CYP2D6 genotype to phenotype. Changes affect phenotype grouping, as well as the value used to calculate activity score for the CYP2D6*10 allele to better reflect the substantially decreased activity of this allele which is the most frequent allele found in Asian populations. This study aimed to evaluate whether the lower value for CYP2D6*10 as recommended, and the revised phenotype groupings improve the relationship between CYP2D6 genotype and risperidone measures. One hundred and ninety-nine children and adolescents with autism treated with a risperidone-based regimen for at least four weeks were included. CYP2D6 genotype was determined using the Luminex xTAG CYP2D6 Kit assay and translated into phenotype using different translation methods. Plasma concentrations of risperidone and 9-hydroxyrisperidone were measured using LC/MS/MS. Plasma levels of risperidone, risperidone concentration/dose ratio, and risperidone/9-hydroxyrisperidone ratio in patients with an activity score < 1 were significantly higher than those ≥ 1 . Plasma risperidone levels and risperidone concentration/dose ratios were significantly higher in intermediate metabolizers (defined as AS = 0.25–0.75) than normal metabolizer (defined as AS = 1–2) patients  as well as risperidone/9-hydroxyrisperidone ratio . This is the first study in an Asian population utilizing the revised CPIC-recommended method for translating the CYP2D6 genotype to phenotype. In addition to validating that CYP2D6 genetic variation significantly impacts risperidone metabolism, we demonstrated that revised value for the CYP2D6*10 was superior for genotype to phenotype translation. However, at least for risperidone, subjects with an activity score of 1 presented as phenotypic normal, and not intermediate metabolizers, suggesting that phenotype classification is substrate dependent.Recently, the Clinical Pharmacogenetics Implementation Consortium (CPIC) have revised recommendations for the translation of  CYP2D6 catalyzes the hepatic metabolism of a large number of clinically important medications, including codeine, amitriptyline, fluvoxamine, risperidone, fluoxetine, aripiprazole, paroxetine, and dextromethorphan3. The CYP2D6 gene is highly polymorphic. To date, over 130 allelic variants have been designated by the Pharmacogene Variation Consortium (PharmVar)5.Cytochrome P450 (P450) 2D6 is a major drug-metabolizing enzyme expressed in the liverCYP2D6 allele frequencies vary substantially among different ethnic and ancestral populations9. The decreased function CYP2D6*10 allele  is the most common allele in East Asian populations, including Thai, Chinese, Taiwanese, Korean, Vietnamese, and Filipino16. This allele is also observed in other populations, including Europeans, Africans, and their descendants, its frequency, however, considerably lower8. Conversely, the nonfunctional CYP2D6*4 allele is more frequent in European populations but is rarely observed in Asian populations8.CYP2D6 genetic variation leads to a wide range of metabolic capacity ranging from no to increased activity. Based on their genotype, individuals are grouped into four phenotype groups, i.e., poor metabolizers (PMs), intermediate metabolizers (IMs), normal metabolizers (NMs), and ultrarapid metabolizers (UMs)17. The activity score system (AS) has been broadly accepted to translate the CYP2D6 genotype into phenotype and the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG) for their respective guidelines19. Briefly, each allele is assigned a value of 0, 0.5 or 1 reflecting no function, decreased or normal function, and the sum of the values provides the AS of a genotype. The previous CPIC translation method classified AS = 0 as PM, AS = 0.5 as IM, AS = 1 to 2 as NM, and > 2 as UM. In an effort to harmonize genotype to phenotype translation, a CPIC-led working group has recently published a revised method and recommends using this new method to translate genotype to phenotype19. One major change was downgrading the value used for activity score calculation of the decreased function CYP2D6*10 allele from 0.5 to 0.25 to more accurately reflect the dramatically decreased function of this allele. Furthermore, an AS of 1 is no longer categorized as NM, but as IM. While the new system has recently been applied to an in vitro study comprising mostly Caucasian liver tissue samples20, there are no investigations to date assessing the performance of the new method on any Asian populations with high frequencies of CYP2D6*10. There is also a paucity of information regarding the impact of substrate specificity on performance of the new translation method.CYP2D6 genotype on plasma concentrations of RIS is well-established25, RIS is a well-suited drug to evaluate whether the new translation method is superior over the previous method.The use of a standardized method to infer phenotype from genotype is essential for test reporting and clinical implementation to prevent confusion and inconsistencies. We applied the new CPIC-recommended method to data obtained from risperidone (RIS)-treated Thai children and adolescents diagnosed with autism spectrum disorders (ASDs) and treated with RIS. Since the impact of CYP2D6*10 indeed improves the relationship between AS and RIS plasma drug levels and to assess whether phenotype groupings, as recommended by CPIC, are appropriate for RIS.The aims of this investigation were to demonstrate whether the revised value for One hundred and ninety-nine participants with ASD, aged 3–18 years, and diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) criteria in the Yuwaprasart Waithayopathum Child Psychiatric Hospital, Samut Prakan, Thailand, were recruited during 2017–2018. All patients were treated with a RIS-based regimen for at least four weeks before blood sample collection. Socio-demographic data were collected by a questionnaire including gender, age at assessment, daily RIS dosage, duration of RIS treatment, and concomitant medication. Patients were excluded if they were receiving concomitant treatments that could potentially affect RIS metabolism. This study was approved by the Ethics Review Committee on Human Research of the Faculty of Medicine Ramathibodi Hospital, Mahidol University, Thailand (MURA2017/556) and conducted in accordance with the Declaration of Helsinki. The study protocol was clearly explained to all participants and/or their legal guardians, and informed consent was given before the study.CYP2D6 based on allele-specific primer extension (ASPE) and hybridization to oligonucleotide bound microspheres26 using the Luminex xTAG CYP2D6 Kit v3  according to the manufacturer's instructions27. The assay interrogates 21 variants including 19 CYP2D6 single nucleotide polymorphisms (SNPs): − 1584C > G, 31G > A, 100C > T, 124G > A, 137_138insT, 882G > C, 1022C > T, 1660G > A, 1662G > C, 1708delT, 1759G > T, 1847G > A, 2550delA, 2616delAAG, 2851C > T, 2936A > C, 2989G > A, 3184G > A, and 4181G > C, as well as gene deletion and duplication)25. The allelic variants called by this array are CYP2D6*1 , *2, *35 , *9, *10, *17, *29 and *41 (decreased function), and *3, * 4, *5, *6, *7, *8, *11 and *15 (no function), as well as the presence of duplications. Patients who were carriers of a CYP2D6 duplication were excluded, because this array did not further characterize gene duplications . For instance, a duplication observed in an individual genotyped as CYP2D6*1/*10 could result in e.g. a CYP2D6*1xN/*10, CYP2D6*1/*10xN or a *1/*36 + *10 genotype call.Genomic DNA was extracted from EDTA blood with the MagNa Pure automated extraction system according to the manufacturer's instructions. A bead array platform genotyped *4, *5) = 0; the decreased function allele *10 = 0.25; other decreased function alleles  = 0.5, and normal function alleles  = 1. The AS of each diplotype is the sum of the assigned value to each allele. Individuals with an AS of 0 were categorized as PMs, those with an AS of 0.25, 0.5 or 0.75 were categorized as IMs, and those with an AS of 1.25, 1.5, 1.75, or 2 were grouped as NMs. To compare translation methods, those with an AS of 1 were either categorized as IM (new CPIC method), or NM (previous CPIC method).To calculate the AS, values were assigned to the alleles identified in the study cohort as follows: no function alleles , which was connected to an AB Sciex API 3200  instrument. Chromatographic separation was achieved on the C18 column . Integration of peak areas and determination of the concentrations was performed with the Analyst 1.5.2 software . Quadratic regression with 1/ × weighted concentrations was used. The mean inter- and intra-assay accuracy for both RIS and 9-OH-RIS was set within ± 15.0% Relative Error of nominal, and precision < 15.0% Relative Standard Deviation.Trough plasma concentration of RIS and its 9-OH-RIS metabolite were quantified, between 8:00 and 10:00 AM, approximately 12 h after the bedtime dose, using a validated, previously published high-performance liquid chromatography procedureP < 0.05 for a two-tailed distribution.Descriptive statistics were used to describe the clinical characteristics of the subjects. Data were expressed as mean  or median  in normal or non-normal distribution data, respectively. The nonparametric Kruskal–Wallis (comparisons more than two groups) and Mann–Whitney U tests (comparisons between two groups) were used to assess the association between plasma drug levels and the studied genotypes or predicted phenotypes at each time point. Statistical analyses were carried out using SPSS v24  for Windows. Statistical significance is reported as Our sample consisted of 199 children and adolescents with a mean age of 9.25  years who had been diagnosed with autism spectrum disorders. Demographic data are presented in Table CYP2D6*10 decreased function allele was the most common allele identified among the 199 subjects at 51.8%. The frequencies of the normal function alleles CYP2D6*1 and CYP2D6*2 were 25.1% and 6.3%, respectively. Another decreased function allele, CYP2D6*41, was observed at 6.8%. CYP2D6*4 and CYP2D6*5, both nonfunctional alleles, were found at frequencies of 1.3% and 8.3%, respectively. We also observed two subjects with the rare CYP2D6*14 allele (0.50%) in this study cohort. CYP2D6 allele frequencies are presented in Table The CYP2D6 genotypes identified, CYP2D6*1/*10 was the most frequent (29.6%), followed by CYP2D6*10/*10, CYP2D6*5/*10, and CYP2D6*10/*41 .Genotype frequencies are summarized in Supplementary Table CYP2D6*1/*10 and CYP2D6*2/*10 genotypes. There were significant differences in RIS, the metabolic ratio RIS/9-OH-RIS, and C/D of RIS plasma concentrations between AS of 0.25, 0.5, 0.75, and 1, 