Conyza bonariensis (Asteraceae) aerial parts (CBEO) was extracted by hydrodistillation in a Clevenger-type apparatus and was characterized by gas chromatography–mass spectrometry. The antitumor potential was evaluated against human tumor cell lines , as well as non-tumor keratinocyte lines using the MTT assay. The effect of CBEO on the production of Reactive Oxygen Species (ROS) was evaluated by DCFH-DA assay, and a protection assay using the antioxidant N-acetyl-L-cysteine (NAC) was also performed. Moreover, the CBEO toxicity in the zebrafish model was assessed. The majority of the CBEO compound was (Z)-2-lachnophyllum ester (57.24%). The CBEO exhibited selectivity towards SK-MEL-28 melanoma cells , and induced a significant increase in ROS production. In addition, the CBEO’s cytotoxicity against SK-MEL-28 cells was reduced after pretreatment with NAC. Furthermore, after 96 h of exposure, 1.5 µg/mL CBEO induced death of all zebrafish embryos. Non-lethal effects were observed after exposure to 0.50–1.25 µg/mL CBEO. Additionally, significant alterations in the activity of enzymes associated with oxidative stress in zebrafish larvae were observed. These results provide evidence that CBEO has a significant in vitro antimelanoma effect by increasing ROS production and moderate embryotoxicity in zebrafish.The essential oil from  Cancer is a relevant public health problem worldwide . In 2020Currently, cancer treatment mainly includes surgery, radiotherapy, chemotherapy, and targeted therapy . NeverthNatural products or their derivatives represent more than 60% of the molecules used for cancer treatment ,8, anti-Conyza (Asteraceae) comprises about 50 species. The secondary metabolites in Conyza plants include alkaloids, terpenoids, steroids, phenolic compounds, flavonoids, and tannins. In most Conyza species EOs, the major chemical compounds found are limonene and β-farnesene [Conyza bonariensis (L.) Cronquist is an invasive plant, native to South America [C. bonariensis aerial parts as anti-aging [Genus arnesene . Conyza  America . Severalti-aging , antibacti-aging , and antti-aging ,21. C. bonariensis aerial parts carried out in various countries have revealed different major components, such as (E)-β-farnesene [Literature data show differences in the chemical composition of EOs even within the same species . Studiesarnesene ,24, germarnesene , caryopharnesene , and metarnesene ,28.C. bonariensis aerial parts (CBEO) collected in João Pessoa, Paraíba, Brazil. Additionally, we evaluate its antitumor effect in human cell lines , and its toxicity on human peripheral blood mononuclear cells (PBMCs) and zebrafish.Here, we present the chemical characterization of the EO from ®; St. Louis, MO, USA), Roswell Park Memorial Institute 1640 (RPMI) medium , Histopaque®-1077 , Buffered phosphate solution (PBS) , doxorubicin (DXR) , penicillin–streptomycin , 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) , N-acetylcysteine (NAC) , hydrogen peroxide (H2O2) , 3--2,5-diphenyltetrazolium bromide (MTT) , sodium chloride (NaCl) , potassium chloride (KCl) , calcium chloride (CaCl2) , magnesium sulfate (MgSO4) , sodium sulfate (Na2SO4) Sigma-Aldrich®; St. Louis, MO, USA), methylene blue Sigma-Aldrich®; St. Louis, MO, USA), dimethylsulfoxide (DMSO) , Sodium Dodecyl Sulfate (SDS) , phytohaemagglutinin , trypsin 0.25% with ethylenediaminetetraacetic acid (EDTA) , Fetal Bovine Serum (FBS) , fish food , hexane .Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-AldrichThe drugs and reagent solutions were prepared immediately before use.2.The SK-MEL-28 (human melanoma), HeLa , HCT-116 (human colon carcinoma), HL-60 (human promyelocytic leukemia), K562 (chronic myeloid leukemia), and HaCat  cell lines were obtained from Rio de Janeiro Cell Bank (BCRJ), Brazil, and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)  or Roswell Park Memorial Institute 1640 (RPMI)  medium supplemented with 10% Fetal Bovine Serum and 1% penicillin–streptomycin at 37 °C with 5% COg, 20 °C, for 30 min) of the blood samples in tubes containing Histopaque®-1077. Subsequently, the interface containing the mononuclear cells was collected and washed with PBS . Twenty-four hours before CBEO or DXR treatment, PBMCs were resuspended in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 2% phytohemagglutinin, and cultured in 96-well plates (1 × 106 cells/mL) at 37 °C with 5% CO2 [Blood samples were collected from healthy donors in tubes containing EDTA. Human peripheral blood mononuclear cells (PBMCs) were isolated after centrifugation (400× h 5% CO2 . Zebrafish embryos (AB wild-type strain) with approximately 1 h post-fertilization (hpf) were provided by the Production Unit for Alternative Model Organisms (UniPOM), Federal University of Paraíba (UFPB), João Pessoa, Brazil. The parents were maintained in a recirculation system with regular monitoring of water quality parameters . The room temperature (26 ± 1 °C) and photoperiod (14:10 light/dark cycle) were controlled. Fish were fed daily with commercial food and freeze-dried spirulina , and were also monitored for abnormal behavior or disease development.2, and 0.33 mM MgSO4) containing 0.005% methylene blue. Only spawning with a fertilization rate of ≥90% was used. Viable embryos  were selected under an inverted light microscope  at 50× magnification.The day before the experiment, zebrafish adults  were transferred to a 7 L spawning tank with a bottom mesh and a quick-opening valve for embryo collection. Embryos were collected on the day of the experiment and cultured in adapted embryonic medium E3  Cronquist branches and leaves (1 kg) were collected from the Medicinal Plant Garden, Institute of Research in Drugs and Medicines of Federal University of Paraíba (UFPB), João Pessoa, Paraíba, Brazil (7°08′30.0″ S–34°50′46.7″ W) in September of 2019. An exsiccate of C. bonariensis was identified by Prof. Dr. Maria de Fátima Agra and deposited at Herbarium Lauro Pires Xavier-JPB of UFPB, under the number JPB 26391 (registry number SISGEN ABB39C8). 2SO4), with a yielding (w/w) of 1.3%.Essential oil extraction was performed by hydrodistillation in a Clevenger-type apparatus. The samples were crushed and subjected to distillation for 2 h . After em/z 50 to 500. The sample solution was prepared in hexane at a dilution of 999:1 (v/v) and 1 μL was injected into the chromatograph at a flow rate (split) of 1:200.Gas chromatography–mass spectrometry (GC-MS) analysis was performed using a Shimadzu QP-2010 Ultra Quadrupole MS system, operating at 70 eV ionization energy. A capillary column RTX-5MS  was used with Helium as a carrier gas at a flow rate of 3 mL/min with a 1:100 split. The injector and detector temperatures were set at 220 °C and 280 °C, respectively. The column temperature was programmed from 40 °C  to 220 °C at a rate of 10 °C/min . Subsequently, the temperature was increased from 220 °C to 280 °C at a rate of 20 °C/min and held isothermally for 5 min at 280 °C. The ions were scanned in scan mode, ranging from ®) was injected. The retention index for each compound was determined based on a similarity index above 89% estimated by the libraries (Nist. 08 and Wiley 9) used for compound identification. The retention index was calculated using the chromatogram obtained through the Van Den Dool and Kratz equation [To calculate the retention indices of compounds, under the same operating conditions, a series of hydrocarbons (C10 to C40) (Sigma-Aldrichequation .2. The cell suspension was added to 96-well plates (100 µL/well) at a density of 3 × 105 cells/mL , 5 × 105 cells/mL (for HL-60 and K562), or 