Aspergillus and Fusarium mycotoxins were the most prevalent compounds identified, especially aflatoxins and fumonisin B1 in 100% and 95% of samples, respectively. Of the emerging toxins, beauvericin and enniatin type B showed high occurrences, with more than 90% of rice bran and maize contaminated, whereas zearalenone-14-glucoside and zearalenone-16-glucoside were found in rice bran in the range of 56–60%. Regulated mycotoxins (DON and ZEN) were the most frequent mycotoxin combination with emerging mycotoxins (BEA and ENN type B) in rice bran and maize. This study indicates that mycotoxin occurrence and co-occurrence are common in raw feed materials, and it is critical to monitor mycotoxin levels in ASEAN’s feedstuffs so that mitigation strategies can be developed and implemented.Raw feed materials are often contaminated with mycotoxins, and co-occurrence of mycotoxins occurs frequently. A total of 250 samples i.e., rice bran and maize from Cambodia, Laos, Myanmar, and Thailand were analysed using state-of-the-art liquid chromatography-mass spectrometry (LC-MS/MS) for monitoring the occurrence of regulated, emerging, and masked mycotoxins. Seven regulated mycotoxins – aflatoxins, ochratoxin A, fumonisin B Animal feed is an important part of the food chains and shows an enormous role in the safety and quality of animal by-products (meat products and dairy products) in the food supply chain . HoweverAspergillus genera [−1 and 12,000 µg·kg −1, respectively [Rice bran and maize are common feed ingredients used by ASEAN’s feed industry, especially for poultry and swine. Proportions of 10–30% rice bran and 48–60% maize are recommended for animal feed to guarantee suitable nutrient contents. Rice bran is an abundant rice by-product of rice grain milling, especially during rice harvest seasons. ASEAN rice production in 2020 (the crop year 2019/2020) was 187.49 million tons, with rice by-products reaching 20.62 million tons annually . In a prFusarium and Aspergillus species [In comparison, ASEAN maize production in 2020 (the crop year 2019/2020) was 44.94 million tons [ species ,10,11. 1 by legislation through the European Commission Directive 2002/32/EC [1 (FB1) and fumonisin B2 (FB2) [Mycotoxins in feed, particularly rice bran and maize, are a serious health concern in animals and humans that must be controlled and minimized. Dietary exposure of farm animals to mycotoxins has a detrimental impact on their health, performance, and productivity, with the severity and observed symptoms dependent on the type of mycotoxin present, concentration, and duration of exposure, gender, age, type of animal and exposure to other feed contaminants ,12. Addi02/32/EC  and limiB2 (FB2)  as shownOf the hundreds of mycotoxins identified to date, regulated mycotoxins are considered the most frequently found feed contaminants that can potentially induce subacute and acute toxic effects in farmed animals and, therefore, have been regulated in many parts of the world ,18. NeveThe occurrence of new and rising combinations of mycotoxins in food and feed shows the capacity of fungi to adapt to changing environmental conditions . Therefo1 (FB1), HT-2, T-2 toxin, ochratoxin A, zearalenone), emerging mycotoxins , beauvericin (BEA), enniatin type A (ENN A and A1), enniatin type B (ENN B and B1), ergocornine (Ergocor), ergocristine (Ergocris), stachybotrylactam (STLAC), sterigmatocystin (STC)), and masked mycotoxins , 15-acetyl deoxynivalenol (15-Acetyl-DON), deoxynivalenol-3-glucoside (DON-3-Glu), alpha-zearalenol (α-ZEN), beta-zearalenol (β-ZEN), zearalenone-14-glucoside (ZEN-14-G) and zearalenone-16-glucoside (ZEN-16-G)). These fungal metabolites are natural products produced by several Aspergillus, Fusarium, and Penicillium species. Emerging and masked mycotoxins are most commonly observed in food and feed [Regulated mycotoxins are routinely analysed and monitored by the ASEAN national laboratories; however, there is a limited analytical testing capability and regulatory framework for the other important mycotoxins in feed raw materials . Moreoveand feed ,29,30, wand feed . However2) for the tested mycotoxins were >0.990, showing good linearity for the calibration curves. Limits of detection (LOD) and limits of quantification (LOQ) for the compounds analysed ranged from 0.05–25 µg·kg−1 and 0.5–50 µg·kg−1, respectively, for rice bran, while for maize LODs and LOQs ranged from 0.04–25 µg·kg−1 and 1–50 µg·kg−1, respectively. The trueness and the precision (RSD%) of the method were satisfactory for all the studied compounds. In rice bran, RDSs of ≤13% and ≤15% were observed for repeatability (RSDr) and reproducibility (RSDR), respectively, for the target mycotoxins. In maize, RSD values of ≤15% and ≤14% for repeatability and reproducibility, respectively, were found. The method performance is shown in The multi-analyte method was validated based on the guidelines recommended by European Commission Regulation No. 2002/657/EC and SANTE/12089/2016. The coefficients of determination  in both rice bran and maize samples. The prevalence percentage of mycotoxin was calculated by the ratio of positive samples to total samples multiplied by 100. The prevalence of mycotoxins in rice bran was higher than in maize, with most samples indicating the presence of multiple mycotoxins, up to 7 mycotoxins, in rice bran samples.A total of 125 rice bran samples and 125 maize samples were analysed to determine contamination levels of mycotoxins. Regulated mycotoxins, including AFs, DON, FBcotoxins . The eme samples . ENN A1,1, AFB2, AFG1, AFG2), FB1, ZEN, OTA, DON, HT-2, and T-2; emerging mycotoxins including BEA, ENN B, ENN B1, STC, and STLAC and masked mycotoxins (ZEN-14-G and ZEN-16-G) as shown in A total of 125 rice bran samples were analysed for the presence the regulated mycotoxins i.e., AFs  and 94% (117 of 125), with maximum concentrations of 43.7 µg·kg−1 and 218.9 µg·kg−1, respectively , with STC found in 98% (122 of 125) of rice bran samples. The maximum concentrations of BEA, ENN B, ENN B1, and STC were 2281.0 µg·kg−1, 15.4 µg·kg−1, 14.7 µg·kg−1, and 272.3 µg·kg−1, respectively . In this study, 113 samples (90.4%) were co-contaminated with major and emerging mycotoxins, with some samples indicating the presence of 4 to 7 mycotoxins.The masked mycotoxins contaminating rice bran were ZEN-14-G and ZEN-16-G and occurred in 60% (75 of 125) and 56% (70 of 125) of the samples at maximum concentrations of 20.7 µg·kgectively . Only on1, ZEN, OTA, DON, HT-2, and T-2); emerging mycotoxins , and masked mycotoxins (ZEN-14-G and ZEN-16-G) were all detected in maize samples. The data were summarised and presented in boxplots  and 90% (113 of 125) of the samples with maximum concentrations of 20,900.9 µg·kg−1 and 65.5 µg·kg−1, respectively  were co-contaminated with both regulated and emerging mycotoxins, with some samples indicating the co-occurrence of five mycotoxins.This study found the co-occurring regulated mycotoxins  combinations in rice bran and maize samples. The positive samples (100%) were simultaneously contaminated with at least two mycotoxins. Rice bran samples were contaminated with four, five, six, and seven mycotoxins at 2.4%, 8.0%, 40.0%, and 49.6%, respectively, and the co-occurrence of 7 mycotoxins was observed in all four countries. Multiple mycotoxins were detected in maize at 2.4%, 19.2%, 24.0%, 40.8%, 13.6%, and 7.2% for two, three, four, five, six, and seven mycotoxins, respectively .1 and AFs+ZEN, which were detected in 100% of the rice bran samples, followed by AFs+OTA and AFs+DON at 99.2% and 93.6%, respectively. Furthermore, co-contamination of AFs+DON+OTA+FB1 was observed in 92.8% of the rice bran samples. In maize, AFs+FB1 and AFs+OTA, were the most frequent co-occurring mycotoxins at 96.8% and 90.4%, respectively. The combination of four regulated mycotoxins (AFs+DON+OTA+FB1) was observed in 37.6% of maize samples.The co-occurrence of regulated mycotoxins was found in rice bran and maize. The greatest co-occurrence was observed for AFs+FBThe co-occurring regulated and emerging mycotoxins found in rice bran and maize samples are presented in 1 was strongly correlated with ZEN (r = 0.702). HT-2 and T2 were also strongly correlated (r = 0.927)  (r = 0.898) and T2 and HT-2 (r = 0.971)  . Beauverctively) . In maiz= 0.971) .2 levels, and rainfall patterns are exacerbated as a result of the changing climate [aw) of more than 0.78, and relative humidity (RH) of 88% to 95% are all regarded as suitable for the growth of a fungal and subsequent generation of mycotoxin [Mycotoxins are harmful fungal metabolites frequently found in cereal grains and feed products. They have been shown to negatively impact food and feed safety because of the potential carry-over of mycotoxins to animal by-products, including meat products and dairy products, leading to possible mycotoxin uptake by humans as a consequence of the animal eating contaminated feed . The cli climate . The wea climate . The maiycotoxin . The Intycotoxin . Climateycotoxin .1 in maize were found in Southeast Asia in 2017 compared to 2013–2016 due to high rainfall and temperature during the spiking period [The studied samples were collected from Cambodia, Laos, Myanmar, and Thailand from August to December 2020. The average temperature range was 25 °C to 28 °C. Most of Southeast Asia experienced above-average rainfall between August and December; for example, the highest rainfall in Cambodia and Thailand was recorded in October of this year, while for Laos and Myanmar, the highest rainfall occurred during August ,39. Climg period , with th1, AFB2, AFG1, AFG2) with concentrations ranging between 0.1–2149.7 µg·kg−1. A prevalence of AFs was observed, with 100% of samples testing positive at values of 0.4–271.1 µg·kg−1 (mean level of 13.0 µg·kg−1) for rice bran and 0.1–2149.7 µg·kg−1 (mean level of 158.1 µg·kg−1) in maize with contamination in samples from Cambodia, Laos, Myanmar, and Thailand. The incidence of AFs, particularly AFB1, indicated that 2% of rice bran and 28% of maize samples positive for AFs were contaminated at a level higher than the European Commission’s maximum levels of 20 µg·kg−1 for feed materials [The results of this study indicated that of the 250-rice bran and maize samples tested, 100% of them indicated the presence of AFs  and 21.9–1728.4 µg·kg−1 (mean level of 183.2 µg·kg−1), respectively. The observed results indicated a higher prevalence than that reported by Kananub et al. [1 and OTA was detected in over 90% of maize samples with concentrations ranging from 1.8–20900.9 µg·kg−1 (mean concentration of 2786.0 µg·kg−1) and 0.3–65.5 µg·kg−1 (mean concentration of 12.0 µg·kg−1), respectively. Previously, researchers found that the varieties of maize significantly impacted the occurrence of OTA [1, ZEN, and OTA were lower than the safety limits. Guidance values set for ZEN and OTA are 2000 µg·kg−1 and 250 µg·kg−1, respectively, in cereals and cereal products for feedstuff. In maize by-product, guidance levels of 3000 µg·kg−1, and 60000 µg·kg−1 were set for ZEN, and the sum of FB1 and FB2, respectively.In this study, FBe of OTA . Comparee of OTA , the detEmerging mycotoxins are a new group of mycotoxins  that are−1 (mean 130.37 µg·kg−1) and 0.02–1459.18 µg·kg−1 (mean 60.67 µg·kg−1), respectively. These levels were higher than those reported in studies performed in Japan, China, and Korea [−1 [−1 [−1. BEA has been observed in corn-dried distiller grains in Thailand (350 µg·kg−1), Irish farm silages (21.8 µg·kg−1), and rice bran from Spain (64.8 µg·kg−1) [n = 83) of feed and raw feed materials analysed were contaminated with BEA, with a maximum level of 2326 g·kg−1 [BEA has been identified as being toxic in vitro to human tissues and cells . BEA hasnd Korea ,52,53. Borea [−1 . An evalµg·kg−1) ,55,56. F6 g·kg−1 .