1.25, 1.5, 2. There was a significant difference between patients when divided into two groups, one with AS < 1 and the other with AS ≥ 1. Plasma levels of RIS and RIS/9-OH-RIS ratio, and plasma C/D of RIS in patients with AS < 1 were significantly higher than those in patients with AS ≥ 1   were NMs, followed by IMs . There was only one patient with a predicted PM phenotype of 0.5%.P < 0.001) and RIS/9-OH-RIS ratio (P < 0.001) when subjects were categorized as described above (Table P < 0.001) and lower when compared to that found in the PM individual (2.67 ng/ml). The RIS/9-OH-RIS ratio in IM subjects was statistically significantly higher than the ratio observed in the NMs . These patients also had a significantly higher C/D of RIS than NMs .There were statistically significant differences for the plasma RIS concentration P < 0.00 and RIS/CYP2D6 genotype to phenotype in an Asian population. This new method is anticipated to have a considerable impact on Asians compared to other populations due to the high frequency of the CYP2D6*10 allele. This allele conveys a considerable decrease in function and thus was downgraded, i.e., now receives a lower value for AS calculation, to improve the accuracy of phenotype prediction. The CPIC recommendations are drug-agnostic, i.e., the phenotype does not take substrate-specificity into account. Thus, in addition to evaluating whether the revised value for CYP2D6*10 improves the relationship between RIS, RIS/9-OH-RIS ratio, and C/D of RIS, we also assessed whether phenotype groupings, as recommended by CPIC, are appropriate for RIS.To the best of our knowledge, this is the first study applying the revised CPIC recommendations for the translation of CYP2D6*10 AS calculation did improve the relationship between AS and RIS, RIS/9-OH-RIS ratio, and C/D of RIS. Similar findings were observed by Brown et al. who showed that systemic exposure of atomoxetine (AUC0-∞) of AS of 1 was not significantly different from that observed for subjects with an AS of 1.5 or 229. In addition, Frederiksen et al30, demonstrated allele-specific metabolism of vortioxetine suggesting substantial differences among decreased function allele. Taken together, these findings raise awareness of the limitations and pitfalls of drug-agnostic genotype to phenotype translation methods. This is further substantiated by the plasma concentrations of RIS and RIS/9-OH-RIS ratios being significantly higher in AS of 0.25–0.75 than AS of 1–2 arguing that the former should be classified as IMs and the latter as NMs. Therefore, to predict CYP2D6 phenotype for RIS treatment, genotype should be translated into phenotype as shown in Table Owing to the revised AS definition, a notable number of subjects would be reclassified as IMs Fig. . SpecifiCYP2D6 genotype (or AS) had a substantial impact on the trough dose-corrected plasma concentration of RIS. In accordance with results we previously reported for a different cohort, there were statistically significant differences in the plasma concentration for RIS (P < 0.001) and the RIS/9-OH-RIS ratio (P < 0.001) among phenotype groups in Thai autism children31. Furthermore, PM patients had significantly higher RIS C/D than those genotyped as CYP2D6*1/*132. The same pattern was also observed in another study33, i.e., the C/D ratio for RIS was significantly different in CYP2D6 PMs. The presence of the CYP2D6*10 allele was also associated with significantly higher levels of C/D of RIS levels at week 12 (P = 0.003) in North Indian patients with schizophrenia34. Moreover, plasma RIS/9-OH-RIS ratios were significantly higher in patients with an AS of 0.5 compared to those with an AS of 2 in an independent cohort of Thai subjects24. Taken together, the RIS/9-OH-RIS metabolic ratio is a biomarker for CYP2D6 activity, which may be useful to guide the treatment of patients in need of psychotropic drugs35.Additionally, the 32. Similarly, the total active moiety, sum of the plasma concentrations of RIS and 9-OH-RIS, corrected for the dose, did not significantly differ between individuals of different genotypes. These findings are consistent with a previous study in another Thai cohort of ASD patients31 that showed no significant differences in 9-OH-RIS and active moiety concentrations. This finding is consistent with a previous study using positron emission tomography scans of healthy volunteers after receiving a single oral dose of RIS showing that plasma concentrations of the sum of RIS and 9-OH-RIS partly overlapped between the NMs and PMs36. Therefore, the plasma concentrations of the 9-OH-RIS and total active moiety are independent of the CYP2D6-related metabolism. It has been suggested that the efflux transporter ABCB1, as well as CYP3A5 can contribute to the steady-state plasma concentration of RIS, 9-OH-RIS, and active moiety38.There were no significant differences in 9-OH-RIS and total active moiety concentrations among the CYP2D6 predicted phenotype groups, as found in an earlier studyAs mentioned above, the CPIC-recommended drug-agnostic method to predict phenotype may not accurately predict phenotype across all drugs and all allelic variants. Regardless of the imperfections and shortcomings of the method, using a standardized system, although imperfect, is preferable because it makes comparisons of results among studies easier. However, it also demonstrates the need to develop more sophisticated algorithms that take substrate specificity, among other patient-specific information, into account.We acknowledge the following limitations of the Luminex platform. This test does not quantitatively determine copy number nor does it determine which allele is duplicated or identify any other structural variants. Furthermore, only the most common alleles are tested. We speculate that some subjects may have rare or novel alleles which may explain some of the outliers shown in Fig. Supplementary Information 1.


N-acetyl-5-methoxytryptamine) is a pleiotropic signaling molecule that plays a crucial role in the regulation of various environmental stresses, including heat stress (HS). In this study, a 100 μM melatonin (MT) pretreatment followed by exposure to heat stress for different time periods was found to efficiently reduce oxidative stress by preventing the over-accumulation of hydrogen peroxide (H2O2), lowering the lipid peroxidation content  content), and increasing proline (Pro) biosynthesis. Moreover, the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were increased substantially in MT-pretreated wheat seedlings. The presence of MT significantly improved the heat tolerance of wheat seedlings by modulating their antioxidant defense system, activating the ascorbate–glutathione (AsA–GSH) cycle comprising ascorbate peroxidase (APX), and increasing glutathione reductase (GR) activities. It also held the photosynthetic machinery stable by increasing the chlorophyll content. Enhancement in the endogenous MT contents was also observed in the MT+HS-treated plants. Furthermore, the expression of reactive oxygen species (ROS)-related genes TaSOD, TaPOD, and TaCAT, and anti-stress responsive genes, such as TaMYB80, TaWRKY26, and TaWRKY39, was also induced in MT-treated seedlings. Due to these notable changes, an improvement in stress resistance was observed in MT-treated seedlings compared with control. Taken together, our findings suggest that MT can play a key role in boosting the stress tolerance of plants by modulating the antioxidant defense system and regulating the transcription of stress-responsive genes.Melatonin ( Triticum aestivum L.) is an important cereal crop whose total global production was 881 million tons in 2016 [2O2), single oxygen (1O2), and superoxide radicals (O2−) [Wheat  , which lls (O2−) ,6. As a ls (O2−) . This anls (O2−) ,8,9. Morls (O2−) ,13,14,15ls (O2−) ,16 in wh2O2, O2−) is considered to be fundamental to enhance stress tolerance of plants [Melatonin (MT) recently attracted the attention of researchers due to its role in protection against numerous environmental stresses. Several in recent reviews in this respect are very interesting ,22,23,24f plants ,27. In af plants ,36,37,38f plants ,40,41,42f plants , kiwifruf plants , onion, f plants , maize [f plants ,47, and f plants , a proteThe current study in wheat seedlings aimed to elucidate (1) the role of MT in the regulation of the expression of stress-specific genes, (2) the influence of MT on chlorophyll content for the purpose of predicting photosynthetic efficiency, and (3) the role of MT in modulation of the antioxidant defense machinery for the purpose of understanding systemic stress-induced injuries.The fresh weight (FW) and dry weight (DW) of shoots and roots from the seedlings significantly decreased under HS (42 °C) . ConversThe lipid peroxidation in plant membranes is usually determined by measuring the MDA content. The high-temperature treatment damaged the cellular membranes in the leaves of wheat seedlings after exposure to HS for up to 6 h. Exogenous application of MT (100 μM) was more effective at overcoming the severe impacts of HS-induced damage. A substantial decline in MDA content in MT + HS and MT leaves at 2 and 6 h was observed . An almo2O2 not only under normal conditions but also under stressful conditions. To determine whether MT alleviates heat-induced oxidative stress, we evaluated the trends of ROS accumulation in MT-pretreated leaves under HS at different time points by detecting H2O2 production  content in different groups at different intervals is summarized in Plants produce Hoduction A. At theThe role of MT in the suppression of oxidative stress was investigated, and the activities of antioxidant enzymes at 0, 2, and 6 h were recorded . The MT A remarkable increase in the GR activity was observed at 2 h, where it reached a maximum of 14.6 µmol/g FW. However, a decrease in the GR activity was observed at 6 h, where it reached a minimum of 10 µmol/g FW in the MT and MT + HS-treated leaves .