1 × 106 cells/mL (PBMC). After culturing for 24 h, cells were incubated with 100 µL of CBEO  dissolved in DMSO. Doxorubicin (DXR) (molecular weight—MW: 543.52 g/mol) was used as a standard drug. After 72 h, 110 µL of the supernatant was discarded and 10 µL of the 3--2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was added and incubated for another 4 h. The deposited formazan was dissolved with Sodium Dodecyl Sulfate (SDS) (100 mL/well) [®, Winooski, VT, USA) at λ = 570 nm and used to calculate the IC50 . Three independent experiments were performed in triplicate.The MTT -2,5-diphenyltetrazolium bromide) assay was performed to evaluate the CBEO cytotoxicity. MTT is a yellow tetrazolium salt that can permeate cell membranes. In viable cells, MTT is converted to insoluble purple formazan crystals, which can be measured spectrophotometrically. The optical density value is proportional to the number of viable cells . The hummL/well)  and the 50 of the non-tumor cell line (HaCaT) and the IC50 of the tumor cell line.The Selectivity Index (SI) was determined from the ratio between the IC5 cells/mL. After 24 h, cells were exposed to CBEO (20 or 40 µg/mL), DXR (4 µM), or hydrogen peroxide (H2O2) (500 µM) in the presence of DCFH-DA (10 µM), and incubated for 30 min, 1 h, or 3 h. After the incubation periods, the cells were trypsinized, washed, and resuspended in PBS. The percentage of fluorescent cells was obtained by flow cytometry from 10,000 events acquired at 530 nm fluorescence and 485 nm excitation wavelengths. Three independent experiments were performed in duplicate.Reactive Oxygen Species (ROS) were quantified by the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) reagent oxidation method . DCFH-DA5 cells/mL and incubated for 24 h . After this incubation period, cells were incubated for another 3 h  in the presence or absence of 5 µM of N-acetylcysteine (NAC). Subsequently, cells were treated with CBEO  or DXR  and incubated for 72 h in an atmosphere of 5% CO2 and 37 °C. Then, the plates were centrifuged, and 110 µL of the supernatant was removed. Then, 10 µL of the MTT solution (5 mg/mL) was added, followed by incubation for 4 h at 37 °C, CO2 5%. The formazan was dissolved with 100 μL of SDS [®) at λ = 570 nm. Three independent experiments were performed in triplicate. To assess the involvement of ROS in CBEO cytotoxicity, SK-MEL-28 cells were added to 96-well plates (100 µL/well) at a density of 3 × 10L of SDS  and the CBEO acute toxicity was determined by Fish Embryo Acute Toxicity (FET) assay. The FET test was conducted independently with CBEO according to OECD’s guideline number 236 [Zebrafish embryos with up to 3 hpf of age were exposed to five increasing concentrations  of CBEO. For each concentration tested, a 96-well plate was prepared, containing 20 fertilized eggs (1 embryo per well) exposed to the test sample, and 4 embryos were exposed only to E3 medium . Two additional plates containing embryos exposed to E3 medium (negative control) and 0.1% DMSO (solvent control) were also assayed. The volume of liquid in each well at the beginning of the exposure was 0.3 mL. The plates were protected from evaporation by using their own lid and incubating them in a humidified chamber with controlled temperature (26 ± 1 °C) and humidity (70%).The exposure was performed for 96 h, and the embryos were analyzed daily for lethality endpoints: egg coagulation; lack of somite formation; lack of detachment of the tail-bud from the yolk sac; and lack of heartbeat. In the presence of these endpoints, the embryo/larva was considered dead. The number of deaths was used to calculate the survival rate . Additionally, non-lethal effects  were also recorded every 24 h. The exposures were under static conditions (without renovation of the test sample or negative and solvent controls). Observations were using a stereomicroscope (50× magnification) and documented with photographs. After 96 h, surviving larvae were euthanized with eugenol and appropriately disposed of. 50  and EC50 (median effective concentration) through probit analysis [The number of deaths and prevalent non-lethal effects (presence in at least three concentrations) was used to calculate the LCanalysis . These vThe FET test was repeated for CBEO under the same conditions described in item 2.8.1., but at this time the embryos were independently exposed to three sublethal concentrations of CBEO  . After 9w/v) solution. Homogenates were centrifuged at 10,000× g for 10 min at 4 °C, and the resulting supernatants were used for measurement of soluble protein content and enzymatic activity. The activities of lactate dehydrogenase (LDH), glutathione transferase (GST), acetylcholinesterase (AChE), glutathione peroxidase (GPx), and catalase (CAT) enzymes were measured according to Domingues et al. (2010) [Subsequently, the larvae were macerated using cold NaCl 0.9% 1:9 (. (2010) . Tests wp < 0.05). The half-maximal inhibitory concentrations (IC50) and their 95% confidence intervals (CI 95%) were obtained by non-linear regression analysis. For embryotoxicity assay, the median lethal concentration (LC50) values and the median effect concentration (EC50) values were calculated by probit regression analysis.Statistical analysis was performed using GraphPad Prism 8.0.2 . Results are expressed as the mean ± standard error of the mean (SEM). Data statistical analysis was performed using Analysis of Variance (ANOVA), followed by Tukey’s test -2-lachnophyllum ester (57.24%) (MW: 176.21 g/mol), and the remaining components were monoterpenes and sesquiterpenes (39.71%), as shown in 50 of 32.20 ± 1.10 µg/mL, while the human melanoma cell line (SK-MEL-28) was the most sensitive to the treatment, IC50 of 18.65 ± 1.16 µg/mL. Regarding the non-tumor human keratinocyte cell line (HaCaT), CBEO presented IC50 of 56.49 ± 1.03, and the standard drug, doxorubicin (DXR), showed high cytotoxicity (IC50: 0.28 ± 0.001 µM) after 72 h of treatment. Then, the Selectivity Indices (SI) of CBEO and DXR were determined using the HaCaT healthy skin cell line as a non-tumor cell model. The CBEO showed the highest SI for SK-MEL-28 cells (3.03), as shown in CBEO induced the least cytotoxicity on the acute promyelocytic leukemia cell line (HL-60), IC50 values were 2.68 ± 1.29 . CBEO induced a concentration-dependent cytotoxicity. The IC8 ± 1.29 A and 0.08 ± 1.29 B for CBEp < 0.05 for both), 1 h , and 3 h  of treatment, compared to the control . DXR, which was used as a standard drug, induced a significant increase in the percentage of fluorescent cells after 30 min (75.64 ± 6.02% p < 0.05) and 3 h  of treatment, compared to the control. As expected, in the group exposed to hydrogen peroxide (H2O2), there was a significant increase in the percentage of fluorescent cells, compared to the control  (In the dichlorodihydrofluorescein 2′7-diacetate (DCFH-DA) assay, CBEO treatment induced a significant increase in the percentage of fluorescent cells after 30 min  .p < 0.05 for both) compared to the control (100.00 ± 2.47%). NAC pretreatment significantly reduced the CBEO cytotoxic effect, compared to the groups treated only with CBEO at the respective tested concentrations  , a significant reduction in cell viability was observed  . p < 0.05) compared to the group treated with DXR in the absence of NAC (47.23 ± 0.81%)  .The embryotoxicity assay was performed using CBEO concentrations ranging from 0.5 to 1.5 µg/mL. After 96 h of exposure, all embryos died at the highest concentration tested. However, the two lowest concentrations (0.5 and 0.75 µg/mL) did not induce any mortality .The