−1 (mean 6.73 µg·kg−1), whereas ENN B1 levels ranged between 0.53-14.70 µg·kg−1 (mean 2.58 µg·kg−1), with rice bran indicating higher levels than maize for these two mycotoxins. These results are higher than those found by Shimshoni et al. [−1 and 0.9 µg·kg−1, respectively, were detected in both corn and wheat samples. Jestoi et al. [1 in barley samples from Finland, at 3980 µg·kg−1 and 3240 µg·kg−1, respectively. According to Uhlig et al. [−1, and Sorensen et al. [−1. Monitoring of wheat in Romania for the presence of ENNs. Researchers reported the occurrence of ENN B in wheat and wheat flour, at 21–407 µg·kg−1 (71% of positive samples) [Fusarium spp. Therefore, this explains the higher concentrations of ENNs in the countries mentioned [Fusarium mycotoxins should be closely monitored particularly because of the possible synergistic effects of these mycoestrogens [Concerning ENNs type B, this study revealed that this mycotoxin was present in over 95% of rice bran samples analysed. Like BEA, ENNs cause cytotoxicity and genotoxicity. Both ENNs and BEA are cyclic hexadepsipeptides with similar biological pathways , however1 mean 2. µg·kg−1,i et al.  reportedg et al. , ENN B wn et al.  reportedsamples)  indicatisamples) ,63. In sentioned . This ststrogens ,64.1. Its hepatotoxicity and nephrotoxicity have been demonstrated in animals however, due to the limit of information on toxicity, the authorities have not prepared a risk assessment [−1. This is probably a result of the proliferation of toxin-producing fungi at the aleurone layer of rice bran [−1 and other foods at 20 µg·kg−1 [The occurrence of STC has been found in many types of food and feed ,67,68,69sessment . In our ice bran . Previouice bran . Althoug µg·kg−1 .There are limited data on the combined toxic effects of mycotoxins, therefore the health risks from multiple mycotoxin exposure are unknown. The natural prevalence of mycotoxins in food and feed is quite not unusual. Some fungi can produce more than one mycotoxin, multiple fungi may infect agricultural commodities, and the influence of climate will have a huge impact on contamination levels . The stu1 was higher than in the study in South America, which reported co-occurrence of AFs and FBs in maize at 7–8% [1+FB1 [In the study, the samples contained at least two mycotoxins, and the co-occurrence of AFs+FB at 7–8% ,75 and 5% [1+FB1 . The com% [1+FB1 . The co-% [1+FB1  in which% [1+FB1 . Previou% [1+FB1 , and aga% [1+FB1 ,82.1 and FB2), ZEA, and the trichothecenes, T-2 and HT-2 were found in 26.2% of maize samples in Croatian fields [Moreover, numerous surveys have revealed the co-occurrence of mycotoxins involving more than two mycotoxins. In 2016, DON, fumonisin . DON and ZEN were assessed in this investigation because it is one of the most highly distributed contaminants in feed. Exposure to high levels of these toxins is associated with function in animals; as an example, the chronic levels of DON and ZEN affect the kidney function in swine that is more sensitive to exposure to DON and ZEN than most other species . ConsumpMoreover, in the same study by Perez-Fuentes et al. , the comThe overall results show that the mycotoxins affecting rice crops and maize in Southeast Asia include AFs, FBs, DON, OTA, and ZEN as well as the emerging mycotoxins BEA, ENNs, and STC. Mycotoxin contamination of Southeast Asia crops will undoubtedly increase due to climate changes, including higher temperatures, increased rainfall, drought, and floods. Most of Southeast Asia has already experienced above-average temperature and above-average rainfall. According to global climate change scenarios, the global temperature will rise by up to 4.8 °C in the 21st century . In addiFactors such as poor-quality control, poor production technology, and poor crop storage condition encourage the growth of fungi and mycotoxin formation in developing countries, resulting in a higher prevalence of mycotoxin-contaminated foods ,93. Crop1, ZEN, OTA, DON, BEA, ENN type B, and STC were detected in greater than 90% of samples. This study demonstrates mycotoxin levels in all samples in compliance with EU regulations (except AFs and one sample for HT-2). Moreover, the results showed co-occurrence of Aspergillus and Fusarium mycotoxins were most commonly found in feed samples with a high prevalence of co-occurring of regulated mycotoxins (DON and ZEN) with emerging mycotoxins (BEA and ENN type B).The regulated mycotoxins, emerging mycotoxins, and masked mycotoxins were analysed in rice bran and maize samples from four Southeast Asia countries, Cambodia, Laos, Myanmar, and Thailand. The most prevalent mycotoxins in Southeast Asia from August to December 2020 were Afs, FBs, DON, OTA, and ZEN, and the emerging mycotoxins . AFs were detected in 100% of rice bran and maize samples. The difference in mycotoxin occurrence has been highlighted in the different sampling zones, the detection levels of AFs in rice bran were highest in samples sourced from Myanmar (Myanmar>Laos>Thailand>Cambodia) whereas the levels in maize were highest in samples from Laos (Laos>Cambodia>Myanmar>Thailand). FBA total of 125 rice bran and 125 maize samples, commonly used to formulate feed to meet the energy requirements of livestock animals were collected from major feed mills in Cambodia, Laos, Myanmar, and Thailand, from August to December 2020. Each aggregate sample had approximately 1 kg and was collected according to the European Commission Regulation (EC) No 152/2009 guidelines. All samples were ground into powder using a Tecator Cyclotec 1093 mill fitted with a 1 mm sieve , then stored at −20 °C before mycotoxin analysis.v/v/v) was added, and the sample was vortexed for 90 min. After centrifugation at 5000 rpm for 15 min, an aliquot of 250 µL of the supernatant was mixed with 750 µL of acetonitrile: water: formic acid  in an Eppendorf tube. The mixture was vortexed for 30 s and filtered through a 0.2 µm PTFE syringe filter into an LC-MS/MS vial for analysis.Sample extraction was performed using a dilute-and-shoot approach ,96, with1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), alternariol, beauvericin (BEA), deoxynivalenol (DON), enniatin A (ENN A), enniatin A1 (ENN A1), enniatin B (ENN B), enniatin B1 (ENN B1), fumonisin B1 (FB1), HT-2 toxin (HT-2), ochratoxin A (OTA), T2 toxin (T-2), and zearalenone (ZEN) were obtained from Romer lab, UK. Ergocornine (Ergocor), ergocristine (Ergocris), sterigmatocystin (STC), and stachybotrylactam (STLAC) were obtained from Sigma-Aldrich . 3-Acetyl-deoxynivalenol (3-Acetyl-DON), 15-Acetyl-deoxynivalenol (15-Acetyl-DON), deoxynivalenol-3-glucoside (DON-3-Glu), zearalenone-14-glucoside (ZEN-14-G) and zearalenone-16-glucoside (ZEN-16-G) were obtained from Enzo science . From the solid standards, individual stock solutions were prepared at a 1 mg·mL−1 concentration in an appropriate solvent and stored based on the manufacturer’s instructions. All standard solutions were kept in amber glass vials at −20 °C and brought to room temperature before use. The stock solutions were renewed every 2 months, while multi-mycotoxin working solutions were prepared weekly.Ammonium hydroxide (≥25% in water), LC−MS grade methanol, and acetonitrile were obtained from Sigma-Aldrich . A Milli-Q system  was used as a source of deionized water. Mycotoxins standards: aflatoxin Bv/v/v) and mobile phase B—methanol/water/acetic acid 97:2:1 (v/v/v), both containing 5 mM ammonium acetate buffer. Mycotoxins were eluted following a gradient elution program as follows: 0 min 1% B, held for 1 min at 1% B, 5 min 65% B, 7 min 80% B, 8.5 min 80% B, 9 min 95% B, 10 min 95% B and 11.5 min 1% B. A mobile phase flow rate was maintained at 0.7 mL·min−1, with sample injection volume set at 3 µL, and the total runtime was 11.5 min.Quantitative analysis of the targeted mycotoxins in each feed raw materials was carried out on an ExionLC™ AD Ultra-High Performance Liquid Chromatography system  coupled with a SCIEX 5500+ QTrap triple quadrupole Mass Spectrometer (MS/MS) equipped with a Turbo V™ electrospray ionisation source . Chromatographic separation was achieved using a Gemini C18-column  maintained at 30 °C. Elution was carried out in a binary gradient mode consisting of mobile phase A—methanol/water/acetic acid 10:89:1  mode. The capillary voltage and source temperatures were set at 4.5 kV and −4.5 kV for ESI+ and ESI- respectively, with the temperature set at 600 °C. Collision gas, ion source gas (GS1), ion source gas (GS2), and curtain gas were set at 9, 60, 60, and 35 psi, respectively. Two MRM characteristic transitions  were monitored for each analyte. The selected MRM transitions and their respective analyte-dependent operating conditions, i.e., declustering potential (DP), collision cell exit potential (CXP), and collision energy (CE), are listed in The optimised LC-MS/MS method for the analysis of mycotoxins in rice bran and maize was validated based on the acceptable performance criteria of analytical methods set by the European Commission regulations No. 2002/657/EC . The per−1), medium (50 