−1 FW, respectively. Similarly, in root, the endogenous MT content respectively reached to its maximum of 71 and 87 ng·g−1 FW in HS and MT + HS-treated seedlings . However, a remarkable shift was observed at 6 h, where the chlorophyll a content reached its peak value of 600 µmol/g FW under MT and MT + HS , jasmonate (JA) [Arabidopsis thaliana is reported to have 204 MYBs, whereas 218 and 244 MYBs were identified in rice (Oryza sativa) [Glycine max) [TaMYB80 gene expression level in MT + HS-treated plants after an extended period of exposure to HS at 6 h  , and ethate (JA) , was preate (JA) ,38. Toge sativa)  and soybine max) , respectS at 6 h E, as alsTriticum aestivum L.) plants. The wheat seeds were pretreated with 1% sodium hypochlorite for 10 min. Then, seeds were washed with distilled water 3–5 times, placed on petri plates with filter paper, and allowed to germinate for three days. For hydroponic culture, wheat seedlings were grown for seven days in 1/4 Hoagland solutions. The seedlings were divided into two sub-groups for challenge under different treatments. In the first sub-group, we sprayed 80 mL of 100 μM MT on each of the wheat seedling leaves each day and for seven days. In the second sub-group, the same volume of water was sprayed on each of the wheat seedling leaves. After one week, half of the water-treated seedlings and half of the MT-treated seedlings were removed and exposed to high temperature (42 °C). This protocol resulted in four groups of plants: (1) a control group (CK); (2) an MT (100 μM) treatment group; (3) an HS (42 °C) treatment group; (4) an HS (42 °C) and MT (100 μM) treatment group. The MT concentration was selected based on a previous experiment [2O (0.01% v/v). Tween-20 was used as a surfactant. All experiments were conducted in a growth chamber at Northwest Agriculture and Forestry University, where environmental conditions were maintained at 25/20 °C (day/night) with a relative humidity of 50% ± 5%, a light intensity of 450 µmoL·m−2·s−1, and a 12/12-h (light/dark) photoperiod. There were at least three biological replicates per treatment.We used Shannong 33  group were moved to growth cabinet  with settings as above but exposed to 42 °C to stress the plants, over 2 h, and then sustained at 42 °C to the 6-h time point. All the control samples were collected at 0 h, which was also used as the starting point before extending the stress periods to 2 and 6 h, respectively . All colTo assess combined effect of MT and HS in wheat seedlings, we measured different growth indicators such as fresh and dry weight of shoots and roots. For dry weight determination, plants were oven-dried at 65 °C for overnight.w/v) trichloroacetic acid solution (TCA) and centrifuged at 10,000× g for 10 min at 4 °C. Then, 2 mL of supernatant and 0.67% (w/v) thiobarbituric acid (TBA) was added, and the solution was sealed, mixed, and heated for 10 min in a bath of boiling water. The absorbance of the supernatant was measured at 450, 532, and 600 nm following the bath and then cooling. The H2O2 concentration was determined based on the method described in Reference [g. Then, 1 mL of supernatant was added to 0.1 mL of 5% (w/v) titanium sulfate and 0.2 mL of concentrated ammonia, and the solution was mixed. Then, the mixture was centrifuged for 10 min at 4 °C. Subsequently, the precipitate was removed and washed 3–5 times with acetone until the plant’s pigment was removed. Finally, the precipitate was dissolved in 5 mL of 2 M concentrated sulfuric acid, and the volume of distilled water was fixed to 10 mL. The absorbance was measured at 415 nm. The H2O2 content was based on a standard curve generated with known H2O2 concentrations. The chlorophyll concentration was measured as described by [The thiobarbituric acid method was used to measure the MDA content . Brieflyeference . Brieflyribed by . The chlribed by . Brieflyribed by ; the quaribed by .w/v) polyvinylpyrrolidone (PVP), 2 mM dithiothreitol (DTT), and 0.1 mM ethylene diaminetetraacetic acid (EDTA). The homogenates were then centrifuged for 10 min at 4 °C and 10,000× g, and the supernatant was saved for further analysis of enzyme activity. The POD activity was measured by using guaiacol as a substrate. The reaction mixture contained 50 mM PBS (pH 5.5), 30% (v/v) H2O2, guaiacol, and the enzyme extract. The variation in absorbance at 470 nm was recorded and the result was expressed as µmol/g (a decrease in absorbance of 0.01 per min is 1 U at 470 nm). The SOD activity was measured based on the photochemical reduction of nitro blue tetrazolium (NBT) and monitoring the absorbance at 560 nm. The result was expressed as µmol/g (the absorbance of 1 g of leaf sample in the reaction mixture at 470 nm is reduced by 0.01 per minute in 1 U). The CAT activity was measured by monitoring the decrease at 240 nm and the result was expressed as µmol/g (the absorbance of 1 g of leaf sample in the reaction mixture at 470 nm is reduced by 0.01 per minute in 1 U). The reaction mixture consisted of 100 mM H2O2, 200 mM PBS (pH 7.8), and the enzyme extract.The CAT, POD, and SOD activities were measured by following the method of . Brieflyw/v) polyvinylpolypyrrolidone, then homogenized with 8 mL of 50 mM phosphate buffer (pH 7.5) containing 1 mM EDTA–Na2 and 0.3% Triton X-100. The reduction in absorbance at 290 nm was measured as H2O2 was oxidized. The GR activity was assayed by using the method described in [−1·cm−1) in a microtiter plate reader using equipment.The APX activity was measured by following the method of . Brieflyribed in . BrieflyTriticum aestivum L.) leaves was used to extract total RNA by using a Total RNA kit  following the manufacturer’s instructions. For the complementary DNA (cDNA) synthesis, 2 mg of total RNA was reverse-transcribed using a PrimeScript TM II First-Strand cDNA synthesis Kit . The quantitative real-time PCR (qRT-PCR) analysis was performed with a Light Cycle 480 II system  using an SYNR Premix Ex TaqTM kit . The UBI-eq gene was used as an internal control. The selected stress-responsive genes were from wheat (Triticum aestivum L.). The primer sequences used for the PCR are given in A 0.1-g sample of wheat .All of the data were statistically analyzed with SPSS software . One-way analysis of variance was performed, and statistically significant differences among the treatments were determined using Tukey’s honest significant differences test at The probable underlying mechanisms via which exogenous application of MT mitigates the induction of oxidative damage and modulates the antioxidant defense system in wheat seedlings are proposed in the schematic diagram shown in


The crystalline phases and morphologies of the Ni nanoparticles, CWO, and CWO–Ni composites were analyzed using X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDAX). The electrochemical properties of CWO and CWO–Ni composite electrode materials were assessed by cyclic voltammetry (CV), and galvanostatic charge–discharge (GCD) tests using KOH as a supporting electrolyte. Among the CWO–Ni composites containing different amounts of Ni1, Ni2, and Ni3, CWO–Ni3 exhibited the highest specific capacitance of 271 F g−1 at 1 A g−1, which was greater than that of bare CWO (128 F g−1). Moreover, the CWO–Ni3 composite electrode material displayed excellent reversible cyclic stability and maintained 86.4% of its initial capacitance after 1500 discharge cycles. The results obtained herein demonstrate that the prepared CWO–Ni3 nanocomposite is a promising electrode candidate for supercapacitor applications.In the present study, the synthesis of CoWO Natural resource depletion and global warming pose a serious threat to both humans and the environment; therefore, developing efficient energy conversion and storage systems is essential . Among d4 (CWO) in the present study.Because of their multiple oxidation states, excellent conductivity, and high specific capacitance (SC), transition metal oxides play important roles in electrochemical processes. Oxides of Ru , Fe 16]16], Co , Ni 18]18], and 4 [4 [3O4@CoWO4/reduced graphene oxide (rGO) [4 [2O4@NiWO4 [4 [4/rGO [2O3 composite electrode exhibiting an SC value of >400 F g−1 at 20 mV s−1. The conducted stability test involving continuous cyclic voltammetry (CV) revealed that the synthesized material retained >50% capacitance after 200 CD cycles [−1 at 5 mV s−1 and 506 F g−1 at 1 A g−1. Lai et al. [−1 for nanorod arrays and 1195 F g−1 for nanosheet arrays at 1 A g−1.In recent years, various metal tungstate oxides, including CoWO4 , reduced4 [4 , Co3O4@Cde (rGO) , three-d(rGO) [4 ,25, NiCoO4@NiWO4 , FeWO4 [NiWO4 [4 , and NiW4 [4/rGO , have be4 [4/rGO ,30. For D cycles . FurtherD cycles  preparedi et al.  synthesi4 and its composites were applied as electrode materials for supercapacitors [4-based electrode materials could be used for electrolysis of water and for generation of green renewable energy storage systems. Hence, we fabricated a composite of CoWO4 and Ni nanoparticles using a wet chemical method. Pleasingly, an improvement in the electrochemical properties and cyclic stability was observed upon the addition of Ni nanoparticles to CoWO4, demonstrating the potential of the composite for supercapacitor applications.Metal tungstates have multiple oxidation states, improving their electrochemical performance in energy storage devices . In thispacitors ,22,23. W2·6H2O), cobalt chloride (CoCl2·6H2O), sodium tungstate (Na2WO4·2H2O), sodium hydroxide pellets (NaOH), ethylene glycol , and potassium hydroxide pellets were purchased from SDFCL, India. Hydrazine hydrate (N2H4), ethanol and polyvinylidene difluoride (PVDF) were obtained from Sigma-Aldrich, USA. N-methyl-2-pyrrolidone (NMP) was acquired from HiMedia, India.Nickel chloride  water and ethanol. The obtained product was dried at 50 °C for 12 h.The Ni nanoparticles were fabricated using the method that was previously reported by Wu et al. . Briefly2·6H2O and 0.659 g of Na2WO4·2H2O were dispersed in a 100-mL beaker containing 40 mL of DD water. The resulting solution was stirred for 30 min at ambient temperature. In another 50-mL beaker, 20 mg of Ni nanoparticles was dispersed in 10 mL of ethanol, followed by sonication for 1 h. The Ni nanoparticle solution was subsequently slowly poured into the first solution, and the reaction mixture was stirred for 3 h at ambient temperature. The resulting precipitate was filtered and washed with DD water and ethanol. The crude material was dried at 80 °C in a hot oven. Pure CoWO4/Ni20 was obtained as a light bluish black powder. A similar procedure was followed for the preparation of CoWO4/Ni40 and CoWO4/Ni80, where 40 and 80 indicate the amount of Ni nanoparticles added into the reaction (mg). Bare CoWO4 was synthesized without the addition of the Ni nanoparticles. The molar ratio of Co:Ni in CoWO4/Ni20, CoWO4/Ni40, and CoWO4/Ni80 was approximately 1:0.18, 1:0.36, and 1:0.71, respectively. Hereafter, CoWO4, CoWO4/Ni20, CoWO4/Ni40, and CoWO4/Ni80 are denoted as CWO, CWO–Ni1, CWO–Ni2, and CWO–Ni3, correspondingly.Approximately 0.475 g of CoCl2) and was dried overnight. The amount of CWO and CWO–Ni composite nanoparticles loaded on each electrode was 1 mg.The CWO and CWO–Ni nanoparticle composite electrodes that contain varying amounts of Ni were prepared by mixing 80% of the fabricated active material, 10% of carbon black (Super-P), and 10% of PVDF as the binder. All