-cysteine is not sufficient in the reaction, phloroglucinol cannot react with L-cysteine thoroughly, resulting in a decrement in the crosslinking density between phloroglucinol and L-cysteine. The region with a low crosslinking density pyrolyzes into gaseous products and leads to the collapse of the carbon matrix, resulting in the lower specific surface area in NSUC-0.3. On the otherwise, increasing the mass of L-cysteine beyond the optimum value increases the contents of N and S in the carbon samples. Due to the lower covalent band energy of C–N compared with C–C, the release of abundant nitrogen and sulfur atoms during the carbonization results in the partial collapse of the carbon framework, thus leading to the decrement in the specific surface area in NSUC-0.5. The Ns, O 1s, N 1s and S 2p, indicating the presence of four elements: C, N, S and O [s state involves four overlapping peaks of pyridine nitrogen oxide (N-X), graphitic nitrogen (N-Q), pyrrolic N (N-5) and pyridinic nitrogen (N-6). Usually, the N-5 with good electro–donor characteristics and high charge mobility is considered to enhance carbon catalytic activity in electron-transfer reactions and thus effectively improves electrochemical performance [p state can be identified as three resolved peaks at 163.5 eV, 164.7 eV and 167.4 eV, being ascribed to S 2p3/2, S 2p1/2 and oxidized sulfur moiety (−SOn−). The two prominent peaks correspond to the S 2p3/2 and S 2p1/2 of the C–S–C covalent bond of the thiophene-S caused by spin-orbit coupling [The surface chemical conditions of NSUC-x were investigated by XPS techniques. All spectra presented in  S and O . As tabuformance . Meanwhiformance . The N−Qformance . The sigcoupling . N, S coAll morphologies of NSUC-x observed in SEM images a–c show −1, indicating the presence of pseudocapacitance induced by N, S co-doping. The capacitive contribution of NSUC-0.4  of the NSUC-0.4 electrode integrated from GCD is dramatically higher than those of NSUC-0.3 (218 F g−1) and NSUC-0.5 (189 F g−1). In −1 at 0.5 A g−1 as well as remains 189 F g−1 with a triangle shape up to 10 A g−1, signaling a high-rate charge–discharge capability. The charging–discharging kinetics of NSUC-0.4 were also discussed according to the formula TC, EC and t are the total capacitance, EDLC and discharging time, respectively [EC of NSUC-0.4 is 155 F g−1 and close to the value (133 F g−1) fitted by CV curves. Thereby, the ultramicropores and N, S doping, which mainly contributed to the slow-kinetic capacitance, are crucial to the enhancement of the electrochemical energy storage. The 10,000 consecutive charge–discharge cycles in −1, indicating an excellent cycling stability. The electrochemical performance of NSUC-0.4 is compared with that of other carbon materials in The GCD profiles of NSUC-x at 1.0 A gectively ,44. As s5 Hz are shown in s, 1.24 Ω) and the charge-transfer resistance  with the smallest quasi-semicircle in the high-frequency region. Abundant micropores, especially ultramicropores, can benefit the fast transportation and diffusion of electrolyte ions to enhance the electrochemical performance. At the same time, the existence of nitrogen and sulfur elements can improve the surface properties and electric conductivity. Meanwhile, the relaxation time (τ) and the diffusive resistance (σ) of NSUC-0.4 obtained from −1/2, respectively, which were lower than those of NSUC-0.3 and NSUC-0.5 [Nyquist plots of NSUC-x electrodes measured in 6 M KOH aqueous solution in a frequency range of 0.01 to 10NSUC-0.5 . These E−1 exhibit the rectangular shapes, implying the rapid and reversible charge–discharge procedure. Moreover, GCD curves detected with 0.2 to 10 A g−1 (−1 at 100 W kg−1 with excellent long cycle life (retention of 92.4%) and coulombic efficiency (98.5%) after 10,000 cycles (s (0.64 Ω) and Rct (0.66 Ω) of the 10,000th cycle are slightly larger than those of the 1st cycle (Rs = 0.52 Ω and Rct = 0.42 Ω), meaning a low electrochemical resistance in the KOH-loaded device with a good stability.10 A g−1 b show sy0 cycles e. Nyquis2 g−1) to be effectively employed in electrochemical processes. Thereby, NSUC-0.4 exhibits an excellent specific capacitance of 339 F g−1 at 0.5 A g−1. The assembled symmetrical supercapacitor based on the NSUC-0.4 electrode delivers an energy output of 7.4 Wh kg−1 at 100 W kg−1 in 6 M KOH with a long cycle life. Our results firmly show a new avenue that the heteroatoms-doped ultramicroporous carbons provide for advanced energy storage devices with a high electrochemical performance. N, S co-doped ultramicroporous carbons were fabricated by applying the L-cysteine as a nitrogen and sulfur source. The high nitrogen (7.35 at.%)/sulfur (2.37 at.%) content as well as the ultramicroporous structure enhance the electrochemical performance of NSUC-0.4 in the following aspects: (i) heteroatom doping provides additional pseudocapacitance and superior hydrophilic surface, and (ii) the superior hydrophilic surface and high graphitization degree make the high specific surface area (891 m