ng·g−1), and high (200 ng·g−1). Spiked samples were placed in the dark overnight to allow evaporation of the solvent and interaction of the analyte with the matrix. The spiked and blank (control or unspiked) samples were extracted as described above. Following sample extraction, blank samples were also spiked with the same levels of multi-mycotoxin solution to determine the extraction efficiency/recovery. Recovery of each analyte was calculated as the ratio of peak area of blank sample spiked before and after sample extraction multiplied, expressed as a percentage (%). Matrix-induced suppression/enhancement (SSE) was determined by comparing the response of the matrix spiked with standards after extraction to a solvent standard at the same concentration. SSE was calculated as the ratio of the peak area of the analyte in the matrix and solvent, expressed as a percentage (%).Extraction and apparent recoveries were determined by spiking homogenised feed raw materials at three different levels with a multi-mycotoxin standard solution  of each analyte. LODs and LOQs determined the sensitivity of the developed method. LOD was defined as the concentration of each analyte that gave a peak with a signal-to-noise ratio (S/N) of 3, which was determined by injecting neat solvent standard solution at different concentrations, while LOQ was defined as the concentration of the analyte in spiked samples at an S/N ratio of 10. Precision was determined by intra-day precision (repeatability) and inter-day precision (reproducibility). Intra-day precision was carried out by analysis of three replicates on the same day at three different concentration levels, while inter-day precision was assessed by repeating the same procedure over three consecutive days. The data were used to calculate within-laboratory accuracy and precision and expressed as relative standard deviation (RSD). The criteria for confirming a positive sample include a retention time within ±0.5% compared with the analyte in a pure solvent, both qualifier and the quantifier with transitions above S/N ratio of 10:1, and the ion ratio of the quantifier and the qualifier transition within ±25%.p < 0.05 and p < 0.01 values were considered statistically significantly different using the SPSS 10.0 software .The Pearson’s correlation coefficient was used to calculate the correlation; data with


S)-Sakuranetin is a 7-O-methylflavonoid that has anticancer, antiviral, and antimicrobial activities. Methylation process is involved in biosynthesizing (2S)-sakuranetin from (2S)-naringenin, in which S-adenosylmethionine (SAM) serves as the methyl donor. In this study, after methyl donor and substrate inhibition were identified as limiting factors for (2S)-sakuranetin biosynthesis, an efficient (2S)-sakuranetin-producing strain was constructed by enhancing methyl donor supply and cell tolerance to (2S)-naringenin. Firstly, PfOMT3 from Perilla frutescens was selected as the optimal flavonoid 7-O-methyltransferase (F7-OMT) for the conversion of (2S)-naringenin to (2S)-sakuranetin. Then, the methylation process was upregulated by regulating pyridoxal 5′-phosphate (PLP) content, key enzymes in methionine synthesis pathway, and the availability of ATP. Furthermore, genes that can enhance cell resistance to (2S)-naringenin were identified from molecular chaperones and sRNAs. Finally, by optimizing the fermentation process, 681.44 mg/L of (2S)-sakuranetin was obtained in 250-mL shake flasks. The titer of (2S)-sakuranetin reached 2642.38 mg/L in a 5-L bioreactor, which is the highest titer ever reported. This work demonstrates the importance of cofactor PLP in methylation process, and provides insights to biosynthesize other O-methylated flavonoids efficiently in E. coli.(2  ConsideS)-Sakuranetin is produced in plants in response to ambient pressures. For example, ultraviolet light or pest infection could activate 7-O-methyltransferase (7-OMT) in Oryza sativa to synthesize (2S)-sakuranetin [S)-sakuranetin. In a previous study, (2S)-sakuranetin was synthesized by chemically utilizing the regioselective deacetylation of naringenin triacetate [S)-sakuranetin could be produced from (2S)-naringenin by flavonoid 7-O-methyltransferases (F7-OMTs) catalysis in E. coli. In a previous report, 40 mg/L of (2S)-sakuranetin was obtained by constructing a de novo biosynthesis pathway in E. coli [S)-sakuranetin was produced in a 2.5-L bioreactor by using an E. coli–E. coli modular co-culture system [(2uranetin . Chemicaiacetate . However E. coli . 79 mg/Le system .S-adenosylmethionine (SAM)-dependent flavonoid methyltransferases that can transfer the methyl group from SAM to the 7-OH of flavonoids [E. coli can restrict the accumulation of (2S)-sakuranetin. Previous studies focused on balancing the biosynthesis of (2S)-naringenin during the biosynthetic process of (2S)-sakuranetin, and there are no studies on strengthening the methylation of (2S)-naringenin to improve the production of (2S)-sakuranetin. In addition, flavonoids can interact with DNA, inhibit DNA gyrase [E. coli. Considering that microbes have developed certain mechanisms to adapt to environmental stress, various genetic strategies can be used to alleviate cell pressure, such as overexpression of molecular chaperones to refold the denatured proteins [E. coli stress response might enhance cell tolerance to flavonoids so as to relieve substrate inhibition.F7-OMTs are avonoids . The limA gyrase  and damaA gyrase , which wproteins  and smalproteins ,19. OverS)-sakuranetin from (2S)-naringenin efficiently. Firstly, the optimal F-7OMT was selected. Then key enzymes  and cofactors  essential for SAM biosynthesis were regulated to promote the methylation reaction. Moreover, the availability of an indispensable cofactor in the transsulfuration pathway, pyridoxal 5′-phosphate (PLP), was enhanced to increase the production of (2S)-sakuranetin. Additionally, sRNA RpoS was identified to be effective in enhancing cell tolerance to (2S)-naringenin. Finally, via the combinatorial expression of genes that were beneficial for (2S)-sakuranetin accumulation, and the optimization of fermentation process, 681.44 mg/L of (2S)-sakuranetin was obtained in shake-flask scale. The titer of (2S)-sakuranetin reached 2642.38 mg/L when scaled up in a 5-L bioreactor.In this study, methylation process and substrate tolerance were enhanced to obtain (222.1E. coli JM109 was used for plasmid propagation. E. coli BL21(DE3) was used for protein expression. The genes  were synthesized by Sangon Biotech . The pET-28a (+), pACYCDuet-1 and pCDFDuet-1 expression vectors were obtained from Novagen . High-fidelity Phusion DNA polymerase from Vazyme  was used for amplification. Seamless cloning kit purchased from Sangon Biotech (Shanghai) was used for plasmid assembly. The process for plasmids construction and primers are listed in the Supplementary File and The strains used in this study are listed in 2.2S)-sakuranetin accumulation. Strains were cultured at 37 °C for 10 h in LB medium. Then a 2% (v/v) of LB strain culture was inoculated into the TB medium with shaking at 220 rpm. Isopropyl beta-D-thiogalactoside (IPTG) was added to the medium at a final concentration of 0.1 mM when OD600 reached 0.8–1, at which time the temperature was shifted from 37 °C to 25 °C. 350 mg/L of (2S)-naringenin was added after adding IPTG for 3 h and 13 h to a final concentration of 700 mg/L. The total fermentation time in shake-flask scale was 26 h.The Luria–Bertani (LB) medium was used for culturing seed liquid. The Terrific Broth (TB) medium was used for (22.3S)-naringenin was dissolved in methanol to a concentration of 50 g/L. The optimal strain was selected for scale-up culture in a 5-L bioreactor. Colonies from the culture plate were inoculated into LB liquid medium and incubated at 37 °C with shaking at 220 rpm for 10 h. Then, 4% (v/v) inoculum of seed culture was inoculated into a 5 L bioreactor containing 2.5 L of the TB medium. In the early stage, the fermentation temperature was kept at 37 °C and the rotating speed was 400 rpm. IPTG was added at a final concentration of 0.1 mM when the OD600 reached 6, at which time the temperature was shifted to 25 °C and the stirring speed was changed to the pattern of depending on dissolved oxygen content (40%). (2S)-Naringenin was fed in batches at 6.5 h, 12.5 h, 18.5 h, 21.5 h, 41.5 h, and 48 h to a final concentration of 4 g/L. 500 g/L of glycerol was fed at a speed of 8 mL/h after substrate was added, and 6 mM Mg2+ was added at 6.5 h. The pH was maintained at 7 ± 0.1 with 6 M NaOH during the fermentation.Bioreactor fermentation was conducted in a 5-L glass bioreactor  containing 2.5 L of TB medium. The (22.4S)-sakuranetin, a 500 μL fermentation sample was mixed with 500 μL methanol. Then, the supernatant was filtered using a 0.22-μm organic phase filter membrane after 12000g centrifugation for 5 min. The samples were detected by HPLC  equipped with a Thermo Fisher C18 column  at 290 nm. The mobile phase was as follows: A phase was water containing 0.1% TFA, and B phase was acetonitrile containing 0.1% TFA. The flow rate was 1 mL/min. The following procedure was used: 0–10 min, 10–60% B; 10–20 min, 40–80% B; 20–25 min, 80–10% B. The determination of the intracellular PLP level followed the procedure described by Cabo et al. [To quantitatively analyze the titer of , and 2 μL of seeds dilution were spotted on solid LB plates containing (2S)-naringenin . These LB plates were then incubated for 12 h at 37 °C.33.1S)-naringenin to (2S)-sakuranetin. In order to select the optimal F7-OMT, the production of (2S)-sakuranetin at different (2S)-naringenin concentrations with strains expressing F7-OMTs were compared. When 300 mg/L (2S)-naringenin was added after cells were induced for 10 h at 25 °C, 26.76 mg/L, 44.54 mg/L and 58.95 mg/L of (2S)-sakuranetin was achieved by strains harboring OsNOMT, SaOMT2 and PfOMT3, respectively (S)-naringenin. Therefore, PfOMT3 was chosen for the following experiments. The strain expressing PfOMT3 is referred as strain 7-FOMT-3. (2S)-Sakuranetin titer of strain 7-FOMT-3 showed a decreasing trend with the increase of (2S)-naringenin concentrations, and (2S)-sakuranetin titer decreased sharply when (2S)-naringenin concentration exceeded 250 mg/L (S)-naringenin was added in methylation enhancement experiments.Previous studies have confirmed that OsNOMT , SaOMT2 ectively A, and Pf250 mg/L B. To rem3.2S)-naringenin to (2S)-sakuranetin (S)-sakuranetin, 250 mg/L (2S)-naringenin and different concentrations of methionine were added after cells were induced for 10 h at 25 °C (S)-sakuranetin was obtained after reaction for 10 h, which was 36.17% higher than that of the strain 7-FOMT-3. In order to enhance methionine production of E. coli, metA (encoding homoserine succinyltransferase) and cysE (encoding l-serine O-acetyltransferase) were overexpressed (strain NS01). (2S)-Sakuranetin titer of strain NS01 reached 142.59 mg/L without the addition of methionine, which was 50.26% higher than the strain 7-FOMT-3. When metK (encoding SAM synthetase) was overexpressed in strain NS01, 135.26 mg/L of (2S)-sakuranetin was obtained, a slight decrease compared with the strain NS01.Methyl donor SAM is involved in the conversion of -sakuranetin titer increased by 62.38% and 124.45%, respectively, compared with the strain 7-FOMT-3 (POS5 from S. cerevisiae was heterogeneously expressed in strain 7-FOMT-3 (resulting in strain NS14), improving the titer of (2S)-sakuranetin to 132.58 mg/L, which was 43.35% higher than that of the strain 7-FOMT-3 (S)-naringenin to (2S)-sakuranetin. Regulating the levels of methionine and ATP are essential for (2S)-sakuranetin production.ATP is consumed in the conversion of methionine to SAM, and is an important cofactor in methionine biosynthesis pathway. 