ingredients were mixed with NMP to obtain a paste. The paste was then uniformly loaded on an Ni foam (surface area of 0.5 cmX-ray diffraction (XRD) analysis was conducted using the Bruker D8 Advance X-ray diffractometer with CuKα radiation (λ = 0.1542 nm) at 4°/min. Fourier transform infrared (FT-IR) measurements were performed using JASCO, FT/IR-4600 . Scanning electron microscopy (SEM) images were obtained using JEOL-JSM 7600F . Transmission electron microscopy (TEM) images were acquired using JEOL-JEM 2100F . The chemical compositions of the fabricated nanocomposite materials were examined by inductively coupled plasma atomic emission spectrometry (ICP-AES) .SC is the specific capacitance in F g−1, Idv is the integrated area, m is the mass (weight) of the electrode material in grams, v is the scan rate in mV s−1, and ∆V is the potential window in V.The electrochemical characterization of the CWO, CWO–Ni1, CWO–Ni2, and CWO–Ni3 nanoparticle composites was performed by CV, galvanostatic charge–discharge tests (GCD), and electrochemical impedance spectroscopy (EIS). All investigations were conducted using an electrochemical system  composed of a standard three-electrode assembly with saturated Ag/AgCl as the reference electrode, a Pt wire as the counter electrode, and CWO or CWO–Ni composites loaded on an Ni foam as the working electrodes. An alkaline solution of KOH (6 M) was employed as the electrolyte solution. The CV analyses were performed in an applied bias window of −0.1–0.5 V, while the GCD evaluation was conducted with the applied bias varying from 0 to 0.42 V. The SC was evaluated using CV via Equation (1) according to the previously described approach ,35.(1)SI (A) and t (s) indicate the discharging current and time, respectively, m (g) refers to the amount of loaded nanocomposite nanoparticles, and ΔV denotes the applied bias difference (V).The SC values of the fabricated electrodes were determined based on GCD using the following Equation (2) ,36:(2)SC4 monoclinic phase, JCPDS # 15-0867). Furthermore, the peak corresponding to CWO exhibited low intensity, which indicates an amorphous structure and poor crystallization of the CWO material synthesized via the chemical precipitation method. Typically, compared to a crystalline compound, an active material displaying a broad peak as a consequence of an amorphous structure exhibits additional ion transfer [transfer . Hence, −1) were attributed to the distinctive Co–O, W–O, and W–O–W bridges. Moreover, the IR region below 500 cm−1 corresponded to the deformation modes of the W–O bonds in the WO6 octahedron or to the deformation of the W–O–W bridges [−1 was ascribed to the O–W–O vibration mode and to the W–O bond stretching frequency, respectively [−1 were attributed to the –OH group of water, which was present in the samples. The band at 604 cm−1 corresponded to the stretching vibration of the Ni–O bond, and its presence confirmed the formation of CWO–Ni3.The structures of CWO and CWO–Ni3 were confirmed using FT-IR analysis . The str bridges . The appectively . The absThe elemental compositions of the Ni nanoparticles, bare CWO, and CWO–Ni were characterized using energy-dispersive X-ray spectroscopy (EDAX) (Electronic Supplementary Information (ESI), −1 . Unlike the discharge curves, the CD plots showed a decrease in the SC values with increasing current density, which was attributed to the decrease in the flow of charged ions into the inner active sites and a reduced rate of the Faradaic redox reaction [4. These outcomes indicated that among the studied composites, CWO–Ni3 exhibited the highest discharge time, making it a suitable candidate electrode material for supercapacitors.reaction . Figure −1. −1) at 5 A g−1 increased to 252 F g−1 up to 400 reversible cycles, corresponding to a 104% rise in SC. The long-term cyclic data demonstrated in We subsequently evaluated the cyclic stability of the CWO–Ni3 composite with respect to the SC and coulombic efficiency . The CWOv, indicating the occurrence of a bulk diffusion process via an electrochemical reaction. It is noteworthy that the CV peak current followed the linear Randles–Sevcik equation [v curve using an equation reported in our previous study [−9 and 1.7484 × 10−8 cm2 s−1, respectively. Hence, the CWO–Ni3 composite showed a significantly higher D value than CWO.Furthermore, equation ,45,46. Mus study . The D v−1 at 1 A g−1, which was higher than that of bare CWO (128 F g−1). Notably, in a long-term cyclic stability test conducted by applying up to 1500 continuous reversible CD cycles, the CWO–Ni3 composite retained approximately 86.4% of its initial capacitance. Thus, the outcomes of our study clearly indicated that the CWO–Ni electrode material could be applied in redox supercapacitors.CWO–Ni nanoparticle electrode materials exhibiting different wt% of Ni were synthesized using a wet chemical method. The phase formation of the synthesized samples was confirmed by XRD and FT-IR spectroscopy analyses. The SEM surface morphologies of CWO–Ni3 confirmed the co-existence of irregular spherical shaped Ni nanoparticles and nanoporous CWO in its structure. The prepared CWO–Ni3 nanocrystals were also investigated by TEM. Moreover, electrochemical tests, including CV, CD, and EIS, were performed on CWO–Ni3. According to the obtained CD profile, the CWO–Ni3 composite displayed a maximum SC value of 271 F g


Dictyostelium discoideumatg5 single, atg5/12 double, and atg5/12/16 triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on Klebsiella aerogenes was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of Escherichia coli was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of E. coli, was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy.  Macroautophagy (hereafter autophagy for simplicity) is the major route for the lysosomal degradation of long-lived proteins, protein aggregates, and mal-functioning organelles in all eukaryotic cells . Most liAutophagy is characterised by the de novo formation of a double-membrane autophagosome, which matures into the autolysosome through fusion of its outer membrane with the lysosome. This complex cellular process, which serves to selectively or non-selectively sequester cargo, can be subdivided into initiation, maturation, and lysosomal degradation phases. It engages around 20 core autophagy (ATG) proteins that act in a sequential way and are organised in different molecular complexes ,8,9. In D. discoideum ATG16, survive the embryonic phase, but die one day after birth, corroborating the importance of an intact ATG12~5/16 complex for postnatal survival [In in vitro assays, it was shown that the yeast ATG12~5/16 complex directly binds to membranes and that this binding is mediated by ATG5, inhibited by ATG12, and activated by ATG16 . The intsurvival .Dictyostelium discoideum has a unique life cycle, with motile unicellular and multicellular stages, and the organism serves as a model for a number of biological problems that are relevant to human health [D. discoideum cells must mobilise a large fraction of the required energy for biosynthetic needs and morphogenesis by autophagy and glycogenolysis [D. discoideum is well-established for the investigation of the autophagic process and powerful methods to monitor and quantify autophagy in this organism have been developed [D. discoideum autophagy machinery is more similar to higher eukaryotes than to yeast [D. discoideum and the analysis of single or double knock-out mutants of core autophagy genes revealed informative phenotypes [The social amoeba n health ,26,27. In health . Since denolysis . Consequeveloped ,30. The to yeast  and ectoto yeast ,32. Furtenotypes ,39,40,41We here generated ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ cells and analysed their phenotypes in development, cell viability, growth, phagocytosis, macropinocytosis, and protein homeostasis. This is, to our knowledge, the first report of the analysis of an ATG5/12 double and ATG5/12/16 triple mutant. We observed complex, and for some cellular processes, opposite phenotypes of varying severity in the generated knock-out strains. We find similar phenotypes for the single, double, and triple knock-out mutants in cellular processes known to depend on canonical autophagy. This implies that deletion of any of the components in the ATG12~5/16 complex destroys its function in these processes. Our results further support autophagy-independent functions of the complex and its individual components, as well as the placement of autophagy upstream of the ubiquitin–proteasome system (UPS).D. discoideum AX2 was used as a wild-type strain. The ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ strains were generated by the replacement of the atg5 gene with the knock-out construct in AX2, ATG12¯ and ATG12¯/16¯ cells, respectively [D. discoideum strains were grown at 22 °C in AX2 liquid nutrient medium [Klebsiella aerogenes-overlaid SM agar plates [6 cells/mL) were used for all experiments.ectively . We haveectively . The strt medium  on platet medium  or on Klr plates ,45. For 4 cells/mL. For each strain, the cell titres of three separate flasks for each independent experiment were determined every 24 h for up to 10 days, and the generation time was calculated from three timepoints within the logarithmic growth phase. Each point in the graph in Figure 5A represents the average generation time of one experiment. Three (ATG5¯/12¯/16¯), four (AX2 and ATG5¯/12¯), and six (ATG5¯) independent experiments were performed. Growth on K. aerogenes was quantitated by measuring plaque size on a bacterial lawn every 24 h for 7 days using a stereomicroscope  and the accompanying Leica LAS X software . We have performed four independent experiments for AX2, three for ATG5¯/12¯ and ATG5¯/12¯/16¯, and seven for ATG5¯ cells. In each independent experiment, we have measured between 10 and 23 plaques for each mutant. The average increase in the plaque size per 24 h was calculated. Analysis of cell survival upon nitrogen starvation and developmental experiments were carried out as described [For growth experiments of wild-type AX2 as well as ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ mutants, cells were inoculated in 25 mL of AX2 medium at a density of 2 × 10escribed ,40.E. coli ArcticExpress RIL , purified using Glutathione Sepharose 4B beads  and released through cleavage with PreScission protease . The purified ATG5 polypeptide was used for immunisation of rabbits . Subsequently, the generated ATG5 anti-serum  was affinity-purified using a purified, recombinant ATG5150–280 polypeptide.For the generation of ATG5-specific polyclonal antibodies, the coding sequence for amino