non-lethal effects observed included pericardial edema, yolk sac edema, delayed egg hatching, and egg and blood clotting. Additionally, coagulation and absence of heartbeats were the only lethality outcomes observed in embryos and larvae after CBEO treatments .50) of 1.20 µg/mL. The Lowest Observed Adverse Effect Level (LOAEL) values were 0.5 µg/mL for delayed hatching and 1.0 µg/mL for yolk sac edema, pericardial edema, and blood clotting. The No Observed Adverse Effect Level (NOAEL) value was 0.75 µg/mL for yolk sac edema, pericardial edema, and blood coagulation. The median effective concentration (EC50) for the non-lethal effects found were 0.99 (delayed hatching) and 1.36 .As observed in Subsequently, the activity of enzymes related to oxidative stress in zebrafish larvae exposed to low concentrations of CBEO (0.12 to 0.50 µg/mL) was studied. The rationale for choosing these concentrations lies in the fact that when conducting tests with sublethal concentrations, we typically observe the endogenous mechanisms of detoxification and neutralization of Reactive Oxygen Species (ROS) in action, rather than observing the final adverse effects such as cell death and necrosis, which are less informative from a mechanistic standpoint.p < 0.05 for all), compared to the control (77.87 ± 1.12 µmol/min/mg). Furthermore, a significant increase in glutathione transferase (GST) activity was observed after treatment with CBEO  compared to the control (61.56 ± 0.34 µmol/min/mg). For catalase (CAT) activity, we observed a significant increase in the activity of this enzyme in larvae treated with CBEO  compared to the control (2.36 ± 0.06 µmol/min/mg). In addition, treatment of larvae with 0.25 and 0.50 µg/mL of CBEO induced a significant increase in lactate dehydrogenase (LDH) activity  compared to the control (314.00 ± 2.65 µmol/min/mg). For glutathione peroxidase (GPx) activity, we observed a significant increase in the activity of this enzyme in larvae treated with 0.5 µg/mL CBEO  compared to the control (9.52 ± 0.10 µmol/min/mg).As shown in Conyza bonariensis (L.) aerial parts (CBEO).Nature represents a significant source of bioactive products. Thus, research around the world has sought to discover and investigate the biological effects of natural products against diseases such as cancer . The preThe biological properties of essential oils (EOs) are due to their chemical composition . GeograpZ)-2-lachnophyllum ester, an acetylenic compound, as the major compound (57.24%). However, it is worth noting that the chemical profile of the EO from Conyza bonariensis (L.) aerial parts can vary depending on the country and regions where the plant grows. Until now, (E)-β-farnesene [H-indene-3-carboxaldehyde,2,6,7,7a-tetrahydro-1,5-dimethyl [allo-aromadendrene [Conyza bonariensis (L.) aerial parts. Lundgren et al. (2021) [C. bonariensis (L.) cultivated in the Medicinal Plant Garden of the Institute of Research in Drugs and Medicines of the Federal University of Paraíba (UFPB), João Pessoa, Paraíba, Brazil. These authors obtained a different profile chemical from that obtained in our work, with sesquicineole as the major compound. This shows the influence of conditions such as time of harvest , mechanical or chemical injuries, genetic factors and evolution, storage, irrigation, herbivory, attack of fungal pathogens, and activity of the pollinators in the production and composition of EOs, as already described in the literature [C. bonariensis such as methyl ester of matricaria. Specifically, acetylenic compounds constitute a group of molecules common in species of the Asteraceae family and the Conyza genus [C. bonariensis cultivated in Tunisia in different seasons. However, the methyl ester of matricaria remained the predominant compound in all of them (63.5–76.4%). Chemical analysis of the CBEO revealed the   showed tterature ,49. In aza genus ,51,52. Bza genus  obtainedza genus  observedZ)-2-lachnophyllum ester has also been described as the major component (21.2%) of Conyza bonariensis (L.) cultivated at the University of Athens, Athens, Greece [Conyza bonariensis (L.) aerial parts with unprecedented chemical profile.Finally, the -2-lachnophyllum ester, exhibited a significant in vitro antitumor effect on the tumor cell lines MDA-MB-231 (human breast carcinoma), MCF-7 (human breast carcinoma), and 5637 (human bladder carcinoma), with half-maximal inhibitory concentration (IC50) values ranging from 7.2 to 53.1 µg/mL [Z)-2-lachnophyllum ester also possesses antifungal, antioxidant [Currently, cancer represents a major public health problem worldwide , and man.1 µg/mL . Additioioxidant , insectiioxidant , and nemioxidant  propertiioxidant , the in ioxidant  and in sioxidant ,77,78,79ioxidant . In 2020ioxidant . Thus, nioxidant  showing ioxidant , cell cyioxidant .Duguetia pycnastera leaves (IC50: 21.28 µg/mL) [Satureja khuzistanica aerial parts (IC50: 28.21 µg/mL) [Xylopia laevigata leaves (IC50: 35.30 µg/mL) [50: 2.68 µg/mL) may be related to the major compound, (Z)-2-lachnophyllum ester, which has no report in the literature.We also analyzed the effect of CBEO against human peripheral blood mononuclear cells (PBMCs). PBMCs, including monocytes and lymphocytes, are frequently isolated for use in preclinical research  as well 8 µg/mL) , EO from1 µg/mL) , and EO 0 µg/mL) . Thus, tWe investigated the effect of CBEO on the redox state of SK-MEL-28 cells. In the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, elevated levels of ROS were obtained after CBEO treatment. In addition, pretreatment with N-acetylcysteine (NAC), an antioxidant molecule , signifiDanio rerio) genome shares 70% similarity with the human genome [50) was considered low (1.20 µg/mL); however, similar results related to high toxicity in the zebrafish model were recorded for other EOs from different species, including Zingiber ottensii (LC50: 1.00 µg/mL) composed mainly of monoterpenes and sesquiterpenes [Cupressus sempervirens (LC50: 6.60 µg/mL), whose major compound is α-pinene, a monoterpene [Z)-2-lachnophyllum ester, the major compound of CBEO. In addition, this is the first toxicity report of essential oil from Conyza species in this experimental model. The assessment of drug toxicity is a critical process in the development of new drug candidates . In thisn genome ,112. Then genome , making n genome ,115. Ourn genome ,116. In terpenes , and Cupoterpene . To our Leonurus japonicus aerial parts, rich in phytol and (−)-caryophyllene oxide, induced effects such as yolk sac edema, curved spine, scattered hemorrhages in the edematous yolk sac, incomplete cardiac development, and pericardial edema [Cymbopogon nardus essential oil. In addition, embryos treated with Cymbopogon winterianus essential oil showed slightly shortened tails and mild cardiac changes. In addition, embryos treated with the EO from Cymbopogon citratus and Cymbopogon martini showed slightly slowed development and shortened tails, respectively. All these EOs have monoterpenes as major compounds, such as geraniol and citronellol. At lower concentrations, they all contain limonene (0.7–10%) [Furthermore, delayed hatching, yolk sac, pericardial edema, and blood clotting were observed in embryos exposed to concentrations of 0.5–1.25 µg/mL. Similarly, the EO from al edema . Piaseckal edema  observed0.7–10%) . Therefo2O2) into water and oxygen [2O2 [We also investigated the effect of CBEO on the activity of lactate dehydrogenase (LDH), glutathione transferase (GST), acetylcholinesterase (AChE), glutathione peroxidase (GPx), and catalase (CAT) enzymes in zebrafish larvae. AChE is responsible for hydrolyzing acetylcholine (ACh) , a neurod oxygen . CAT is gen [2O2 . CBEO ingen [2O2 . GPx andgen [2O2 . Therefogen [2O2 . Taken tConyza bonariensis (L.) that demonstrates significant in vitro antimelanoma activity through the induction of oxidative stress. Regarding the toxicity of CBEO, significant cytotoxicity towards PBMCs was observed. Additionally, an oxidative imbalance appears to be involved in the moderate toxicity observed in the zebrafish model. Therefore, further assays should be conducted to better understand the pharmacological and toxicological effects of this EO. Furthermore, these findings stimulate the isolation of (Z)-2-lachnophyllum ester and subsequent in vitro and/or in vivo tests to understand whether the oil’s activity is related to the phytocomplex or the isolated major component.Our results provide the characterization of an unprecedented EO from