7-FOMT-3 B. NADPH 7-FOMT-3 C. These 3.3S)-sakuranetin production, 250 mg/L (2S)-naringenin and different concentrations of PLP were added after cells were induced for 10 h at 25 °C. The result showed that 173.61 mg/L (2S)-sakuranetin was obtained when 90 μM PLP was added, which increased by 79.85% compared with the group without PLP -sakuranetin titer reached 185.35 mg/L without the addition of PLP, which increased by 92.39% compared with strain 7-FOMT-3. In addition, RFC4 and RPS18B from S. cerevisiae [S)-sakuranetin titer of strain NS06 (expressing RFC4 in strain 7-FOMT-3) and NS07 (expressing RPS18B in strain 7-FOMT-3) increased by 59.59% and 45.69%, respectively, compared with the strain 7-FOMT-3. Then SNZ3, RFC4 and RPS18B were co-expressed in strain 7-FOMT-3, resulting in strain NS11; the titer of (2S)-sakuranetin reached 260.45 mg/L, which was 170.34% higher than that of the strain 7-FOMT-3 (S)-sakuranetin production.PLP is known to be a cofactor in the transsulfuration pathway . To verihout PLP A. In ord7-FOMT-3 B, and th3.4S)-naringenin and obtain a higher titer of (2S)-sakuranetin, (2S)-naringenin was added in batches , in which the half quantity of (2S)-naringenin was added after adding IPTG for 1 h, and another 50% of (2S)-naringenin was added after 10 h. The result showed that strain NS17 was the best to accumulate (2S)-sakuranetin, and 451.47 mg/L of (2S)-sakuranetin was obtained when added 500 mg/L (2S)-naringenin (POS5 was expressed with PfOMT3 (strain NS14), this could promote the production of (2S)-sakuranetin, but (2S)-sakuranetin titer decreased when POS5 was co-expressed with genes that enhanced the methylation reaction. Strain NS18 (expressing POS5 in NS17) accumulated only 191.56 mg/L of (2S)-sakuranetin when added 500 mg/L (2S)-naringenin, resulting in a 57.57% decrease compared with the strain NS17. Based on the (2S)-sakuranetin titer, genes combination in strain NS17 was the best for strengthening the methylation process.Genes that can upregulate methylation reaction were expressed by combination. To avoid the negative effect of high concentration of (2ringenin . When PO3.5S)-sakuranetin titer decreased with increasing (2S)-naringenin concentrations, which may due to the low tolerance of strain to (2S)-naringenin. Ribosomal subunits -naringenin. (2S)-Naringenin was supplemented in batches to a final concentration of 400 mg/L, 500 mg/L and 600 mg/L (substrate was added as described in Section S)-sakuranetin titer with strain NS21, NS23 and NS24 was obviously increased at different (2S)-naringenin concentrations compared with the strain 7-FOMT-3 (S)-naringenin was added, the production of (2S)-sakuranetin increased by 108.56%, 122.63%, and 104.26% compared with the strain 7-FOMT-3, respectively.The (2and RpsL ), molecuand RpsL , Ycdy [2and RpsL , Nfua [2and RpsL , and Yajand RpsL ), sRNAs and RpsL , NusB [3and RpsL , AcrR [3and RpsL , and Asrand RpsL ), and thand RpsL , which c7-FOMT-3 A–C; whenPfOMT3-rpsQHis31Pro, pET-28a (+)-PfOMT3-rpoS and pET-28a (+)-PfOMT3-secB, respectively. Genes rpsQ, rpoS and secB can relieve cell pressure to a certain extent; the gene rpsQ can reduce protein mistranslation [rpsQHis31Pro has been proven to have better stress tolerance [secB can prevent protein aggregation [rpoS can increase energy metabolism under stress [rpsQHis31Pro, rpoS and secB to (2S)-naringenin, spot assay was conducted. Strain NS21, NS23 and NS24 all grew better than strain 7-FOMT-3 on solid LB plates containing (2S)-naringenin -PfOMT3-rpoS-rpsQHis31Pro plasmid (NS32) accumulated less (2S)-sakuranetin, resulting in a 36.62% and 40.62% decrease compared with strain NS21 and NS23, respectively, when added 500 mg/L (2S)-naringenin -nslation  and rpsQolerance , the genregation  and promregation ,40, and r stress  and regur stress . To evalringenin D. Then, ringenin E, which 3.6S)-sakuranetin was further improved when substrate tolerance enhancement genes rpoS, secB and methylation enhancement genes were co-expressed. The result showed strain NS34 accumulated the highest titer of (2S)-sakuranetin; 545.98 mg/L of (2S)-sakuranetin was obtained when 600 mg/L (2S)-naringenin was added -naringenin was first supplemented after IPTG was added for 1 h, 3 h, 5 h, 7 h and 9 h , respectively. The result showed that the optimal substrate addition time was 3 h after the addition of IPTG (2+ can activate the leaving group (ATP and giving phosphate) in the biosynthesis of SAM [2+, different concentrations of Mg2+ were added. 681.44 mg/L of (2S)-sakuranetin was obtained when added 6 mM Mg2+ (S)-sakuranetin was obtained at 70 h from 4 g/L (2S)-naringenin -naringenin to (2S)-sakuranetin. Given that exogenously supplied of methionine could improve (2S)-sakuranetin titer and that the conversion efficiency decreased with increasing concentration of (2S)-naringenin, methyl donor and cell tolerance to substrate were considered as limiting factors for (2S)-sakuranetin biosynthesis. Experiments were performed to upregulate methionine, cofactor ATP and PLP content to enhance methylation process, and to select the best molecular chaperone, sRNA or ribosomal subunits to improve strain tolerance to (2S)-naringenin. No previous studies have focused on enhancing the methylation process and improving the bacterial resistance to achieve the efficient synthesis of (2S)-sakuranetin.Previous studies have revealed that metK in E. coli), plays an important role in methylation process. As the direct precursor of SAM, methionine content was enhanced by upregulating key enzymes MetA [S)-sakuranetin increased by 50.26% compared with strain 7-FOMT-3. (2S)-Sakuranetin titer decreased when metK was overexpressed, which may due to the overproduction of metK would consume large amount of ATP and cause a methionine deficiency [The methyl donor SAM, biosynthesized from methionine and ATP by SAM synthetase -sakuranetin increased by 162.38% in the ydaO-expressing strain compared with strain 7-FOMT-3. POS5 is known to be involved in NADPH regeneration in S. cerevisiae and can increase the intracellular NADPH availability in E. coli [S)-Sakuranetin titer increased when POS5 was expressed in strain 7-FOMT-3. However, the titer of (2S)-sakuranetin decreased when POS5 was co-expressed with metA, cysE, ydaO, and the PLP biosynthesis genes. Considering that the disturbance of NADPH pool would trigger the redistribution of metabolic flux in E. coli [S)-sakuranetin titer. In addition, the extreme metabolic burden of overexpression too many foreign genes may also result in the decline of (2S)-sakuranetin production.Methylation proceess was further upregulated by regulating ATP and NADPH contents, which are important cofactors for SAM biosynthesis. Previous studies proved that  E. coli ,48. The  E. coli . (2S)-Sa E. coli , it was S)-sakuranetin was explored. RFC4 and RPS18B are known can increase intracellular pyridoxine and pyridoxamine concentrations [E. coli and S. cerevisiae [RPS18B and RFC4 from S. cerevisiae were heterologously expressed in E. coli. The (2S)-sakuranetin titer increased by 180.72% when SNZ3, RPS18B, and RFC4 were co-expressed compared with the strain 7-FOMT-3. This result provides a valuable insight to biosynthesize other methylated products in E. coli, and demonstrates the significance of PLP in methylation process.Enzymes involved in the transsulfuration pathway utilize PLP as cofactor , whethertrations , and pyrtrations . Given trevisiae , RPS18B S)-sakuranetin titer decreased with increasing concentrations of (2S)-naringenin. Therefore, substrate resistance was considered as another limiting factor. Genes that can enhance cell tolerance to (2S)-naringenin were identified in this study. Strains expressing rpsQHis31Pro, rpoS, and secB showed better performance on (2S)-sakuranetin production and cell growth. The spot assay reflected more intuitively that RpsQHis31Pro, RpoS, and SecB protein could improve the (2S)-naringenin tolerance of host cells. This result provides an idea to alleviate cell stress in biosynthesizing other natural products with antibacterial effect in E. coli, and the selected genes may also be used to relieve cells pressure in the conversion of other flavonoids.Flavonoids are known to have antimicrobial properties; they can inhibit DNA gyrase , alter tS)-sakuranetin-producing strain was constructed by F7-OMTs selection, methylation process enhancement, cell resistance improvement and fermentation process optimization. The titer of (2S)-sakuranetin reached 2642.38 mg/L in a 5-L bioreactor, which is the highest titer reported to date. Given that flavonoid O-methyltransferases (FOMTs) have substrate specificity [In conclusion, an efficient (2cificity , other vQiumeng Sun: Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Song Gao: Formal analysis, Writing - review & editing. Shiqin Yu: Methodology, Supervision. Pu Zheng: Methodology, Supervision. Jingwen Zhou: Methodology, Supervision, Funding acquisition, Writing - review & editing.The authors declare that they do not have any financial or commercial conflict of interest in connection with the work submitted.