acids 150 to 280 of ATG5 was amplified by PCR and cloned into the pGEX-6P-1 expression vector. The GST fusion protein was expressed in For immunoblotting, the affinity-purified ATG5 antibody was used at a 1:2500 dilution. ATG12 was detected with mouse monoclonal antibody K89-141-1 at a 1:10 dilution , ATG16 wFor immunofluorescence analysis, the ubiquitin antibody P4D1, as well as a rabbit polyclonal antibody directed against ATG8a , were used at a 1:1000 dilution in PBG buffer (1× PBS containing 0.5% BSA and 0.045% fish gelatin).5 cells per lane as described [SDS-PAGE and Western blotting of total cell lysates were performed with 4 × 10escribed ,50,51. TE. coli bioparticles, D. discoideum cells were prepared as described for fluorescence microscopy without fixation [E. coli (K-12 strain) BioParticles labelled with Alexa-Fluor 594  were added to the cells at a 50-fold excess relative to D. discoideum cells. After 30 min of incubation, cells were washed four times with Soerensen’s phosphate buffer . Cells were fixed, nuclei were stained with DAPI and immunofluorescence microscopy was performed as described [Quantitative macropinocytosis of TRITC-labelled dextran and quantitative phagocytosis of TRITC-labelled heat-killed yeast cells were performed as described . For phafixation . E. coliescribed .D. discoideum strains were conducted using the ibidi 2D μ-slides for chemotaxis according to the manufacturer’s instructions with minor changes. D. discoideum log phase cells were harvested, washed with Soerensen phosphate buffer and starved for 4 h in Soerensen phosphate buffer. A total of 6 µL of a cell suspension with 3 × 106 cells/mL was injected into the central channel of the ibidi μ-slide. The central channel connects two large reservoirs and also represents the observation area. Cells were allowed to attach for 10 min; non-adherent cells were removed by two injections of 10 µL Soerensen phosphate buffer and the right and the left reservoir were filled with 65 µL Soerensen phosphate buffer. The random movement of D. discoideum cells in the central channel was documented in a TIRF microscope, and the accompanying Leica LAS X software (v.3.3.0) over a period of 30 min by taking images every 30 s. After the experiment, the images were transferred to Image J (https://imagej.nih.gov/ij/), and for each strain cell tracking and determination of the velocity of 20 cells were carried out with the “manual tracking” plugin.Cell motility measurements of the different Proteasomal activity measurements were performed using the established protocol from skeletal muscle tissue  with minD. discoideum cells were incubated with the indicated primary antibodies; secondary antibodies were Alexa-Fluor 488 conjugated goat anti-mouse and Alexa-Fluor 568 conjugated goat anti-rabbit IgG at a 1:10,000 dilution . Nuclei were stained with 1 µg/mL 4′,6-diamidino-2-phenylindole . Images of fixed cells were taken with an inverted Leica TCS SP5 confocal laser scanning microscope  with a 100 × HC Pl APO 1.4 oil immersion objective. Excitation of DAPI was at 405 nm, emission at 460–480; excitation of Alexa-Fluor 488 was at 488 nm, emission at 515–535 nm; excitation of TRITC and of Alexa-Fluor 569 and 594 was at 561 nm and emission at 570–580 nm, 590–610 nm, and 605–625 nm, respectively. Images were processed using the Leica MMAF Acquisition- and Analysis software, Adobe Photoshop CS, and Corel Draw 2017.Immunofluorescence microscopy was essentially done as described . Fixed DD. discoideum ATG5 were identified using the SMART webtool [D. discoideum ATG5 was created with SWISS-MODEL ProMod3 version 1.3.0 [D. discoideum cells for RNAseq experiments were performed as described [seq analysis was done as described by using the DESeq2 package [seq results will be published separately.Multiple sequence alignments were performed with Clustal Omega version 1.2.4 , and seq webtool . To anal webtool  was usedon 1.3.0  and visuon 1.3.0 . The stron 1.3.0 . Isolatiescribed ,61. Six escribed , alignedescribed , and evaescribed . RNAseq  package ,64. The p-value: <0.05 = significant *; <0.01 = very significant **; <0.001 = highly significant ***. Details are given in the figure legends. For data with parametric statistics (ANOVA), mean values and the standard error of the mean were used for data visualisation. For nonparametric statistics , median and the interquartile range were used.Unless otherwise indicated, all data shown are derived from at least three independent experiments. The analysis of the statistical significance of experimentally detected differences was carried out with the software environment R, version 3.5.2  and the Saccharomyces cerevisiae [D. discoideum ATG5, the ubiquitin-like domains are interrupted by asparagine-rich (N-rich), low-complexity regions  as template [D. discoideum ATG5  adopt a functional fold despite the interruption of the two UblDs by low-complexity regions (in red). The UblDs mediate the interaction with the N-terminal part of ATG16 (in rose) containing the ATG5 interaction motif (AFIM), and the HRD is responsible for the interaction with ATG12 (in yellow)  B. The D.β-sheets . The lysβ-sheets B,C.atg5 in the AX2 wild-type, ATG12¯ and ATG12¯/16¯ background. In addition, we included in some assays the previously generated ATG12¯, ATG16¯ and ATG12¯/16¯ cells [To analyse the cellular functions of the ATG12~5/16 complex and its individual proteins, we generated knock-out mutants of 6¯ cells . The ideD. discoideum; LC3s and GABARAPs in mammals) to PE of the autophagosomal membrane. Therefore, we next investigated the presence of ATG8a-positive autophagosomes in AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells by immunofluorescence analysis. We found that cells lacking ATG5 contained no ATG8a-positive autophagosomes, while these were clearly detectable in AX2 wild-type cells  on a lawn of iability B. Again,iability .D. discoideum strains are capable of consuming liquid nutrients by macropinocytosis [D. discoideum cells. All three mutant strains showed significantly reduced macropinocytic activity in comparison to AX2 wild-type cells. After 2 h, the relative fluorescence of ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells was 31%, 27% and 52%, respectively, lower than for AX2 , while this enigmatic activity is not seen with ATG5¯ cells. On the contrary, this strain and the ATG5¯/12¯/16¯ strain grew much faster on a lawn of K. aerogenes.We next investigated the clearance of  smaller B. The di smaller C. This rE. coli BioParticles labelled with Alexa-Fluor 594 and quantitated phagocytosed and plasma membrane adherent bacteria by fluorescence microscopy  revealed no significant changes in the differential regulation of proteasomal genes  to the headgroup of the membrane lipid PE and is essential for canonical autophagy. In this complex, ATG16 is responsible for binding to the correct membrane via protein–protein and protein–lipid interactions, ATG12 mediates binding of the ATG3~8 conjugate, and ATG5 holds the E3 ligase activity ,76,77. Arocesses .D. discoideum protein sequences suggests a highly similar structure, despite the interruption of each UblD with a low-complexity, asparagin-rich sequence stretch. In the predicted structure, the ubiquitin-like protein ATG12 and the N-terminal α-helix of ATG16 are located on opposite sides of ATG5 and there is no common interface of ATG12 and ATG16N  confirmed the ubiquitin-like fold for ATG12 and revealed for ATG5 two ubiquitin-like domains (UblDs) that flank a helix-rich domain (HRD), and an additional N-terminal α-helix . Modellid ATG16N B. Hence,d ATG16N B,C. In min mouse ,78. Simium cells B. These atg12 mRNA [Our Western blot analyses of single, double and triple knock-out mutants of components of the ATG12~5/16 complex revealed very efficient conjugation of ATG12 to ATG5 in vivo, because we could only detect the ATG12~5 conjugate of around 68 kDa, but not monomeric ATG12 or ATG5 in wild-type AX2 and ATG16-deficient cells. In addition, the presence of ATG5 appears to be required for the stability of ATG12, because unconjugated ATG12 was not detectable in ATG5-deficient cells A. In cong12 mRNA . A possig12 mRNA ,85. Of nATG5, ATG12 and ATG16 act in canonical autophagy together as a heterohexameric complex, which provides the E3-like ligase activity for conjugation of ATG8 to PE . The sinK. aerogenes for ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ cells (Dictyostelium is apparently able to discriminate between different types of bacteria and other microorganisms [We could distinguish three additional main classes of phenotypes. A basically similarly severe phenotype of the single and double mutant, and an increase in the defect in the triple mutant A. We pro6¯ cells . These crganisms ,97. FinaD. discoideum, we and others found an increase in ubiquitinated proteins and the appearance of ubiquitin-positive protein aggregates in all studied autophagy-compromised strains [seq, and quantitation of the 20S proteasomal subunit psmA7, was unchanged in the mutant strains (Protein homeostasis is crucial for cell welfare and is accomplished by a delicate balance between protein synthesis and degradation. Autophagy and the UPS are of utmost importance for the clearance and recycling of proteins and other cellular material, and defects in either system cause serious cellular problems. Although the two degradation machineries have different substrate preferences and separate molecular mechanisms, it is clear that the two pathways are interrelated and share molecular determinants and substrates ,99,100.  strains ,39,40,53 strains ,39,40. O strains . The marD. discoideum the UPS is dependent on intact autophagy for full activity. The exact molecular principle is not clear; however, an attractive possibility is that proteasomal subunits experience post-translational modifications, e.g., ubiquitination, during their lifetime, which cause a more or less pronounced decrease in the proteolytic activity of the proteasome. These less active or inactive proteasomes are sensed and delivered for degradation by proteaphagy, a recently described novel type of selective autophagy [We conclude that in utophagy ,111,112.Our results disclose novel, diverse, and complex phenotypes for different cellular processes in ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ cells. They are consistent with autophagy-independent functions of the ATG12~5/16 complex and its components, in addition to its function in canonical autophagy. Further work is needed to fully unravel all independent activities of ATG5, ATG12, and ATG16, as well as possible additional roles of the ATG12~5, ATG12~7, and ATG12~10 conjugates.