Apis mellifera bees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate produce green propolis. Among the examples of bioactivity of this matrix are antioxidant, antimicrobial, and antiviral actions. This work aimed to verify the impact of the experimental conditions applied in low- and high-pressure extractions of green propolis, using sonication (60 kHz) as pretreatment to determine the antioxidant profile in the extracts. Total flavonoid content (18.82 ± 1.15–50.47 ± 0.77 mgQE·g−1), total phenolic compounds (194.12 ± 3.40–439.05 ± 0.90 mgGAE·g−1) and antioxidant capacity by DPPH (33.86 ± 1.99–201.29 ± 0.31 µg·mL−1) of the twelve green propolis extracts were determined. By means of HPLC-DAD, it was possible to quantify nine of the fifteen compounds analyzed. The results highlighted formononetin (4.76 ± 0.16–14.80 ± 0.02 mg·g−1) and p-coumaric acid (<LQ—14.33 ± 0.01 mg·g−1) as majority compounds in the extracts. Based on the principal component analysis, it was possible to conclude that higher temperatures favored the release of antioxidant compounds; in contrast, they decreased the flavonoid content. Thus, the obtained results showed that samples pretreated with 50 °C associated with ultrasound displayed a better performance, which may support the elucidation of the use of these conditions.The demand for bee products has been growing, especially regarding their application in complementary medicine.  Apis mellifera bees using Baccharis dracunculifolia D.C. (Asteraceae) as a substrate produce green propolis, classified by Park et al. [B. dracunculifolia, also widely known as “Alecrim do campo”, is native to the Southeast and South regions of Brazil and has been the subject of different investigations for ethnomedicinal, phytochemical and pharmacological purposes [The demand for apiculture products has been growing, especially regarding their application in complementary medicine , as is tk et al.  along wipurposes .Candida albicans isolates. The bioactivity of this matrix is mainly due to the presence of phenolic compounds  in its chemical composition [Over the years, records in the literature have been demonstrating the bioactive potential of this resin . Among tposition .These bioactive compounds present in different types of propolis can be extracted using different methods and solvents , which cAmong the non-conventional methods, the extraction with supercritical fluid presents desirable characteristics in what concerns the extraction of thermosensitive compounds. Once it allows the use of low temperatures, providing a minor degradation of sample constituents, besides eliminating eventual problems with residual solvents ,20,21, tEfforts have been made to improve conventional methods, applying alternatives that improve the yield and reduce the time and costs of the extraction step ,25. One In this context, the present work aimed to verify the impact of different experimental conditions applied to low-pressure (ethanolic) and high-pressure  extractions of green propolis, using sonication (60 kHz) as pretreatment, as well as to determine the profile of phenolic compounds in the extracts obtained.p < 0.05) was observed for TPC, FT and CA among the green propolis extracts obtained by different methods. TPC showed a variation of 57.5% among the extracts   (50) varied by 83%  a, while ctively) b, wherea−1, samples B10 and B20, respectively) while those obtained by SFE varied by approximately 4% . Therefore, on average, LPE extracts showed 45% higher TPC than SFE. Since the low polarity of supercritical CO2 provides it with a high power to solubilize compounds with similar polarity, such as waxes, this may explain the lower TPC yield in the extracts obtained by SFE. Waxes are poorly soluble in ethanol, which contributes to the increased interaction of this solvent with the phenolic compounds present in the propolis sample, increasing the yield in this type of extraction. In contrast, CO2 displays weaker interaction with the phenolic compounds, leading to a decrease in the extraction yield. Although solvent polarity is an important parameter, this is not the only preponderant characteristic in the extraction process, since aspects such as solute/solvent interaction are of equal importance [B. dracunculifolia, Casagrande et al. [−1, respectively).Importantly, the extracts obtained by LPE showed a 29% variation in TPC (311.74 ± 3.43 to 439.05 ± 0.90 mgGAE·gportance . In thei−1) of approximately 4% higher than the ESC (186.81 ± 0.32 mgGAE·g−1). Of the LPE extracts, the sample with the highest TPC was B20 (439.05 ± 0.90 mgGAE·g−1), pretreated with ultrasound under the same conditions as the UESC extract, and this was 27% higher than the ST sample (320.97 ± 3.07 mgGAE·g−1). De Souza et al. [The UESC sample  showed a TPC value  when compared to the SFE extracts (between 29.68 ± 0.26 and 50.47 ± 0.77 mgQE·g−1). Among the samples obtained by LPE, the one that obtained the highest FT concentration was A10  (29.14 ± 3.98 mgQE·g−1). In contrast, sample C30  (18.82 ± 1.15 mgQE·g−1) obtained 35% lower yield and was pretreated at higher temperature and treatment time conditions, which can be explained by the thermosensitivity of flavonoids [Scutellaria baicalensis  had decreased yield in extractions conducted with temperature higher than 60 °C due to loss of activity and degradation of flavonoids.Regarding the FT, a different behavior was observed, where the LPE samples obtained 36% lower yield . The authors De Andrade et al.  indicate50 value was lower in ethanolic sample B10  (33.86 ± 1.99 µg.mL−1). This value was approximately 2.8 times lower than the extract with the highest antioxidant capacity reported by Zhang et al. [−1). Considering that, the IC50 expresses the sample concentration required to neutralize the DPPH radical by 50%; the lower this value, the higher the antioxidant potential of the sample. Within this perspective, the sample with the lowest antioxidant capacity, represented by the highest IC50 value, was the supercritical ESC (201.29 ± 0.31 µg.mL−1). These data can be explained since the TPC of the B10 extract is approximately 1.6 times higher than that of the ESC sample. This indicates that the phenolic compounds comprising this pretreated sample, or even the presence of other compounds released in the pretreatment, contribute to the antioxidant potential [The research for natural matrices with high content of compounds with antioxidant capacity has increased considerably in recent years, especially due to the potential benefits that these components present considering biological environments ,39. Withg et al.  for Brazotential .50: 133.17 ± 1.09 µg.mL−1) when compared to its ESC control (IC50: 201.29 ± 0.31 µg.mL−1). This effect is consistent with the FT concentration of these samples, since in the pretreated sample the concentration of these compounds was approximately two times higher than that in the control sample. Flavonoids are biocompounds that are oxidized by free radicals, resulting in a more stable and less reactive radical, providing these compounds with the antioxidant potential [Additionally, in the samples obtained by SFE, it was possible to notice that the UESC sample had higher AC , flavonoids (mgQE·g−1) and DPPH radical scavenging capacity  through Principal Component Analysis (PCA) in order to detect the principal component that best describes the highlighted influences of this study. The principal components  differentiate the extraction methods according to TPC, FT and AC properties. 