Ocimum campechianum Mill. (Peruvian basil) is an essential oil-bearing plant of the Lamiaceae family. Volatile oil produced through steam distillation of Peruvian basil was examined to establish the aromatic and stable isotope profiles of samples (n = 9) from three different cultivated plots in Peru. The resulting essential oils were analyzed by GC/FID, GC/MS, and GC/IRMS. In accordance with findings from other researchers, multiple chemotypes, defined by the most abundant aromatic compounds, exist within these populations. Overall, 55% of samples are the eugenol chemotype , 33% are the methyl eugenol chemotype , and a single sample is a mixture of both chemotypes, containing high levels of both eugenol (38.1%) and methyl eugenol (8.6%). Stable isotope ratios, δ2H and δ13C, performed on prominent compounds provide supporting data for distinguishing chemotypes. Complete aromatic profiles, stable isotope ratios, and essential oil yield are established for each sample. This study confirms the existence of multiple chemotypes and, for the first time, to the author’s best knowledge, establishes stable isotope ratios for O. campechianum essential oil, which proves a useful tool in further investigating plant metabolism and determining essential oil authenticity.  Ocimum campechianum Mill. , is an essential oil-bearing plant of the Lamiaceae family that is native to the Americas, including a wide geographic region in North and South America and several islands in the Caribbean islands [ islands ,2. Given islands ,4,5. The islands ,7. Other islands ,9,10.Many morphological parts of the Peruvian basil plant have associated ethnobotanical significance, with use of the leaves as the most common practice. In some areas, this plant is viewed as a valued and prized remedy  and/or uMany of these traditional uses have been substantiated through recent studies. While most studies reviewed here did not show strong antibacterial properties of Peruvian basil essential oil ,15,16, aE-cinnamate [E)-caryophyllene [The active properties of Peruvian basil essential oil are contingent on the aromatic profile. Multiple chemotypes, defined by the most abundant aromatic compounds in the essential oil, exist within this species including those with prominent aromatic compounds being methyl eugenol ,25, 1,8-innamate , (E)-carphyllene , methyl phyllene , or eugephyllene ,26,29,30phyllene ,29,30, sphyllene , and plaphyllene . O. campechianum essential oil. Distillation yields are also reported. Results provide further insight into chemical variation and provide fundamental data for continual substantiation of ethnobotanical applications. This study confirms the existence of multiple chemotypes within cultivated populations in Peru and, for the first time, to the author’s best knowledge, establishes stable isotope ratios for Consistent with previous findings ,26,29,30δ2H and δ13C, were established for prominent compounds in essential oil samples. Prominent compounds are defined by the relative area % of a given compound being >5% for all samples from a region, including 1,8-cineole (samples D–I), cis-β-ocimene (samples D–I), eugenol (samples D–I), methyl eugenol (samples A–C), and (E)-caryophyllene (samples A–I). Stable isotope data is detailed in Stable isotope ratios, Essential oil yield is detailed in Ocimum campechianum essential oil. According to the aromatic profiles, two chemotypes exist within these samples (n = 9). The three samples from the Pueblo Libre Region (samples A–C) conform to the methyl eugenol profile chemotype, the samples from the El Diamante (samples D–F) and Villa Rica  regions conform to the eugenol profile chemotype, and sample H, which contains a high relative area % of both eugenol (38.1%) and methyl eugenol (8.6%), could be considered a mixture of the two chemotypes. In the scientific literature previously reviewed, Peruvian basil chemotypes included both the eugenol [This study provides the essential oil profiles and, for the first time, to the author’s best knowledge, establishes stable isotope ratios for  eugenol 8.1% and ples n = . The thr eugenol ,26,29,30 eugenol ,25 chemoδ13C results of all four compounds analyzed from sample H are outliers within the Villa Rica group. As a result, the standard deviation for δ13C according to collection area is up to 24× larger in the Villa Rica compared to the El Diamante collection areas -caryophyllene is the only data measured for all 9 samples, due to the abundance of this compound in each sample. (E)-caryophyllene δ13C ranges for the Pueblo Libre collection area are −32.850‰ to −32.688‰ and the ranges for the El Diamante and Villa Rica collection areas, excluding sample H, are −29.838‰ to −28.059‰. The (E)-caryophyllene δ13C value for sample H sits in between each range, at −31.612‰. These data provide further support that stable isotope ratios may be a useful tool in chemotyping by providing additional insight into the metabolism and carbon sequestration of the plant. The stable isotope data for  × 3 (distilleries) = 9 ). Plant material was collected and distilled during the first and second week of January 2022. Plant material was collected within a 200 m radius of the following three locations: Pueblo Libre: 5°52′46.8″ S 77°7′27.6″ W, 830 m elevation; El Diamante: 5°44′19.9″ S 77°30′0.2″ W, 950 m elevation; Villa Rica: 5°47′2.7″ S 77°28′52.3″ W, 935 m elevation. Collected plant material is representative of the above ground portions of the plant, primarily consisting of the aromatic leaves and inflorescence , 41 and 93 (β-pinene), 93 and 119 (α-trans-bergamotene), and 93 and 121 (γ-elemene). Volatile compounds were quantified and are reported as a relative area percent by GC/FID using an Agilent 7890B and Agilent J & W DB-5, 60 m × 0.25 mm, 0.25 μm film thickness, fused silica capillary column. Operating conditions: 0.1 μL of sample , 25:1 split injection, initial oven temperature at 40 °C with an initial hold time of 2 min, oven ramp rate of 3.0 °C per minute to 250 °C with a hold time of 3 min, helium carrier gas. Essential oil samples were analyzed in triplicate by GC/FID to ensure repeatability . Compounds were assigned using retention indices coupled with the retention time data of reference compounds . Essential oils were analyzed, and volatile compounds identified, by GC/MS using an Agilent 7890B GC/5977B MSD  and Agilent J&W DB-5, 60 m × 0.25 mm, 0.25 μm film thickness, fused silica capillary column. Operating conditions: 0.1 μL of sample , 150:1 split ratio, initial oven temperature of 40 °C with an initial hold time of 5 min, oven ramp rate of 4.5 °C per minute to 310 °C with a hold time of 5 min, helium carrier gas. The electron ionization energy was 70 eV, scan range 35–650 amu, scan rate 2.4 scans per second, source temperature 230 °C, and quadrupole temperature 150 °C. Volatile compounds were identified using the Adams volatile oil library  using ChThe hydrogen and carbon stable isotope ratios of essential oils were analyzed by GC/IRMS using a Thermo TRACE 1310 gas chromatograph coupled to a Thermo Delta V Advantage Isotope Ratio Mass Spectrometer , with an Agilent J & W DB-5, 60 m × 0.25 mm, 0.25 μm film thickness, fused silica capillary column, and a 10.0 μL syringe. 2H/1H analysis. From the second sample vial prepared for 2H/1H analysis, a 90 uL aliquot was removed and placed into a third vial. The resulting solution was brought to 1 mL in hexane and used for 13C/12C analysis.Essential oil samples were prepared for GC/IRMS analysis in the following manner: 35 mg of sample was weighed into a 2 mL clear glass vial and brought up to 1 mL with hexane. A 100 µL aliquot was removed and placed into a second vial. The second vial was then brought up to 1 mL with hexane. This first aliquot was then used for 2H/1H analysis or the combustion reactor for 13C/12C analysis. HTC reactor temperature was set to 1410 °C and was regularly conditioned by injecting 1 µL of hexane in backflush mode. Combustion reactor temperature was set to 1000 °C. This reactor was also conditioned with oxygen at regular intervals.GC operating conditions: splitless injection of 1 μL of sample with splitless time set at 0.25 min, injection port 270 °C, initial oven temperature of 50 °C with initial hold time of 2.0 min, oven ramp rate of 6.0 °C per minute to 250 °C with a hold time of 2.0 min, then an oven ramp rate of 10.0 °C per minute to 310 °C with a hold time of 7.0 min, helium carrier gas with constant flow 1.55 mL/min. After passing through the capillary column, samples were sent through the HTC reactor for δ2H isotope ratios are expressed relative to VSMOW and δ13C isotope ratios to VPDB. The following three reference materials, along with their known values, were used to normalize results: hexadecane #C (USGS69), δ2H: 381.4‰, δ13C: −0.57‰; nonadecane #2, δ2H: −56.3‰, δ13C: −31.99‰; and tetradecanoic acid methyl ester #14M, δ2H: −231.2‰, δ13C: −29.98‰. To normalize IRMS results, reference materials were purchased from Dr. Arndt Schimmelmann at Indiana University and from the United States Geological Survey (USGS)—Reston Stable Isotope Laboratory. cis-β-ocimene (samples D–I), eugenol (samples D–I), methyl eugenol (samples A–C), and (E)-caryophyllene (samples A–I). δ2H values are reported with a standard deviation < 2.8‰ and δ13C values are reported with a standard deviation < 0.2‰.Samples were analyzed in triplicate to ensure repeatability. Isotope ratios were determined for the following prominent volatile compounds: 1,8-cineole (samples D–I),


Further preclinical and clinical investigations are warranted.Cancer can develop due to abnormal cell proliferation in any body’s cells, so there are over a hundred different types of cancer, each with its distinct behavior and response to treatment. Therefore, many studies have been conducted to slow cancer progression and find effective and safe therapies. Nutraceuticals have great attention for their anticancer potential. Therefore, the current study was conducted to investigate the anticancer effects of curcumin (Cur), thymoquinone (TQ), and 3, 3′-diindolylmethane (DIM) combinations on lung (A549) and liver (HepG2) cancer cell lines’ progression. Results showed that triple (Cur + TQ + DIM) and double  combinations of Cur, TQ, and DIM significantly increased apoptosis with elevation of caspase-3 protein levels. Also, these combinations exhibited significantly decreased cell proliferation, migration, colony formation activities, phosphatidylinositol 3-kinase (PI3K), and protein kinase B (AKT) protein levels with S phase reduction. Triple and double combinations of Cur, TQ, and DIM hindered tumor weight and angiogenesis of A549 and HepG2 implants in the chorioallantoic membrane model. Interestingly, Cur, TQ, and DIM combinations are considered promising for suppressing cancer progression  Cancer ranks as a leading cause of death worldwide . The IntCurcuma longa L.  - validated), is the most studied compound described as a potential anticancer agent due to its multi-targeted signaling/molecular pathways  seeds, is a well-known natural bioactive compound used for the management of several cancer types (Nigella sativa L. is shown in Thymoquinone (TQ) , the bioer types . TQ induer types . In the er types  is preseer types . The taxhttps://clinicaltrials.gov/) for cancer therapy, as stated in Cur, TQ, and DIM were incorporated in clinical trials (Lung (A549) and liver (HepG2) cancer cell lines were purchased from ATCC . A549 and HepG2 cells were grown in low-glucose Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution.50) of Cur, TQ, and DIM was determined by seeding A549 or HepG2 cells in 24-well plates (3 × 104 per well in 1.5 ml) and incubated for 48 h at 37°C in a 5% CO2 incubator. Cells were treated with 1 ml of Cur , TQ , and DIM  and incubated for 24 h, then treated with MTT reagent (1.25 mg/ml) and incubated for 2 h. The resulting formazan crystals were dissolved in 1 ml DMSO, and the optical density was determined using a microplate reader at 570 nm  to determine the IC50 of Cur and TQ against A549 and HepG2 cells. These experiments were repeated three times  and incubated for 48 h at 37°C in a 5% CO2 incubator. Control, Cur, TQ, and DIM groups of A549 cells were treated with Fa 95, 0.75 (Fa 95), 0.50 (Fa 95), and 0.25 (Fa 95) in 1 ml of the medium. Similarly, control, Cur, TQ, and DIM groups of HepG2 cells were treated with Fa 95, 0.75 (Fa 95), 0.50 (Fa 95), and 0.25 (Fa 95) in 1 ml of the medium  and propidium iodide (PI) detection kit . A549 and HepG2 cells were cultured in T25 flasks for 48 h. A549 cells were treated with 0.75 (Fa 95) of Cur, TQ, DIM, and their double  and triple (Cur + TQ + DIM) combinations in 10 ml of medium for 24 h . Also, HUsing a FACSCalibur™ flow cytometer  and Cell Quest software, the cell cycle status was analyzed in A549 and HepG2 cells that were cultured and treated with Cur, TQ, and DIM by the same method mentioned in the annexin-V assay.4 per well in 1.5 ml) and incubated for 48 h at 37°C in a 5% CO2 incubator. Cells were treated with 0.75 (Fa 95) of Cur, TQ, DIM, and their double and triple combinations in 1 ml for 24 h (A549 or HepG2 cells were cultured in 12-well plates (3 × 10for 24 h . Cells wfor 24 h . The tot4 per well in 1.5 ml) and were cultured to 100% confluence in a 12-well plate. Cells were then wounded by denuding a strip of the monolayer with a 200 μl pipette tip. Cells were washed twice with PBS and then incubated with 0.75 (Fa 95) of Cur, TQ, DIM, and their double and triple combinations in 1 ml for 24 h  per well were plated in 12-well plates and were allowed to grow for about 4–5 days until small colonies could be seen. Cells were treated with 0.75 (Fa 95) of Cur, TQ, DIM, and their double and triple combinations in 1 ml for 24 h. Also, 0.50 (Fa 95) and 0.25 (Fa 95) of Cur, TQ, and DIM were used to determine colony formation in double and triple combinations . Cells w4 were cultured in a T25 flask for 48 h. Seeded cells were treated with 10 ml 0.75 (Fa 95) of Cur, TQ, DIM, and their double and triple combinations for 24 h. Also, 0.50 (Fa 95) and 0.25 (Fa 95) of Cur, TQ, and DIM , allowing direct access to the underlying CAM.The effect of Cur, TQ, DIM, and their combinations on A549 and HepG2 implants’ Chorioallantoic membrane model (CAM) of tumor weight and tumor angiogenesis were carried out as previously described by ® in the 7-day-old fertilized chick egg. Treatment effects on tumor weight and angiogenesis were determined on day 7 after tumor cell implantation. For these studies, Matrigel was thawed overnight at 4°C and placed on ice. In the exponential growth phase, cells were harvested using 0.25% trypsin–EDTA, washed, and suspended in the medium. Only suspensions of single cells with viability exceeding 95% were used. Approximately 1 × 106 cells in 30 μl of medium mixed with the same volume of Matrigel were implanted on the CAM. The treatment groups were Matrigel with A549 or HepG2 cells (control), Matrigel/A549 or HepG2 with Cur (3.5 μg/CAM and 5.8 μg/CAM), TQ (6 μg/CAM and 3 μg/CAM), DIM (11.7 μg/CAM and 10.4 μg/CAM), and their combinations of Cur + DIM, Cur + TQ, TQ + DIM, and Cur + TQ + DIM (A549 or HepG2 cells were implanted at 1 million cells per CAM in MatrigelTQ + DIM .n = 5 eggs per group) and tumor hemoglobin (Hb) (mg/dl) ± standard deviation of the mean (n = 5 per group).After incubation at 37°C with 55% relative humidity for 3 days, the CAM tissue directly beneath each filter disk was resected from control and treated CAM samples. Tissues were washed 3 times with PBS and placed in 35-mm Petri dishes. Results are presented as mean tumor weight (mg) per treatment group  are set out in Migration assay was performed by induction of a wound of A549 monolayer before and 24 h after treatment with Cur, TQ, DIM, and their combinations . Cur-, TMTT assay has been used to determine the Fa values of Cur, TQ, and DIM against HepG2 cells . Fa valuCytotoxicity percentages of Fa 95, 0.75 (Fa 95), 0.50 (Fa 95), and 0.25 (Fa 95) of Cur, TQ, DIM, and their double and triple combinations against HepG2 cells are shown in Results shown in Cell cycle analysis of HepG2 cells treated with Cur, TQ, DIM, Cur + TQ, Cur + DIM, TQ + DIM, and Cur + TQ + DIM by 0.75 (Fa 95) doses showed S phase inhibition . ConcentCur-, TQ-, DIM-, Cur + TQ-, Cur + DIM-, TQ + DIM-, and Cur + TQ + DIM-treated HepG2 cells with concentrations of 0.75 (Fa 95) exhibited significant decreases in migration activity as presented in It can be seen from the data in Caspase-3 protein levels were significantly increased in the Cur-, TQ-, DIM-, Cur + TQ-, Cur + DIM-, TQ + DIM-, and Cur + TQ + DIM-treated HepG2 cells with concentrations of 0.75 (Fa 95), Cur + TQ + DIM at a concentration of 0.50 (Fa 95), and Cur + TQ + DIM at a concentration of 0.25 (Fa 95) . On the Tumor weights and Hb concentrations of A549 (r2 = 0.99) between tumor growth inhibition and tumor angiogenesis inhibition was demonstrated for the various natural bioactive compounds and their combinations, which suggest a key role for their anti-angiogenesis activities in suppressing tumor progression , we stated Cur, TQ, and DIM’s anticancer effect against numerous cancer types targeting numerous genes represented in By searching in the comparative toxicogenomics database  and double  combinations increased apoptosis and decreased proliferation and migration colony formation activities with S phase reduction. In addition, triple and double combinations significantly reduced tumor weight and Hb concentrations in A549 and HepG2 cells’ implants in the CAM model. Therefore, Cur, TQ, and DIM combinations are promising agents for suppressing tumor growth and angiogenesis. Further preclinical and clinical studies are warranted trying to use their approved doses as stated in Ki), and the ligand efficiency of common targets with Cur, TQ, and DIM have been determined using InstaDock software (Cur, TQ, and DIM have common targets as stated in software  and repr


Ventricular muscle protein expression and structure were investigated with Western blot and electron microscopy, respectively. (3) Results: The amplitude of shortening was increased in ZF compared to ZL but not compared to ZDF myocytes. Resting Ca2+ was increased in ZDF compared to ZL myocytes. Time to half decay of the Ca2+ transient was prolonged in ZDF compared to ZL and was reduced in ZF compared to ZL myocytes. Changes in expression of proteins associated with cardiac muscle contraction are presented. Structurally, there were reductions in sarcomere length in ZDF and ZF compared to ZL and reductions in mitochondria count in ZF compared to ZDF and ZL myocytes. (4) Conclusions: Alterations in ventricular muscle proteins and structure may partly underlie the defects observed in Ca2+ signaling in ZDF and ZF compared to ZL rat hearts.(1) Background: Cardiovascular complications are a leading cause of morbidity and mortality in diabetic patients. The effects of obesity and diabesity on the function and structure of ventricular myocytes in the Zucker fatty (ZF) rat and the Zucker diabetic fatty (ZDF) rat compared to Zucker lean (ZL) control rats have been investigated. (2) Methods: Shortening and intracellular Ca As of 2021, there were around 573 million adults between the ages of 20–79 who were suffering from diabetes mellitus (DM), an illness characterized by high blood glucose levels, and this number is expected to increase to 643 million by 2030 and around 783 million by the year 2045  compared to ZL (0.31 ± 0.007 RU) mean rank [74.83] in myocytes (2+ transient was not significantly (p > 0.05) different between ZF (70.86 ± 1.42 ms), ZDF (69.69 ± 1.92 ms) and ZL (72.42 ± 1.84 ms) myocytes (2+ transient was significantly (H(2) = 26.26, p < 0.001) prolonged in ZDF (123.23 ± 2.68 ms) mean rank [115.66] compared to ZL (112.54 ± 3.46 ms) mean rank [84.03] and shortened in ZF (106.08 ± 2.30 ms) mean rank [68.02] compared to ZDF myocytes (2+ transient was not significantly (p > 0.05) different between ZF (0.034 ± 0.0013 RU), ZDF (0.035 ± 0.0017 RU) and ZL (0.037 ± 0.0016 RU) myocytes  different in ZDF and ZF compared to ZL myocytes. Records of shortening and Cap = 0.027) lower in ZDF (0.53 ± 0.07) mean rank [2.0] compared to ZF (1.45 ± 0.02) mean rank [8.0] but not compared to ZL (1.06 ± 0.2) mean rank [5.0] ventricle tissue. Interestingly, Ryanodine expression was only modestly elevated (p > 0.05) in ZDF and ZF compared to ZL ventricle tissue and SERCA2 expression was modestly (p > 0.05) lower in ZDF (1.01 ± 0.1) and ZF (0.85 ± 0.04) compared to ZL (1.45 ± 0.11) ventricle tissue. Tropomyosin expression was significantly (H(2) = 6.49, p = 0.039) higher in the ZF (0.51 ± 0.05) mean rank [8.0] group compared to ZDF (0.09 ± 0.02) mean rank [2.33] and ZL (0.16 ± 0.03) mean rank [4.67] controls. Troponin C expression was modestly lower (p > 0.05) in ZDF (0.16 ± 0.08) and in ZF (0.28 ± 0.05) compared to ZL (0.64 ± 0.05) ventricle tissue. Expression of Troponin I was significantly (H(2) = 6.49, p = 0.039) reduced in ZDF (0.13 ± 0.05) mean rank [2.33] compared to ZL (0.84 ± 0.06) mean rank [8.0] and Troponin T was significantly (H(2) = 6.49, p = 0.039) reduced in ZDF (0.09 ± 0.03) mean rank [2.0] compared to ZL (0.82 ± 0.19) mean rank [7.67] ventricle tissue. Connexin Cx45 was significantly (H(2) = 7.2, p = 0.027) reduced in ZF (0.007 ± 0.004) mean rank [2.0] compared to ZL (0.4 ± 0.17) mean rank [8.0] ventricle tissue.Western blot analysis revealed significant differences in the expression of proteins that are involved in the process of cardiac muscle contraction. Typical blots are shown in p < 0.001) was shortest in ZF (1.21 ± 0.02 µm) mean rank [17.45], intermediate in ZDF (1.61 ± 0.03 µm) mean rank [76.41] and longest in ZL (1.71 ± 0.01 µm) mean rank [99.6] myocytes (p < 0.001) was lowest in ZF (86.23 ± 7.26) mean rank [12.85], intermediate in ZDF (100.81 ± 10.50) mean rank [27.09] and highest in ZL (122.15 ± 4.45) mean rank [36.13] myocytes with significant difference (p < 0.001) between ZL and ZF , a mediator of cardiac contractility, plays an important role in cardiac muscle contraction and relaxation as well as in the performance of the diabetic heart and is linked to cardiac hypertrophy [2+ signaling and Ryanodine receptor (RyR) SR Ca2+ release [Interestingly, blood insulin levels were significantly higher in ZF rats than they were in ZL and ZDF rats. ZDF rats were previously found to have elevated fasting serum glucose, insulin, cholesterol and triglyceride levels as well as impaired glucose tolerance . Analyziertrophy ,24,25,26 release , whereas release . 