Cucumis sativa L.) seedlings. We stored cucumber seedlings under four different photosynthetic photon flux densities  at 12°C, and examined how the morphological, physiological, and photosynthetic changes in seedlings during storage affected their ability to recover after transplanting. Our results indicated that at least 15 μmol·m-2·s-1 PPFD was needed for cucumber seedlings stored in the dark for 2 or 4 d, and at least 30 μmol·m-2·s-1 PPFD was needed when the ST was extended to 6 d. Overall, our results showed that cucumber seedlings require light-emitting diode (LED) illumination during storage to maintain their quality and recovery ability.Continuous darkness decreases seedling quality during storage, whereas appropriate light quality and intensity can overcome these negative effects. In this study, we determined the light intensity, storage time (ST), and storage temperature suitable for cucumber ( Cucumis sativus L.; Cucurbitaceae) is an important crop worldwide. China is the largest producer of cucumber, accounting for approximately 77% of global cucumber production ..7].1, x2, x3, and xn represent the number of cucumber seedlings at each level; and a1, a2, a3, and an represent the different levels.The leaf degradation index (LDI) of cucumber seedlings was calculated using the following equation:The leaf integrity index (LII) of cucumber seedlings was estimated as follows:Leaf disks (0.84 cm diameter) were collected from six plants, and eight disks per plant were weighed and used for the measurements. Chlorophyll was extracted from the cotyledon and first true leaf using 95% ethyl alcohol in the dark for 24 h. The chlorophyll content was determined by measuring the absorbance at 665 and 649 nm using an ultraviolet-visible (UV-Vis) spectrophotometer, as described previously . Subsequ2 and then thawed for a soluble sugar content analysis, as described previously [The first leaves were frozen in liquid Neviously , using aThe central portion of the leaf blade, without veins, was cut into rectangles (approximately 2 × 1 mm) and used to observe the chloroplast ultrastructure as described previously .2 concentration (Ci), and transpiration rate (Tr) were measured using the LI-6400xt system  equipped with an LED light source at 250 μmol·m-2·s-1 PPFD. The leaf area clipped by the chamber was 6 cm2. The temperature, relative humidity, and CO2 concentration were maintained at 25°C, 40 ± 5%, and 400 μmol·mol-1, respectively, during the measurements. At least three plants from each treatment were used for these measurements.The Pn, stomatal conductance (Gs), intercellular COAfter transplanting, 18 plants from each treatment were numbered. The location of the heart of each plant was marked and outlined using a transparent paper to obtain the heart leaf area . After 8The shoot length, stem diameter, shoot and root fresh weights, and shoot, root, and total dry weights were significantly influenced by the ST and light intensity treatment (T) and their interaction. In the dark (T0), most seedling growth parameters, including stem diameter, shoot fresh and dry weights, and total fresh and dry weights, showed decreasing trends with increased ST . HoweverThe leaf health index, but not the chlorophyll content, was significantly influenced by ST, T, and the ST × T interaction . The chlThe ultrastructure of cucumber leaf chloroplasts was significantly influenced by ST and T. Before storage (0 d), leaf chloroplasts were elliptical in shape and were filled with starch grains, and the chloroplast grana thylakoids were closely arranged . In contThe soluble sugar content of cucumber seedlings was significantly affected by ST, T, and the ST × T interaction. The soluble sugar content of seedlings in the T0 and T15 treatments decreased with increasing ST. However, providing seedlings with appropriate light during storage alleviated the adverse effects. After 4 d of storage, T0 seedlings had a significantly lower soluble sugar content than T250 seedlings, whereas there were no significant differences in T15, T30, and T45 seedlings compared with T250 seedlings. After 6 d of storage, seedlings in all treatments had a significantly lower soluble sugar content than T250 seedlings, although T30 and T45 seedlings maintained a higher soluble sugar content than T0 seedlings.Both Pn and Gs were significantly affected by ST, T, and the ST × T interaction . Both PnThe RGR of new leaves was significantly influenced by ST, T, and their interaction . In the Shoot length, stem diameter, and shoot, root, and total fresh and dry weights of transplanted seedlings were significantly influenced by ST, T, and their interaction . In the Both the Pn and Gs of transplanted seedlings were significantly affected by ST, T, and their interaction . The Pn -2·s-1 PPFD is necessary for maintaining seedling dry weight and leaf health.Low temperature and darkness are commonly used to maintain seedling quality during short-term storage . HoweverLight affects chloroplast development, chloroplast structure, and consequently photosynthesis –24. In t-2·s-1 PPFD is necessary for maintaining photosynthesis during 6 d of storage.With an increase in dark ST, the seedlings showed a decrease in carboxylation efficiency and net photosynthetic rate , 25. In -2·s-1 PPFD  under dim light during storage tended to show better growth after transplanting than those grown under darkness , 5. In o-2·s-1 PPFD is necessary for maintaining photosynthesis after the transplantation of seedlings subjected to 6 d of storage.Exposure of seedlings to light during storage is one of the main factors that preserves their photosynthetic ability; thus, ensuring the transplant quality of post-storage seedlings , 28, 31.-2·s-1 PPFD, and when the ST was extended to 6 d, at least 30 μmol·m-2·s-1 PPFD was required to maintain a normal chloroplast ultrastructure, healthy leaves, and optimal photosynthetic performance during storage. The provision of these light intensities would enable cucumber seedlings to recover quickly after transplanting, which would increase the RGR of new leaves, plant growth, and photosynthetic ability.Overall, this study demonstrates that to preserve the quality of cucumber seedlings during storage and to improve their recovery post-transplantation, appropriate LED illumination should be provided when the storage duration changes. Our data showed that cucumber seedlings stored at 12°C in darkness for 2 or 4 d required at least 15 μmol·mS1 Data(XLSX)Click here for additional data file.