50 value. The ESC treatment, despite using higher temperature and time, was also grouped in the same quadrant due to its high IC50 value; however, the lower phenolic content of this sample explains its low antioxidant capacity. Inversely located on the left side (negative), samples B10 and C20 were grouped together  the UESC treatment was grouped along with the flavonoids variable (Treatments such as A10 and A20 were grouped in the right quadrant (positive) along with the DPPH variable , indicattogether , indicatvariable . This mavariable b.Conversely, on the opposite left side (positive and negative) are samples C10, C20 and C30, that, despite sonication, showed the lowest flavonoid contents b. This s−1); p-coumaric acid (<LQ—14.33 ± 0.01 mg·g−1); quercetin (from 0.67 ± 0.02 to 2.45 ± 0.00 mg·g−1); gallic acid (<LQ—2.78 ± 0.01 mg·g−1); kaempferol (from 0.34 ± 0.01 to 2.51 ± 0.03 mg·g−1); caffeic acid (0.19—3.02 ± 0.30 mg·g−1); catechin (from 0.52 ± 0.00 to 1.37 ± 0.03 mg·g−1); epicatechin (from 0.22 ± 0.03 to 0.98 ± 0.01 mg·g−1) and rutin (from 0.15 ± 0.01 to 10.00 ± 0.03 mg·g−1)  . Determi−1) and p-coumaric acid (<LQ—14.33 ± 0.01 mg·g−1) . Many of these effects are directly associated with its AC [In most samples obtained by LPE, the majority compounds were formononetin (from 4.76 ± 0.16 to 14.80 ± 0.02 mg·g mg·g−1) . Formonocompound . The precompound . These fcompound . p-Coumapropolis ,57. Ferrpropolis  showed tpropolis  showed th its AC .50: 33.86 ± 1.99 µg.mL−1) and C20 (IC50: 67.1 ± 0.86 µg.mL−1). From the results of the chromatographic analyses, it was possible to note the high concentration of formononetin and p-coumaric acid simultaneously in these samples, which may have directly influenced the ability to neutralize the DPPH free radical. In their in vitro study to determine the antioxidant potential of formononetin, Vishnuvathan et al. [p-coumaric acid against the DPPH radical.Among the extracts obtained by LPE, those with the highest AC were B10  and p-coumaric acid (7.87 ± 0.03 mg·g−1) as the majority compounds. In contrast, the UESC sample showed lower levels of these compounds , but the high amount of rutin (10.00 ± 0.03 mg·g−1) may have directly interfered with the antioxidant potential of this sample , while PC2 y-axis had the lowest score value (4.71%). Observing the right side (positive), it is possible to notice the grouping of treatments B10, C10, C20 and C30 together with the variable formononetin and p-coumaric acid. On the other hand, on the left side (negative) are opposed treatments such as A10, A20 and A30 that have lower content of these biocompounds, which may be an indication that higher temperatures favored the extraction of these phenolic compounds.The PCA presented in Both the ESC and UESC samples were positioned to the left-positive side, showing that it is possible that supercritical extraction and/or pretreatment will have disfavored the extraction of the caffeic acid compound. However, the samples obtained by SFE occupied this position along with the variable rutin due to the higher concentration obtained for this compound. It is possible, analyzing the control sample (ESC), that the pretreatment with ultrasound may have been responsible for the release of kaempferol and rutin in the UESC sample. Since most of the samples obtained by LPE are opposed to the rutin variable, it may be an indication that SFE may have been more selective for obtaining this compound.2) used in the extraction presented 99.9% purity . The gallic acid (CAS number 149-91-7), catechin (CAS number 7295-85-4), epicatechin (CAS number 490-46-0), trans-cinnamic acid (CAS number 140-10-3), narigenin (CAS number 67604-48-2), caffeic acid (CAS number 331-39-5), p-coumaric acid (CAS number 501-98-4), resveratrol (CAS number 501-36-0), formononetin (CAS number 485-72-3), rutin hydrate (CAS number 207671-50-9), quercetin (CAS number 117-39-5), kaempferol (CAS number 520-18-3), and myricetin (CAS number 529-44- 2) were purchased from Sigma-Aldrich Chemical Co. , and trans-ferulic acid (CAS number 537-98-4) was purchased from Fluka . Ethanol (HPLC grade) and acetic acid (HPLC grade) were purchased from Merck Co. , methanol (HPLC grade) and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich Chemical Co. . Cellulose ester membrane filters of 0.45 μm  were used. The carbon dioxide . The sample was processed in a mill  to obtain a diameter between 52 and 92 μm, thus facilitating the extraction process and the uniformity of the material. The sample was kept at −10 °C in inert atmosphere conditions (N2) in a fractional form to avoid the oxidation of the material.Propolis extracts were prepared from the homogenization of 2 g of raw green propolis in 15 mL of ethanol (80%). The system was pretreated in an ultrasonic bath at a frequency of 60 KHz, varying the temperature conditions  and exposure time  . Then, t2 flow—6 g.min−1. The extraction time was approximately 2.5 h. The extracts, collected in glass vials, were stored under the shelter of light, in inert atmospheric conditions (N2) to avoid degradation of the constituents. A control (ESC sample) was obtained without pretreatment in an ultrasonic bath. The extracts were kept at 5 °C until the time of use [To obtain propolis extracts by supercritical extraction, an SFT-110 Supercritical Fluid Extractor  was used. In each experiment, the extraction cell was composed of 5 g of ground green propolis with 5.5 mL of ethanol (80%) as co-solvent, as well as wool and glass beads. The system was pretreated in an ultrasonic bath at the frequency of 60 KHz, 50 °C for 20 min, since this treatment condition obtained higher TPC yield in the extracts obtained by LPE . The exte of use .