2+ transient was not significantly different between groups. THALF decay of the Ca2+ transient was significantly prolonged in ZDF compared to ZL and was shortened in ZF compared to ZDF myocytes. AMP of the Ca2+ transient was not significantly different between groups. Contraction and relaxation are mainly regulated by intracellular Ca2+. During the process of Ca2+-induced Ca2+ release there is a small entry of Ca2+ through L-type Ca2+ channels and this small entry of Ca2+ triggers a large release of Ca2+ from the SR [2+ leakage, lower caffeine-evoked Ca2+ release and decreased rate of SR Ca2+-ATPase (SERCA)-mediated Ca2+ uptake in myocytes from db/db diabetic mice [2+, SR store and release rates of Ca2+ and depressed resequestration into SR in myocytes from streptozotocin (STZ)-induced diabetic rats [2+ release may result from structural defects in the SR Ca2+ release channel as well as decreased expression of Ca2+ release channel mRNA [2+ ions, and the terminal cisternae, which are large regions in close proximity to the ends of the transverse tubules known as “T-tubules” that sequester and concentrate Ca2+. Evidence suggests that loss of T-tubule integrity can profoundly affect excitation–contraction coupling (ECC) in myocytes, which is evident during action potential propagation [2+ signaling in the ZDF group.Fura-2 ratio 340/380 nm) was significantly increased in ZDF compared to ZL myocytes. The mean TPK Cam the SR . Researctic mice ,30 as wetic rats . In typenel mRNA ,33,34,350/380 nm pagation ,37,38. Ipagation . Our res2+ is not altered. Myofilament activation is modulated by protein phosphorylation, and myofilament proteins are substrates for PKC, PKA and CAMK, which in turn can induce changes in myofilament sensitivity to Ca2+ [2+, and although no changes have been found in Fura-2 length trajectory, we have found changes in expression of proteins of Troponin I and T proteins between groups, which may have a bearing on myofilament sensitivity to Ca2+. Our previously published paper reported that diabetic Goto-Kakizaki (GK) rat Fura-2 cell length trajectory was steeper in GK compared to control myocytes, which suggested an increase in myofilament sensitivity to Ca2+ [The Fura-2 cell length trajectory during the late stages of relaxation of the twitch contraction was not significantly different in myocytes between groups, which indicates that myofilament sensitivity to Ca to Ca2+ . We have to Ca2+ . 2+ release [2+ handling. Tropomyosin expression was higher in ZF compared to ZDF and ZL ventricle tissue. Tropomyosin has been previously linked to hypertrophic cardiomyopathy [2+ signaling and ECC coupling [Ventricular muscle protein was assessed with Western blot. Myosin expression was modestly lower in ZDF compared to ZF ventricle tissue, which is consistent with a previous study . Myosin  release ,43. Inte release ,45,46. Amyopathy ,48. In tcoupling , and thecoupling , and diacoupling . In thiscoupling . In thiscoupling . Interescoupling . Morphometric analysis of the sections indicated that sarcomeres are longest in ZL, intermediate in ZDF and shortest in ZF myocytes. Similarly, the mitochondrial count is highest in ZL, intermediate in ZDF and lowest in ZF myocytes, with significant differences between ZL and ZF. The differences in mitochondrial count may reflect differences in size or metabolic activity between the groups. These morphological changes in mitochondria and signs of injury in the ZDF have also been similarly described in previous diabetic studies ,54, whic2+ while identifying protein and structural defects. We have found evidence of molecular and structural defects in the ZF and ZDF rat heart. Our results suggest that SR Ca2+ handling, as well as energy utilization, is compromised in ZDF as well as in ZF. Myocyte contraction and relaxation may also be affected in the ZDF and ZF rat due to muscle protein structural defects, which lead to an affected heart rhythm and poor cardiac function. To our knowledge, this is the first study to investigate the effects of obesity and diabesity in the ZDF and ZF compared to ZL controls with simultaneous measurement of myocyte shortening and intracellular Ca+ and K+ currents, which are fundamental for the generation of action potentials. Imaging techniques including CT, MRI and echocardiography could be used to further understand the hemodynamic changes associated with obesity and diabesity. The results of this study provide a strong foundation on which to develop functional and structural studies to further understand the effects of obesity and diabesity on electro-mechanical dysfunction in the Zucker rat heart. The number of rats was limited by the availability of grant funding, and experiments were conducted at a single time point. It would have been interesting to carry out experiments at different time points to evaluate the progression of electro-mechanical dysfunction in obesity and diabesity. Future experiments might include action potential experiments in individual myocytes from different regions of the heart, and at different ages, to assess the progress of obesity and diabesity on the electrophysiology of the heart. In addition, experiments might also include whole cell patch clamp experiments in ventricular myocytes to investigate other ionic currents, including NaOur research demonstrates that ageing and prolonged diabesity lead to adverse clinical outcomes. It is important to implement strategies that will prevent disease progression, such as lifestyle changes—including exercise training and improved diet—and therapeutic interventions.


Oreochromis niloticus) was cloned and its function in resisting pathogen infection was characterized. The full length of OnC3 open reading frame is 4974 bp, encoding 1657 aa, and the predicted protein mass weight is 185.93 kDa. The OnC3 amino acid sequence contains macroglobulin domains. The expression pattern of OnC3 mRNA in the tissues of healthy fish was detected, with the highest in the liver and the lowest in the muscle. After challenged with Streptococcus agalactiae and Aeromonas hydrophila, the expression of OnC3 mRNA was significantly up-regulated in the liver, spleen, and head kidney. Further, the recombinant OnC3 protein alleviated the inflammatory response and pathological damage of tissues after infected with S. agalactiae. Moreover, the OnC3 promoted the phagocytosis of monocytes/macrophages to S. agalactiae. The data obtained in this study provide a theoretical reference for in-depth understanding of C3 in host defense against bacterial infection and the immunomodulatory roles in teleost fish.The complement system is composed of a complex protein network and is pivotal to innate immunity. Complement 3 (C3) is a critical protein in the complement cascade and participates in complement activation and immune defense. In this study, C3 from Nile tilapia ( Complement, a vital constituent of the innate immune system, is a complex protein network composed of more than 50 kinds of proteins and protein sub-fragments ,2,3,4. Tβ2 glycoprotein composed of α and β peptide chains and mainly synthesized by the liver [IL-1 and stimulates respiratory burst such as macrophages and the chemotaxis of mast cells [C3 is a he liver ,13. The he liver . When sthe liver . Both C3he liver ,16,17. Cst cells ,19. C3b st cells ,21. Intest cells . In addist cells ,23.Ctenopharyngodon idella) [Paralichthys olivaceus) [Larimichthys crocea) [Misgurnus anguillicaudatus) [Oncorhynchus mykiss) [A. hydrophila and repairing tissue damage caused by bacterial infection [S. agalactiae and A. hydrophila infection causes serious damage to Nile tilapia tissues and affects the aquaculture industry negatively [The sequence information and functions of C3 have been successfully characterized in several bony fishes including the grass carp ( idella) ,25, Japaivaceus) , large y crocea) , dojo loaudatus) , and rai mykiss) . In grasnfection . In Japanfection . These snfection . The S. gatively ,32. Thergatively ,30,31,32OnC3) was successfully cloned, and the expression pattern after S. agalactiae and A. hydrophila stimulation was also confirmed. Furthermore, the recombinant OnC3 protein was expressed, and it was discovered that it could resist S. agalactiae infection by mediating inflammation and opsonizing the phagocytosis of monocytes/macrophages (MO/Mø). Moreover, (r)OnC3 was found to be able to reduce the tissue damage induced by S. agalactiae infection. Above all, this study proposes a new reference for understanding the functional properties of C3 in teleost host defense against pathogen infection.In the present study, the open reading frame (ORF) of the Nile tilapia complement C3 gene (OnC3 is 4974 base pairs (bp) in length, encoding 1657 amino acids (aa). Different domain sequence information was marked with different colors according to the prediction of SMART OnC3 .IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-10 and TGF-β transcripts in the liver and spleen were detected. The results illustrated that (r)OnC3 upregulated the transcripts of IL-1β, IL-6, TNF-α, and TGF-β in the liver and spleen at 12 h after stimulation (To detect the effects of (r)OnC3 on inflammatory response, the expression of pro-inflammatory cytokines mulation A.IL-1β, IL-6, TNF-α, and IL-10 was about 5-fold to 7-fold higher than the Trx groups and PBS groups at 12 h, 24 h, and 48 h after stimulation (TGF-β in the (r)OnC3 group also was 5-fold higher than that in other groups at 12 h after stimulation OnC3 protein. In the (r)OnC3 groups, the transcription of mulation A–D. The mulation E.Before the experiment, the cells were loaded with the DCFH-DA fluorescent probe, as the fluorescence intensity of DCF can represent the content of ROS. The ROS in MO/Mø was assayed by flow cytometric analysis. The results showed that the DCF intensity in the (r)OnC3 group was 60.6%, which was apparently stronger than the PBS group (50.9%) and Trx group (51.4%) F. The blIL-1β, IL-6, TNF-α, IL-10, and TGF-β in experimental groups were apparently higher than the PBS group after 12 h of stimulation. However, in the S. agalactiae + (r)OnC3 group, the transcripts of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were lower than the S. agalactiae group and S. agalactiae + Trx group, while the expression of IL-10 and TGF-β was augmented compared to the S. agalactiae group and S. agalactiae + Trx group OnC3 stimulated group, the expression of IL-1β, IL-6, and TNF-α was lower than the S. agalactiae and S. agalactiae + Trx stimulated group, while the expression of IL-10 and TGF-β was higher than the two groups.Meanwhile, the expression of inflammation factors confirmed a similar changing tendency in MO/Mø C. In theS. agalactiae were infected, the (r)OnC3 significantly alleviated the inflammatory response and injury score OnC3 group OnC3 group were obviously lower in S. agalactiae and the S. agalactiae + Trx group OnC3 significantly induced the inflammatory infiltration of the liver and spleen B and grery score D,E. The C3 group D. Additirx group E.CD11b, CD18), enzymes , as well as lysosome-related factors  were detected by RT-qPCR. The results showed that in head kidney tissue, the transcripts of these factors in S. agalactiae + (r)OnC3 groups were significantly higher than in S. agalactiae, the S. agalactiae + Trx group, and the PBS group OnC3 group was higher than S. agalactiae, the S. agalactiae + Trx group, and the PBS group OnC3 group was 61.5%, much higher than the 48.0% and 51.0% in the PBS and Trx groups (To further examine the effect of (r)OnC3 in phagocytosis, the expressions of phagocytosis-related receptors . The OnC3 were predicted conserved with seven domains: A2M_N, A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains. Moreover, a highly conserved thioester region (GCGEQ) in OnC3 was also predicted. The phylogenetic analysis indicated that OnC3 clustered with C3 of other bony fish, and also has high similarity with mammals and invertebrates, which further reflects the high conservation of C3 in the evolutionary process [The results of this study revealed that OnC3 is composed of  process ,28,34.OnC3 was the highest in the liver tissue of Nile tilapia [OnC3 mRNA in the head kidney, thymus, and spleen is also at a high level. After S. agalactiae and A. hydrophila infection, the expression levels of OnC3 mRNA in the liver, spleen, and head kidney were significantly up-regulated. These organs with high expression of OnC3 are important immune organs of Nile tilapia in antibacterial infection and are also suitable pathological models in vivo [OnC3 mRNA in head kidney MO/Mø remarkably rose after infected by the two pathogens as well. The S. agalactiae and A. hydrophila are currently the main pathogens of Nile tilapia, causing huge economic losses to the aquaculture industry [OnC3 would increase after pathogen infection and that it is involved in the process of resisting pathogen infection, which further helps to explore the important mechanism of