Bacillus velezensis obtained from fermented food that would generate heme oxygenase-1 (HO-1) expression and lead to nuclear factor erythroid 2-related factor-2 (Nrf2) nuclear translocation. We explored the ability of kinetics and potency for the bacterial killing to work against various pathogenic bacteria. A bioassay showed the lysis zone of MS15 by tricine SDS-PAGE near at 6 kDa. MALDI-TOF/MS verified molecular weight, and the existence of a molecular mass of 6091 Da was reported by purity. The MIC of MS15 ranged from 2.5–160 μg/mL for many pathogenic bacteria, showing greater potency. In macrophage RAW 264.7 cells, MS15 was exposed to assess its inhibitory effect against the generation of reactive oxygen species (ROS) in oxidative stress. In the sample treated group, the translation, and transcriptional levels of CAT , GPx (glutathione peroxidase), and SOD (superoxide dismutase) were significantly greater. In short, MS15 has significant antioxidant properties, reducing ROS production in RAW 264.7 cells, and raising the translation and transcriptional rates of antioxidant enzymes with stimulating HO-1 induction facilitated by Nrf2.The efficient culture and purification of antimicrobial peptides (AMPs), along with intense antioxidant activity, have drawn the interest to study antioxidant activity mechanism. We report the culture conditions optimization, efficient biosynthesis, and purification of an antioxidant peptide MS15 from  Bacterial strain CBSMS15 was obtained by the isolation process from Kimchi . DMEM (Dulbeco's modified eagle medium), Griess reagent , and DCF-DA (dichlorofluorescein diacetate) process have been used for experiments. RAW 264.7 macrophase cells were procured from ATCC . Sephadex G-50 column and DEAE Sephadex A-50 column , and spin column  were used for purification. Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine SDS-PAGE) and MADLI TOF/MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometer)  process were used to check the purification product confirmation. Bacillus sp., was isolated from Kimchi. It could produce AMP , which was highly active against a wide range of pathogenic microorganisms. Kimchi is a distinctive typical fermented Korean food characterized by different microorganisms species such as Lactobacillus and Bacillus subtilis. We used cabbage kimchi for our study to isolate Bacillus strain. The isolation process was described briefly in our previous report [−8 in Mueller–Hinton (MH) broth. The proper CFU/mL (colony forming units) were assessed by streaking from those diluted aliquots. Hereafter, for the further stock, glycerol was used with appropriately diluted aliquots by 20% and 80%, respectively. The strain was discovered as Bacillus bacterial strain, based on morphologically characterized bacteria. It was analyzed following Bergey's manual of systematic bacteriology [The bacterial strain CBSMS15, which had a first nucleotide sequence similarity with s report , whereaseriology . MoreoveMycobacterium smegmatis [2PO4, Na2HPO4, ZnSO4, MgSO4, KH2PO4, MgCl2, NaCl, FeSO4, CaCl2) were utilized in various amounts. Culture optimization was accomplished with diverse nitrogen sources (1%), and carbon sources (1%), with numerous metal ion sources (0.01%) till the last optimization. Finally, a metal ion, nitrogen, and carbon source were utilized to verify the final percentage of culture media by response surface methodology.Antimicrobial activity was established by the method of disc diffusion. Briefly, paper disc filter  soaked on the face of the petri dish with AMP (40 µL) was positioned, including the MH agar plate, overlay with megmatis . Optimizmegmatis . In briep ˂ 0.05) discovered the effect of every single considerable variable on the activity of bacteriocin. A response surface optimizer was applied to verify the optimum environments for soluble starch as carbon sources, yeast extract as nitrogen sources, and NaCl as metal ion sources compositions . Design-g)  for 30 min at 4 °C. Active precipitation fraction was recovered by mixing diammonium sulfate at the saturation of 20–80% using centrifugation at 10,000× g at 6 °C for 50 min. Aliquots dialyzed with 10 mM Tris-HCl (pH 7.2) buffer and subjected to ultrafiltration  with 1 kDa and 10 kDa membrane. Following dialysis and filtration, the biologically active fraction was applied to a Sephadex G-50 (3 × 90 cm) column. The active fractions were combined and employed to ion-exchange separation using the DEAE Sephadex A-50 column (1.2 × 35 cm) and eluted, including the equal buffer. Further, active aliquots were utilized to a spin column named the Pierce spin desalting column and were investigated for purity check. Seed culture produced on De Man, Rogosa, and Sharpe (MRS) media was continuously shaken at 160 rpm for 12 h at 37 °C. The purification process was staged for the culture of peptide MS15 for 32 h at 37 °C, and shaking speed was 170 rpm, including 1.5% soluble starch, 1% yeast extract, 0.01% NaCl obtained by response surface methodology. Then, cell-excluded supernatant was accumulated after centrifugation  by covering the treated gel [The protein content was assessed using BSA  as a reference protein by the Bradford method . Tricineated gel . The gelm/z 500–40,000.The MALDI-TOF method was utilized to check the molecular weight of peptide MS15 as well as the purity check of the final peptide sample . To asseEscherichia coli KCTC 1923 predictor microbial strain. Likewise, a large variety of buffers such as sodium phosphate-citric acid (pH 4.2–6.5), Tris-HCl (pH 7.2–9.4), and KCl-NaOH (pH 10.8–12.4) were tested for pH stability. By investigating the temperature stability of MS15, aliquots were subjected to different treatments of temperature 0–100 °C and 121 °C for 60 and 15 min, respectively, before assessing the residual activity. The specimens were evaluated aligned with the 7 CFU/mL). Gram-negative bacteria: Salmonella typhimurium KCTC 1925, Pseudomonas aeruginosa KCTC 1637, Extended-spectrum beta-lactamase W1, Alcaligenes faecalis ATCC 1004, ESBL 31, Escherichia coli KCTC 1923, ESBL V4 (Escherichia coli); Gram-positive bacteria: VRE 4, VER 89, VRE98, Staphylococcus aureus KCTC 1928, Mycobacterium smegmatis ATCC 9341, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 9341, MRSA B15, VRSA, MRSA 5-3, MRSA B15. The broth dilution method was employed to get the minimum bactericidal concentration (MBC) value [The agar dilution method utilized to test the minimum inhibiting concentration (MIC) of peptide MS15 was used to assess the antimicrobial inhibitory range . Vancomy10), in time-kill kinetics findings analyzed with Escherichia coli cells (5.5 × 105 CFU/mL), which was prepared in LB broth medium [Escherichia coli cells was established. The GC (growth control) study was presented without peptide for the species. The logarithm graph (CFU/mL) was designed alongside the time of growth.Bacteriocin activity of MS15 was studied in the logarithmic growth phase . All analyses were studied in triplicate.The scavenging capacity of DPPH radical was evaluated, applying the procedure portrayed by Nanjo et al. with a minor modification . A 100 µ•+ radical decoloration action method was applied to determine the ABTS•+ radical scavenging capability of peptide MS15 with a slight modification of the method portrayed by Carrasco-Castilla et al. [•+ solution was resuspended in ethanol and equilibrated to offer an absorbance of 0.705 ± 0.04, which was achieved at 734 nm. Variable concentrations (2.5–20 µM) of the peptide fraction (10 µL) were let to mix with 990 μL of the ABTS•+solution for 5 min and the optical density was noted at 734 nm. Ascorbic acid (2–16 µM) was analyzed as a reference standard antioxidant molecule. The % (percentage) inhibition of the ABTS•+ radical was evaluated as: samp is the optical density of the sample and Acont is the optical density of the control. All analyses were studied in triplicate.The ABTSa et al. . The stoThe capability assay of superoxide anion radicals was checked in the riboflavin/EDTA system by UV exposure . The rea3·6H2O; 20 mM), and acetate buffer  with a ratio of 1:1:10. A 10 μL aqueous sample was mixed at different concentrations and 990 μL of FRAP mixture and incubated at 37 °C for half an hour. The optical density was assessed at 595 nm. Besides, ascorbic acid as a standard antioxidant compound was also studied.The FRAP assay was brought with a minor alteration for the measurement of reduction power, as previously described by Benzie et al. . In brie2 (10 mM), and the aliquots were combined to the ultimate solution. After one hour of incubation at 37 °C, the optical density was measured at 450 nm. Moreover, ascorbic acid was employed as a reference antioxidant compound.CUPRAC assay of MS15 was established using the process explained by Apak et al., with minor modifications . A solutFollowing the previous article , the ORA5 cells per well, RAW 264.7 cells were inoculated into a 96-well microplate and incubated at 37 °C for 1 h. At different concentrations of MS15 (2.5–20 µM), the cells were then treated with 1 µg/mL LPS (lipopolysaccharide). NO concentration of the supernatants was assessed with the addition of Griess reagent after 24 h incubation. The absorbance of the mixtures was assessed at a wavelength of 520 nm, employing a fluorescence microplate scanner.Griess reagent was utilized to calculate the concentration of nitric oxide (NO) in the medium as an indicator of NO output as our previous report . At a co5 cells/well) for 24 h. Various concentrations of MS15 (2.5–20 µM) subsequently pretreated in the cells after incubation and incubated for one hour. Moreover, cells were activated by LPS (1 µg/mL) and set for an extra 24 h. The cells were resolute two times with phosphate-buffered saline (PBS) buffer and treated with DCF-DA (25 μM) in a dark place at 37 °C for 30 min. Gallic acid (1.5–18 µM) was analyzed as a reference standard antioxidant compound. The fluorescence intensity was assessed employing a fluorescence microplate reader at an emission and excitation wavelength of 520 nm and 485 nm, correspondingly.The DCF-DA method was applied to determine the cellular oxidative pressure produced by LPS because of reactive oxygen species (ROS) . The amo2 as our earlier report [5 cells/well . It was cultured and treated with various concentrations of MS15 according to the treatment plan. After incubation, 20 µL MTT -2,5-diphenyltetrazolium bromide) reagent (5 mg/mL) was put in to each well. After two hours of incubation, 100 µL of DMSO was dissolved and incubated for 12 h. Absorbance was noted using a microplate reader  at 570 nm.Cells (RAW 264.7 cells and HeLa cells) were cultured in DMEM medium supplied with FBS (10%) and penicillin/streptomycin (100 µg/mL) at 37 °C under COr report . For cytTM protein extraction kit (cytoplasmic/nuclear) from Bio-Rad  was used to extract nuclear protein. Protein aliquots (20 µg) were separated using SDS-PAGE (10%) and electrotransfer to polyvinylidene fluoride membranes . Hereafter, the membranes were incubated with primary antibodies in skim milk (5%). A series of primary antibodies named β-actin, anti-Nrf2, anti-HO-1, and lamin B were utilized. Horseradish peroxidase (HRP)  was used as secondary antibodies. The ECL (electrochemiluminescence) solution system (Perkin Elmer) was applied to discover the antibody–antigen reaction. Cell lysates of RAW 264.7 cell were added with sample buffer , 2-mercaptoethanol (5%), glycerol (50%), bromophenol blue (0.5%), SDS (10%), DTT (0.5 M)), and boiled