−1. Then, 0.5 mL of the extract solution was withdrawn and mixed with 2.5 mL of aqueous Folin–Ciocalteu solution (10%) and 2.0 mL of 7.5% sodium carbonate. The solution was placed in a thermoregulated bath at 50 °C for 5 min and then the absorbance was measured in a spectrophotometer  at 765 nm. The results of the concentrations of total phenolic compounds were compared with an analytical curve of gallic acid (mgGAE·g−1)  under the same conditions. All analyses were performed in triplicate and expressed as gallic acid equivalents (mgGAE·g−1).The analyses for determination of total phenolic compounds in green propolis extracts were performed by the Folin–Ciocalteu’s spectrophotometric method using gallic acid as standard . Ethanolv/v) solution [−1). All analyses were performed in triplicate.The determination of the total flavonoid content of the green propolis extracts was performed in a spectrophotometer  at 415 nm. A solution with the propolis extracts was prepared using 2.0% aluminum chloride in methanol in 1:1  was calculated using the straight line equation obtained by constructing the curve based on the concentrations of the extracts , according to the methodology described by Brand-Williams et al. . The greextracts .p-coumaric acid, resveratrol, formononetin, rutin hydrate, quercetin, kaempferol, myricetin, O-dianiside, and trans-ferulic acid) were analyzed from the green propolis extracts. First, 10 mg of green propolis extracts obtained from the different extraction methods were prepared and dissolved in ethanol (HPLC grade). A 0.45 μm cellulose ester membrane filter (Micropore) was used to filter the samples, before injection into the chromatographic system. The chromatographic analyses were performed using a High-Performance Liquid Chromatography (HPLC) equipment , composed of a pump (LC-20AT), supplied with an automatic injector (SIL-20AHT), degasser (DGU-205), diode array detector (DAD) (SPD-M20A) and a column oven (CTO-20A). The method used to promote chromatographic separation was adapted from Daugsch (2007) and Machado et al. (2015). A NUCLEODUR® 100-5 C18 column (150 × 4 mm ID) (5 μm) was used in conjunction with a ZORBAX Eclipse Plus C18 pre-column (4.6 × 12.5 mm) from Agilent.The phenolic compounds  and acetonitrile (solvent B), for 0–35 min (0–92% B); 35 to 40 min (92–0% B); 40 to 42 min (0% B), for a total time of 42 min. The furnace was operated at 40°C. The injection volume was 20 μL, analyzed in triplicate, and the chromatographic acquisition was set at wavelengths in the 280 nm, 300 nm, and 320 nm regions . Fifteen−1), flavonoids (mgQE·g−1) and DPPH radical scavenging capacity , using the software PAST  version 3.26. Since the averages for the aforementioned characterization tests use different units of measurement, the data were normalized in the range of 0 to 1.The results were evaluated using ANOVA (one-way) analysis of variance and Tukey’s test to identify whether the changes in the parameters evaluated were significant at 95% confidence level. The principal component analysis (PCA) was performed to evaluate the influence of extraction methods with respect to the content of phenolic compounds (mgGAE·g50 up to 33.86 ± 1.99 µg.mL−1) and total phenolic content (concentrations up to 439.05 ± 0.90 mgGAE·g−1). Based on the principal component analysis, it was observed that the samples obtained by SFE tended to have higher flavonoid contents (concentrations up to 50.47 ± 0.77 mgQE·g−1), an effect that was enhanced when this extraction technique was associated with sonication. In contrast, treatments with higher temperature (75 °C) showed lower total phenolic values (concentrations up to 23.38 ± 2.3 mgQE·g−1). In addition, by means of PCA, it was possible to observe that extracts obtained by SFE favored the obtainment of phenolic compounds such as rutin and kaempferol, with the SFE method being more selective in obtaining rutin (concentrations up to 10.00 ± 0.03 mg·g−1), an effect that was also strengthened with the use of sonication. Thus, it is hoped that these findings can support the elucidation of the use of pretreatment and type of extractive methods in the process of obtaining natural extracts composed of bioactive molecules.The results showed that samples pretreated with medium temperature (50 °C) associated with ultrasound had higher antioxidant capacity (IC


The nitro group constitutes a significant functional moiety within numerous valuable substances, such as nitroimidazoles, a class of antimicrobial drugs exhibiting broad spectrum activity. Conventional chemical methods for synthesizing nitro compounds suffer from harsh conditions, multiple steps, and environmental issues. Biocatalysis has emerged as a promising alternative to overcome these drawbacks, with certain enzymes capable of catalyzing nitro group formation gradually being discovered in nature. Nevertheless, the practical application is hindered by the restricted diversity and low catalytic activity exhibited by the reported nitrifying enzymes.Saccharothrix sp. Phylogenetic tree analysis revealed that SaRohS belongs to the heme-oxygenase-like diiron oxygenase (HDOs) family. SaRohS exhibited optimal activity at pH 5.5 and 25 ℃, respectively. The enzyme maintained relatively stable activity within the pH range of 4.5 to 6.5 and the temperature range of 20 ℃ to 35 ℃. Following sequence alignment and structural analysis, several promising amino acid residues were meticulously chosen for catalytic performance evaluation. Site-directed mutations showed that threonine 75 was essential for the catalytic activity. The dual mutant enzyme G95A/K115T exhibited the highest catalytic efficiency, which was approximately 5.8-fold higher than that of the wild-type and 22.3-fold higher than that of the reported N-oxygenase KaRohS from Kitasatospora azatica. The underlying catalytic mechanism was investigated through molecular docking and molecular dynamics. Finally, whole-cell biocatalysis was performed and 2-aminoimidazole could be effectively converted into azomycin with a reaction conversion rate of 42% within 14 h.A novel N-oxygenase SaRohS harboring higher catalytic capability of transformation 2-aminoimidazole to azomycin was characterized from Saccharothrix sp., its phylogenetics and enzymatic properties were analyzed. Through site-directed mutation, enhancements in catalytic competence were achieved, and the molecular basis underlying the enhanced enzymatic activity of the mutants was revealed via molecular docking and dynamic simulation. Furthermore, the application potential of this enzyme was assessed through whole cell biocatalysis, demonstrating it as a promising alternative method for azomycin production.An efficient N-oxygenase that catalyzes 2-aminoimidazole to azomycin was screened form The online version contains supplementary material available at 10.1186/s13068-023-02446-5. The samples were obtained by centrifugation of the reaction mixture at 10,000×g for 10 min, then filtered through a 0.22 μm Tuffryn membrane. The filtered samples were analyzed by HPLC using an Agilent 1260 HPLC apparatus equipped with a Luna C18, 5 μm, 4.6 mm ID × 250 mm column (Phenomenex). Elution was performed at a flow rate of 0.5 mL·min−1 using a mobile-phase consisting of a linear gradient of water and acetonitrile ((v/v):90:10, 0 to 5 min, 90:10 to 0:100, 5 to 6 min; 0:100, 6 to 10 min, 90:10, 10 to 11 min; 90:10 11 to 15 min), with both solvents containing 0.05% (v/v) trifluoroacetic acid. For 2-aminoimidazole detection, the elution was performed at a flow rate of 0.5 mL·min−1 using a mobile-phase consisting of a linear gradient of water and methanol ((v/v): 95:5 to 85:15, 0 to 5 min; 85:15 to 75:25, 5 to 15 min; 0:100, 20 to 25 min; 0:100 to 95: 5, 25 to 35 min; 95:5 35 to 40 min), with both solvents containing 5 mM ammonium acetate. The LC–MS analysis was conducted using a Ulimate 3000 UHPLC system coupled to a Compact Q-TOF LC/MS system, performed in positive ion mode with a mass scan range of 30 to 800 and an ESI ion source.The cell density at ODAdditional file 1: Figure S1. Verification of the proteins’ expression and purification by SDS-PAGE.Additional file 2: Figure S2. Verification of the azomycin product by different enzymes using LC–MS. A, the specific ion flow of the standard and production with different enzymes; B, the mass spectrum of the azomycin standard; C, the mass spectrum of the azomycin production with KaRohS; D, the mass spectrum of the azomycin production with SaRohS; E, the mass spectrum of the azomycin production with AtRohS; F, the mass spectrum of the azomycin production with KvRohS.Additional file 3: Figure S3. The predicted 3D structure of SaRohS (A). The definitely