OnC3 in Nile tilapia resisting pathogen infection.In mammals, the liver is considered as the foremost organ of C3 synthesis . Consist tilapia ,28. The  tilapia . Further in vivo . Monocyt in vivo . Similarindustry ,32. ThesIL-1β, IL-6, TNF-α, IL-10, and TGF-β) were apparently ascended after injection of the (r)OnC3 protein in liver and spleen of healthy fish. The inflammatory factors and intracellular reactive oxygen species that increased significantly were also up-regulated in head kidney MO/Mø after (r)OnC3 challenge. Meanwhile, slight tissue damage was detected in liver and spleen by H&E staining. These results suggested the content and function of C3 in Nile tilapia is tightly regulated, preventing damage to the immune system due to the imbalance of complement [Complement components are present in an inactive state to prevent the negative effects of complement imbalances under normal circumstances. The imbalances of the complement activation are the important causes of tissue damage and end organ damage in autoimmune diseases, such as systemic lupus erythematosus and C3 glomerulopathy in humans ,21,38. Tmplement .S. agalactiae, the liver and spleen of Nile tilapia were congested, with swelling and severe water accumulation [IL-6, IL-1β, and TNF-α) and promoting the expression of anti-inflammatory cytokines (IL-10 and TGF-β), thereby slowing down the tissue damage caused by S. agalactiae. These results suggested that C3 plays a vital role in host immune defense and has antibacterial effects against pathogen infection in bony fishes.The complement components will be activated and function in turn when stimulated ,4. Aftermulation ,43,44,45C3 gene could significantly reduce the phagocytosis of Vibrio alginolyticus by blood cells, which also showed the importance of C3 in invertebrate phagocytes [β2 integrin, which plays a prominent role in recognizing bacteria. It can bind to inactivated C3b (iC3b) and activate phagocytosis [S. agalactiae by Nile tilapia head kidney MO/Mø. The expression of CR3-mediated phagocytosis-related genes was detected as well. Notably, CR3 (CD11b/CD18), RhoA, and ROCK were all up-regulated. Significant up-regulation also occurred in association with phagolysosome formation such as EEA, M6PR, and vATPase [Phagocytic cells are chief components of innate immunity and act as a bridge between innate and adaptive immunity . In mammagocytes . Phagocyagocytes ,37. The agocytes ,16,46. Tagocytes . CR3 , while the control group was treated with 100 µL 10 mM sterile PBS. Three Nile tilapia were selected for each group of experiments to obtain samples. The time of obtaining sample was 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after challenge.Challenges and sampling methods refer to published articles, and the brief steps are as follows . During The total RNA of Nile tilapia tissue and cell samples were extracted with Trizol Reagent  according to the instructions. The tissue samples mixed with 200 µL Trizol were ground to homogeneity with an electric homogenizer, and then the Trizol was added to a final volume of 1 mL. The mixture was then placed on ice for 10 min and centrifuged at 12,000 rpm for 15 min at 4 °C. Then, the supernatant was mixed with 450 µL of chloroform in a new centrifuge tube. The above procedure was then repeated for standing and centrifugation. The upper organic phase was mixed with 600 µL of isopropanol in a new centrifuge tube. After standing and centrifugation, the bottom RNA pellet was collected. RNA samples were then washed with 75% ethanol solution twice. The resulting total RNA was confirmed by 1% agarose gel electrophoresis and the RNA concentration was determined using a Nanodrop 2000 spectrophotometer . All RNA samples were stored at −80 °C until use .® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) , and the brief steps were as follows. The reaction contained 1 µg of total RNA, 2.0 µL of 5 × gDNA digestion buffer, and 1.0 µL of gDNA digestion, and these were finally added to a total volume of 10 µL with RNase-free ddH2O. The mixture was maintained at 42 °C for 2 min. Then, 2 µL primescript RT enzyme mix, 1 µL RT primer mix, and 2.0 µL 5 × Hifair® II  Buffer plus were mixed with the reaction solution from the first step, then RNase-free ddH2O was added to bring the total volume to 20 µL. After that, the mixture was maintained at 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min. Prepared cDNA templates were stored at −20 °C until use. The cDNA template was diluted to a total volume of 200 µL with RNase-free ddH2O before being used for RT-qPCR.The cDNA template synthesis was performed with Hifairhttps://www.ncbi.nlm.nih.gov/ (accessed on 19 November 2020)) was acquired with XP_019203045.1. The sequencing primers were designed with Primer Premier 5 based on the predicted sequence and synthesized by BGI, China. All primers used in this study are listed in OnC3 ORF was confirmed by polymerase chain reaction (PCR). The PCR reaction volume was 25 µL, consisting of 12.5 µL LA Taq Mix , 7.5 µL ddH2O, 3 µL cDNA template, 1 µL forward primer, and 1 µL reverse primer. The PCR reaction process contained 5 steps, pre-denaturation (95 °C) for 5 min, denaturation (95 °C) for 1 min, annealing (50 °C) for 30 s, extension (72 °C) for 2 min, and final extension (72 °C) for 5 min. In those steps, denaturation, annealing, and extension were performed for 35 cycles. After amplification, the PCR product was detected with 1% agarose gel electrophoresis and sequenced by BGI, China.The predicted OnC3 sequence from NCBI (C3 were carried out with Blast X. The nucleotide sequence of OnC3 was translated into amino acid sequence with ExPASy tools (http://expasy.org/tools/ (accessed on 14 May 2021)) and the protein domain was analyzed with the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/ (accessed on 14 May 2021)). The signal peptide was predicted with SignalP 4.1 ). The phylogenetic tree was constructed with MEGA 7 based on the neighbor-joining (NJ) algorithm. The structure of OnC3 was predicted by the server equipped with A100 GPU. The Github source code from AlphaFold2.0 ) [In this study, the multiple sequence alignment and identity of r 2021)) .OnC3 transcripts in the tissues was performed with QuantStudio 5 Real-time PCR system . The volume of the reaction was 20 µL, including 10 µL Hieff® qPCR SYBR Green Master Mix , 2 µL forward primers, 2 µL reverse primers, 3 µL cDNA template, and 4 µL ddH2O. The program was a two-step method comprising pre-denaturation (95 °C) for 5 min, denaturation (95 °C) for 10 s, and annealing/extension (60 °C) for 30 s. The denaturation and annealing/extension were performed for 40 cycles. The melt curve was acquired in the last stage of RT-qPCR. Nile tilapia β-actin was selected as the internal reference for calculating the relative expression of target genes based on the 2−ΔΔCT method [The expression of T method . In thisg for 40 min, then the MO/Mø fraction of the 31–54% interface was collected and washed three times with RPMI 1640 medium. The cell concentration was adjusted to 5 × 106 cells/mL by the L-15 medium with 1% penicillin/streptomycin and 10% FBS. The isolated cells were cultured in 96-well plates (100 µL/well)  and incubated at 25 °C. After 3 days, the non-adherent cells were removed. The adherent Nile tilapia MO/Mø were resuspended and washed with 10 mL 1640 medium 3 times. The cells were diluted to 1 × 106 cells/mL and incubated in 96-well plates for experiments.The isolation of Nile tilapia head kidney MO/Mø was developed based on the references ,61. The OnC3 in MO/Mø after bacterial challenge, the head kidney (1 × 106 cells/mL) cells were challenged with formalin inactivated S. agalactiae or A. hydrophila (1 × 107 CFU/mL) while the control was treated with sterile PBS (10 mM). After stimulation, the cell samples were collected at 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h.In order to explore the expression pattern of β chain of OnC3 was amplificated and inserted into the E. coli expression vector Trx-pET-32a (Trx). The recombinant Trx-OnC3 construct was transformed into E. coli. BL21 (DE3) competent cells and coated on LB medium plates consisting of 1% ampicillin. On the next day, the positive clones were selected, identified with PCR reaction, and sequenced by BGI, China. The cells were cultured with 250 mL LB-ampicillin medium until the OD value at 600 nm was 0.6–1.0. Then, the bacteria liquid was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) at 37 °C for 6 h. After that, the target recombinant protein was purified with His-Band Resin columns  according to the prot °Cols. Finally, the purified protein was detected by 10% SDS-PAGE. The Trx vector protein was prepared through the same methods for the follow-up experiments [The eriments ,55.IL-1β, IL-6, and TNF-α and anti-inflammatory cytokines IL-10 and TGF-β were analyzed with RT-qPCR based on the methods in In order to explore the effects of (r)OnC3 on inflammatory response in vivo, the experimental group was intraperitoneally injected with 100 µL (r)OnC3 protein (50 µg/mL), 100 µL Trx protein (50 µg/mL), and 100 µL PBS (10 mM). The liver and spleen of tilapia were detected at 12 h after stimulation. Sample collection and template preparation are referred to S. agalactiae (1 × 107 CFU/mL), S. agalactiae + Trx protein (50 µg/mL), or S. agalactiae + (r)OnC3 protein (50 µg/mL). The liver and spleen were harvested 12 hours after stimulation, and IL-1β, IL-6, TNF-α, IL-10, and TGF-β were detected. The isolation and culture methods of MO/Mø were the same as in 6 cells/mL) MO/Mø were treated with sterile PBS (10 mM), S. agalactiae (1 × 107 CFU/mL), S. agalactiae + Trx protein (50 µg/mL), or S. agalactiae + (r)OnC3 protein (50 µg/mL), and the cells were collected for RNA extraction after 12 h. The same inflammatory factors were detected by RT-qPCR.Moreover, the regulation of inflammation by (r)OnC3 after pathogen infection was also examined. Nile tilapia were intraperitoneally injected with 100 µL sterile PBS (10 mM), All in vivo stressed fish livers and spleens in 7 cells/mL) were treated with 200 µL DCFH-DA and incubated at 37 °C for 20 min. Cells were washed three times by serum-free cell culture medium. The MO/Mø in the experimental group was treated with 200 µL (r)OnC3 protein (50 µg/mL). The other groups were treated with 200 µL sterile PBS (10 mM) or Trx protein (50 µg/mL). After 30 min of stimulation at 37 °C, the DCF fluorescence was detected by FACS Aria III  flow cytometer with an argon-ion laser of 488 nm. A total of 10,000 individual cells were analyzed in each of the samples.In this research, MO/Mø were isolated and cultured using the method in CD11b, CD18), enzymes , and lysosome-related factors  in the head kidney and head kidney MO/Mø were detected at time of 6 h after challenge. The RNA extraction, cDNA synthesis, and RT-qPCR detection were performed according to the above sections.In S. agalactiae (1 × 108 CFU/mL) labeled with fluorescein isothiocyanate (FITC)  were prepared while the forward scatter (FSC) and side scatter (SSC) were measured [7 cells/mL) and incubated for 60 min with shaking. The solution above was centrifuged with over 3% BSA in PBS supplemented at 100× g, 4 °C for 10 min to eliminate the non-ingested bacteria. The extra fluorescence was quenched by mixing 1 µL trypan blue (0.4%). Finally, the cell suspension was detected by FACS Aria III  flow cytometer with a 488 nm argon-ion laser [Before experiments, the Nile tilapia head kidney MO/Mø and measured ,57. Afteon laser . A totalp < 0.05, ** p < 0.01). The figures were made with GraphPad Prism 7 .All data in the present research were repeated three times and shown as mean ± standard deviation (SD). Then, the data were analyzed by one-way ANOVA with SPSS 17.0. The statistical significance was indicated by letters and asterisks (* 0.01 ≤