at 100 °C for 5 min to denature the mixture portrayed in the earlier study . ReadyPrSOD1, CAT, Nrf-2, GPx-1, GAPDH, HO-1) were amplified with a denaturation step.TRIzol reagent  was used to extract total RNA from RAW 264.7 cells by following our prior study . Total RSOD1 (NM_011434.2): forward, 5′-CAG CAT GGG TTC CAC GTC CA-3′; reverse, 5′-CAC ATT GGC CAC ACC GTC CT-3′; CAT (NM_001752.4): forward, 5′-AG GCT CAG CTG ACA CAG TTC-3′; reverse, 5′-GC CAT TCA TGT GCC GAT GTC-3′; Nrf-2 (NM_010902.4): forward, 5′-CTT TAG TCA GCG ACA GAA GGAC-3′; reverse, 5′-TCC AGA GAG CTA TTG AGG GACT-3′; GPx-1 (NM_001329503.2): forward, 5′-TTC CCG TGC AAC CAG TTTG-3′; reverse, 5′-GGA CGT ACT TGA GGG AAT TCA GA-3′; GAPDH (NM_001357943.2): forward, 5′-GCG AGA TCC CGC TAA CAT CA-3′; reverse, 5′-AGT GAT GGC ATG GAC TGT GG-3′; HO-1 (XM_002743721.5): forward, 5′-CCA GAA AGT GGG CAT CAG CT-3′; reverse, 5′-GTC ACA TTT ATG CTC GGC GG-3′. Ethidium bromide staining was used to visualize the PCR products after electrophoresis. Sequences of the primer designed for RT-PCR were stated as p < 0.05, ** p < 0.01, *** p < 0.001.The experimental data were presented with three replications per inspection, and every single point signifies the mean ± SD (standard deviation). Statistical analysis was completed employing one-way predictive analysis ANOVA . Difference was measured significant in statistics among the treatment represent as, * Bacillus strains with 16S rRNA gene sequences analysis. The 16S rRNA analysis showed the most intimate nature to Bacillus velezensis CR-502T (Accession no. AY603658) with a homologous resemblance of 99.9%. A phylogenetic tree was shown in Microbial strains that were obtained and isolated from Kimchi  that have the capability of making AMP was preserved. Strain CBSMS15 was initially discovered based on its morphological and biochemical nature, and according to the results, the strain exhibited a close identity with some Following the RSM theory, the higher value derived from the degree of the independent amount of choice, soluble starch (A), yeast extract (B), and NaCl (C) were additionally inspected at three distinctive levels. The bacteriocin production was substantially different in the medium from 7622 to 12367 AU/mL at varying levels of multiple elements . p < 0.001; F-value 31.18). 2D dimensions were produced with cross term BC  and reflector mode (500–6000 Da) to detect the purity and molecular weight of MS15. The molecular weight was at the peak point of 6.091 kDa for peptide MS15 d that deResidual activity was examined corresponding to the preserved bacteriocin activity to verify the temperature stability of MS15, and it persisted in being stable up to 60 °C. However, its activity was declining slowly and retained about 84% of the bacteriocin activity after 30 min of incubation at 70 °C. Total loss of activity appeared for 15 min at 121 °C when it got exposure to autoclave conditions. The capability to maintain the MS15 activity in a wide selection of pH values can result in the denaturation of the variable protein. The peptide MS15 was incubated at various pH buffers to evaluate the stability of the pH. MS15 has been found to tolerate an extensive range of pH values (4.2–12.4). MS15 exhibited a wide pH and temperature array. Enterococci 98, Vancomycin-resistant Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus 5-3, Staphylococcus aureus, Vancomycin-resistant Enterococci 89, Methicillin-resistant Staphylococcus aureus B15, and Gram-negative pathogens, for instance, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Extended-spectrum beta-lactamase . The bactericidal activity was assessed by using the broth dilution method, and it kills bacteria in a range concentration of 5–320 µg/mL. The MICs of peptide MS15 were assessed against different pathogenic bacteria, such as MIC of Gram-positive pathogens (2.5–160 µg/mL) and MIC of Gram-negative pathogens (MIC of 40–160 µg/mL). The concentration of MS15 concerning MIC was contrasted to the value of MIC, which represents the standard antibiotic of bacitracin and vancomycin . AntimicEscherichia coli. A logarithm of viable colony count was analyzed with time intervals on multiple concentrations. Peptide MS15 decreased Escherichia coli count at 40 µg/mL (concentration of MIC) up to 12 h. An initial decrease in the amount of surviving Escherichia coli, at 40 µg/mL peptide concentrations was detected. E. coli cells recovered from the peptide activity after 12 h remaining and began to develop again. Mostly regrowth was noticed up to 120 µg/mL, although 160 µg/mL demonstrated a total decline of the E. coli count  by the concentration variation of peptide MS15 and reference standard ascorbic acid. Peptide MS15 exhibited concentration-dependent antioxidant activity in diverse in vitro antioxidant assay, containing scavenging of ABTS•+ and DPPH radicles. The results of the scavenging of DPPH and ABTS•+ were compared with reference standard ascorbic acid. In contrast, the constant free DPPH radical receives hydrogen radical or an electron from antioxidant and diminishes to develop a steady non-radical form DPPH-H, converted to diphenyl-picrylhydrazine molecule through resulting yellow in color and decline in absorbance. As displayed in ABTS•+ discoloration assay directly associates, including the significance of the scavenging capability of an antioxidant compound involving its hydrogen donating capacity, which was profoundly colored and was concluded by evaluating absorbance at 734 nm. Furthermore, in ABTS•+ scavenging assay, the ABTS•+ radicle was pre-formed by the oxidation process along with potassium persulfate, which could be decreased in the existence of hydrogen-providing antioxidants. Peptide MS15 showed ABTS•+ radical scavenging up to 68% in a concentration of 20 µM  in a concentration of 20 µM (p < 0.001) in a concentration of 20 µM  of MS15 was measured in RAW 246.7 cells, and human epithelial HeLa cells were thoroughly examined. The outcome demonstrated no cytotoxic effects at a concentration up to 40 µM, whereas the significantly less cytotoxic impact of up to 100 µM a.p < 0.001), 33.2% (*** p < 0.001), 45.1% (*** p < 0.001), and 65.4% (*** p < 0.001) at dosages of 2.5, 5, 10, and 20 µM, correspondingly  and detoxifying enzymes of phase II (HO-1), RT-PCR analysis was used to assess a concentration-dependent rise in the mRNA expression of GPx-1, CAT, and SOD1 a. RAW 26Escherichia coli cells after 12 h, which is consistent with the kinetics of other antimicrobial peptide reports [The principal goal of the study was to purify a potential antioxidant peptide that has potent antimicrobial activity and evaluate its antioxidant activity pathway. In the current study, we described the impact of the different components of carbon, nitrogen, and metal-ion sources of several culture conditions and growth media to get optimum bacteriocin activity. Growth media composition, temperature, and pH of dissolved peptide played a crucial part in the production and stability of bacteriocin activity. Peptide MS15 imposed cyclic structure so that the amino acid sequence could not be detected by using the Edman degradation method, although purification was detected with MALDI TOF/MS. The peptide was purified by two-step column chromatography with the 24.49-fold and recovered activity of 5.92%, that of total bacteriocin activity was found at 16000 AU, which was consistent with our previous report ,22. Prim reports ,39.•), superoxide anion (O2•), and hydrogen peroxide (H2O2) and are by-products of cellular metabolic effects, and pathways are intracellular, toxic organisms, partially formed by oxygen reduction (O2) [2• and thus, ROS was assumed to perform primarily as cellular damaging agents, responding extensively to lipids and proteins [• and ABTS•+ methods for evaluating the antioxidant potential of any molecule under study. Peptide MS15 exhibited a high ABTS•+ and DPPH• scavenging activity. The finding revealed that scavenging activity was performed by transferring hydrogen reaction (DPPH assay) or electron transfer reaction (ABTS assay). As presented in Oxidative stress is characterized as a discrepancy among free radical development and its removal by mechanisms of cellular defense. ROS consists of hydroxyl radical (OHion (O2) . ROS is proteins . From thproteins . The mosThe variation of color evaluated antioxidant power in the FRAP from the reduction of the yellow ferric complex to the blue ferrous complex. Otherwise, due to the smaller redox potential of CUPRAC reactive, citric acid was not oxidized, and sugar decline with the CUPRAC reactive. CUPRAC and FRAP are significant measures of reducing the ability of an antioxidant that is correlated with the earlier report  with the2 reduction forms the superoxide of cytosol (O2−) through the slipping of electrons from the electron transport chain of the mitochondrial electron carriers. It was entirely proven that SOD promptly transforms O2− into H2O2. Besides, the antioxidant enzymes, GPx, and CAT, will detoxify H2O2. Those enzymes function collectively as free radicals in the metabolic pathway [The power of cellular antioxidants derives from their immediate power, described as the ability to quench free radicals, with either electrons or hydrogen contributing to ROS. Simultaneously, indirect capability includes defending alongside oxidant stress by stimulating detoxify the expression and antioxidant enzymes in Phase II . During  pathway ,47. In t2O2. Such findings are in the substantial arrangement among other latest results indicating that HO-1 reestablished cell persistence by preventing apoptosis induced by oxidative damage. The antioxidant role of HO-1 performs by turning heme into the strong biliverdin prooxidant and, ultimately, bilirubin, which is a powerful antioxidant [Under normal conditions, the Keap1 protein binds Nrf-2 in the cytoplasm and performs a crucial part in Phase II enzyme stimulation when released and translocated to the nucleus . MS15 trioxidant . The intioxidant . The antDifferent synthesized antioxidant peptide analogs will investigate the antioxidant mode of action after elucidating the completed structural details of MS15 and assessing the subsequent findings, for instance, the phosphorylation of PI3K/Akt and MAPKs, and the luciferase assays. Thus, while further studies should be undertaken on the significance of several GSH-dependent enzymes, the present findings imply that MS15’s cellular protecting capacity against oxidative stress depends on, however, on the signaling pathway of Nrf2/HO-1 in RAW 264.7 cells.2O2 in RAW 264.7 cells. It was done through increasing the amount of cellular thiol, GST activity, and antioxidant enzyme expression and redox-sensitive transcription factor Nrf-2 expression. In conclusion, the present study showed that peptide MS15, which was purified from Bacillus velezensis, had significant and dose-dependent antioxidant capacity. The proposed underlying mechanism through which MS15 mitigates oxidative stress via translational and transcriptional control of oxidoreductase (phase I) and detoxifying enzyme (phase II).The identification of novel antimicrobial and antioxidant peptide MS15 against oxidant damage, throughout its potential effects, might assist well in understanding its action against the various pathophysiological situations related to oxidant stress. This analysis clearly shows that MS15 has a presence of antioxidant operation, the capability to move hydrogen atoms, and the capacity to avoid oxidative stress caused by H