conserved sequence was colored with red, the relatively conserved sequence was colored with green, and the variation sequence was colored with white. The selected sites for site-directed mutation of T75, K115, D198, L212, D266 were displayed as B, C, D, E, and F, respectively.Additional file 4: Figure S4. The enzyme–substrate molecular interaction network of wild SaRohS and each mutants. Conventional hydrogen bond with bright green, Van del Waals with medium green, Carbon hydrogen bond with light green, Metal acceptor with grey, Pi-alkyl with pink, Pi-sigma with purple, unfavorable acceptor–acceptor with red, Pi-anion with brown.Additional file 5: Figure S5. Dynamic simulation of G95A mutant. A, Root-mean-square deviation (RMSD) value; B, Root-mean-square fluctuation (RMSF) value; C, Radius of gyration (Rg) value; D, the amino acid residues with increased flexibility after mutation; E, the conformation change of the substrate in the catalytic center, the blue stick is the conformation before mutation and the white stick is the conformation after mutation.Additional file 6: Figure S6. Dynamic simulation of K115T mutant. A, RMSD value; B, RMSF value; C, Rg value; D, the amino acid residues with increased flexibility after mutation; E, the conformation change of the substrate in the catalytic center, the blue stick is the conformation before mutation and the white stick is the conformation after mutation.Additional file 7: Figure S7. Dynamic simulation of G95A/K115T mutant. A, RMSD value; B, RMSF value; C, Rg value; D, the amino acid residues with increased flexibility after mutation; E, the conformation change of the substrate in the catalytic center, the blue stick is the conformation before mutation and the white stick is the conformation after mutation.Additional file 8: Figure S8. The profile of the residual 2-aminoimidazole during the whole-cell synthesis with G95A/K115T mutant.Additional file 9: Table S1. Information of proteins given > 60% sequence identity with KaRohS. Table S2. Organic solvent tolerance of SaRohS. Table S3. The binding free energy calculated using MM/PBSA. Table S4 Strains, plasmids and primers used in this study.


T isolated from human feces. 16S rRNA gene-based phylogenetic analysis showed that strain KD22T was found to be most closely related to species of the genus Gabonibacter. At the 16S rRNA gene level, the closest species from the strain KD22T corresponded with Gabonibacter massiliensis GM7T, with a similarity of 97.58%. Cells of strain KD22T were Gram-negative coccobacillus, positive for indole and negative for catalase, nitrate reduction, oxidase, and urease activities. The fatty acid analysis demonstrated the presence of a high concentration of iso-C15: 0 (51.65%). Next, the complete whole-genome sequence of strain KD22T was 3,368,578 bp long with 42 mol% of DNA G + C contents. The DDH and ANI values between KD22T and type strains of phylogenetically related species were 67.40% and 95.43%, respectively. These phylogenetic, phenotypic, and genomic results supported the affiliation of strain KD22T as a novel bacterial species within the genus Gabonibacter. The proposed name is Gabonibacter chumensis and the type strain is KD22T (= CSUR Q8104T = DSM 115208 T).A polyphasic taxonomic approach, incorporating analysis of phenotypic features, cellular fatty acid profiles, 16S rRNA gene sequences, and determination of average nucleotide identity (ANI) plus digital DNA–DNA hybridization (dDDH), was applied to characterize an anaerobic bacterial strain designated KD22The online version contains supplementary material available at 10.1007/s00203-023-03671-0. Despite recent therapeutic advances, lung cancer remains the number one cause of cancer-related death worldwide  and was first cultured from a human fecal sample of a healthy young Gabonese . The stool samples collected were stored at −80 ºC in November 2019 prior to initiating culturomics. The patient had not received any antibiotics treatment within the last 3 months before fecal collection. He gave a signed informed consent at the time of sampling and the study was approved by CHUM Research Ethics Committee 20.300. To isolate gut bacteria, 1 g of the fecal sample was injected into an anaerobic culture bottle  enriched with 4 ml filter-sterilized rumen fluid and 5% sheep blood , and then incubated at 37 ºC. After 7 days of incubation, 100 µl culture broth was sampled and plated on sheep blood-enriched Columbia agar (BioMérieux). The agar plates were incubated in an anaerobic chamber  at 37 ºC for 48 h. Each emerging colony was purified and identified using MALDI-TOF mass spectrometry (MS) with a Microflex LT spectrometer  that compared the spectra with those present in the library , as previously reported as previously described  . Phylogenetic neighbors of KD22T were identified using the BLASTn program .The MALDI-TOF MS identification of strain KD22T cell morphology was assessed by transmission electron microscopic as previously reported  under different atmospheres . The pH range growth was also tested at pH 5, 6, 6.5, 7, 7.5, and 8.5. Tolerance of NaCl was determined for concentrations ranked between 0 and 100 g/l. Catalase and oxidase productions were also detected (BioMérieux). Enzymatic and biochemical properties of strain KD22T were determined in duplicate using the API® 20A, API® ZYM, and Rapid ID 32A identification systems (BioMérieux). Short-chain fatty acids were analyzed using both a gas chromatograph (Hewlett Packard) and Microbial Identification System software.Strain KD22® AmpFREE Low DNA Library Kit Library Preparation Kit (Lucigen) according to the manufacturer’s recommendations, with 700 ng of genomic DNA as starting material. Dual-indexed adaptors were purchased from IDT. Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). The average size fragment was determined using a LabChip GX II (PerkinElmer) instrument. The libraries were normalized and pooled, denatured in 0.05 N NaOH, and neutralized using HT1 buffer. The pool was loaded at 225 pm on an Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer’s recommendations. The run was performed for 2X150 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Base calling was performed with RTA v3. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads. Genome assembly was performed as previously reported . The genomic similarity among all compared genomes was evaluated by calculating two parameters: digital DNA–DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) values using Genome-to-Genome Distance Calculator (GGDC) . Subsequently, an almost-complete 16S rRNA gene sequence of strain KD22T was determined , and database searches using nucleotide BLAST revealed highest sequence similarity to that of 2 uncultured bacteria , to ‘Sanguibacteroides justesenii’ OUH 308042 T (97.72%) not validly published and to members of the genus Gabonibacter (97.58% to G. massiliensis GM7T and 96.20% to ‘Gabonibacter timonensis’ Marseille-P3388T). Phylogenetic analysis of KD22T and other related type strain, performed using the neighbor-joining method, confirmed that strain KD22T was phylogenetically closest to G. massiliensis GM7T detected from human feces . Bacteria occurred as single rods or in a short chain. Bacterial colonies were circular, white to pale cream with 0.6–1.7 mm of diameter after 48 h of incubation at 37 ºC in anaerobic conditions on sheep blood-enriched Columbia agar. Growth occurred between 28 and 37 ºC with an optimal at 37 ºC. No growth was observed in the microaerophilic and aerobic atmosphere or at 45 and 55 ºC. The strain grew at a pH ranging from 6 to 7.5, with optimal growth at pH 7.0 and a NaCl concentration of less than 5%. The biochemical and enzymatic characteristics of KD22T, obtained using API® ZYM, API® 20A, and Rapid ID 32A strips, are given in the species description section and the features that differentiate strain KD22T from its close neighbors are shown in Table 15:0 was the major fatty acid (51.65%). The cellular fatty acid profile of strain KD22T and those of its closest related species are mentioned in Table Cells of strain of KD22T resulted in 14 scaffolds (composed of 14 contigs) with a total sequence length of 3,368,578 bp . The genomic comparison of KD22T with its neighbors is presented in Fig. T  are in the range reported for type strains of phylogenetically related species but very close to those of G. massiliensis . Strain KD22T and G. massiliensis show high level of AAI values . Nevertheless, the dDDH values between species ranged from 18.70% with ‘S. justeseni’i and O. laneus to 84.40% between G. massiliensis and ‘S. justesenii’. Strain KD22T shared dDDH values from 18.50% with Butyricimonas virosa to 67.40% with G. massiliensis (Table G. massiliensis and ‘S. justesenii’). Strain KD22T shared ANI values ranging from 65.63% with Parabacteroides distasonis to 95.43% with G. massiliensis  for more distant populations, because the resolution is progressively lost at the nucleotide level  higher than the limit set for species demarcation suggest that these two strains belong to the same species.A polyphasic approach based on a combination of 16S rRNA gene sequence analysis, phenotypic features, chemotaxonomic properties, and genomic data demonstrated that strain KD22Gabonibacter chumensis .15:0 and C16:0 3-OH.Cells are strictly anaerobic, Gram-negative, not mobile, and non-spore-forming coccobacilli with up to 0.5 µm in diameter and 1.5 µm in length. They occur as single rods or in a short chain. After 48 h anaerobically at 37 ºC on sheep blood-enriched Columbia agar, bacterial colonies are circular, white to pale cream with 0.6–1.7 mm of diameter. Growth occurs between 28 and 37 ºC with an optimum at 37 ºC. NaCl concentration and pH range allowing growth are 0–5% and 6–7, respectively. Catalase, oxidase, and urease are not produced. Indole is detected, and nitrate is not reduced to nitrites. Aesculin and gelatin are not hydrolyzed. Acid is produced from D-glucose, D-mannitol, salicin, D-mannose, D-sorbitol, and D-trehalose, but not from lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, glycerol, D-cellobiose, D-melezitose, D-raffinose, and L-rhamnose. Using API® strips (ZYM and Rapid 32A), the strain exhibits alkaline phosphatase, acid pyroglutamic arylamidase, alanine arylamidase, α-chymotrypsin, esterase, esterase lipase, glutamyl acid glutamic, leucyl glycine arylamidase, and naphthol-AS-BI-phosphohydrolase activities. On the other hand, galactosidase (α and β), glucosidase (α and β), β-glucuronidase, N-acetyl-β-glucosaminidase, α-arabinosidase, α-manisidase, and α-fucosidase are negative. The most abundant fatty acids are iso-CT (= CSUR Q8104T = DSM 115208), was isolated from the feces of a patient suffering from lung cell small cancer. Its draft genome measures 3,368,578 bp and exhibits a DNA G + C of 41.99 mol%. The GenBank/EMBL accession numbers of 16S rRNA gene and genome sequences are OP221267.1 and JANSKB000000000.1, respectively.The type strain, KD22Supplementary file 1—Figure S1. Main spectra library (MSP) dendrogram of MALDI-TOF mass spectral profiles from strain KD22 and its neighbors generated by the MALDI Biotyper 3.0 software. Distance is displayed in relative units.Supplementary file 2—Figure S2. Tamura-Nei model maximum likelihood phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain KD22T and closest related taxa. GenBank accession numbers are shown in parentheses. Numbers at nodes indicate bootstrap percentages (based on 100 replicates). Bar, 0.05 substitutions per nucleotide position.Gabonibacterchumensis strain KD22T using a 375 Tecnai G20 transmission electron microscope.Supplementary file 3—Figure S3. Transmission electron microscopy of Supplementary file 4—Table S1. Genome comparison of closely related species to strain KD22T.Supplementary file 5—Table S2. Number of genes associated with the 25 general COG functional categories.Gabonibacterchumensis KD22T and closest related species.Supplementary file 6—Table S3. Average amino acid identity (AAI) between Below is the link to the electronic supplementary material.


For both oral and intravenous administration, the area under the concentration–time curve (AUC0-∞) was determined to be 4.72 ± 1.86 and 11.76 ± 3.25 h·µg/mL, respectively. The corresponding elimination half-life (t1/2λz) was measured as 11.24 ± 3.90 and 10.17 ± 3.72 h. Moreover, the mean residence time (MRT) was calculated as 10.20 ± 2.47 and 7.05 ± 1.97 h for these respective routes. Based on the calculated AUC/MIC ratio values, it can be inferred that the 5 mg/kg BW dosage of danofloxacin, whether administered orally or intravenously, is expected to effectively treat Escherichia coli and Pasteurella multocida infections in Gushi chickens.This study aimed to determine the pharmacokinetics of danofloxacin in Gushi chickens after a single oral (PO) and intravenous (IV) dose at 5 mg/kg body weight (BW). Thirty-two Gushi chickens, aged 20 weeks, were selected and divided into two groups at random, with each group consisting of 16 chickens, evenly distributed between males and females. Following danofloxacin administration, blood samples were taken at predetermined time intervals and the plasma was separated. The concentrations of danofloxacin in plasma were quantified by HPLC with a fluorescence detector. Then the concentrations versus time data were subjected to non-compartmental analysis (NCA) using Phoenix software (version: 8.1.0). After administering danofloxacin orally at a dose of 5 mg/kg BW to Gushi chickens, our results demonstrated that the peak concentration reached 0.53 μg/mL at 4 h. The half-life of absorption (t Gushi chickens, a popular breed in China known for their high-quality meat and eggs, play a crucial role in poultry production . Due to Fluoroquinolones are a rapidly developing class of synthetic antibacterial compounds . They woDanofloxacin, a third-generation veterinary fluoroquinolone, is frequently used in veterinary clinics in its mesylate salt form. It possesses broad-spectrum antibacterial activity against most Gram-negative bacteria, some Gram-positive bacteria, as well as certain mycoplasma and chlamydia species . DanofloNumerous studies have examined the pharmacokinetics of danofloxacin in various bird species, including California brown pelicans, common pheasants, guinea fowls, Japanese quails, and Bilgorajska geese ,10,11. H3PO4) was purchased from Shanghai Boer Chemical Reagent Co., Ltd. , and tri-ethylamine was acquired from Shanghai Yien Chemical Technology Co., Ltd. . Methanol (chromatographic grade) and acetonitrile (chromatographic grade) were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. . Additionally, a 0.9% sodium chloride injection was purchased from Anhui Fengyuan Pharmaceutical Co., Ltd. . All other reagents were obtained from commercial sources.The danofloxacin mesylate reference substance  was obtained from the China Institute of Veterinary Drugs Control , while the danofloxacin mesylate raw material  was sourced from Guobang Pharmaceutical Co., Ltd. . Phosphoric acid  at the age of 20 weeks from a commercial farm in Luoyang City. Each chicken was individually placed in a wire cage measuring 55 × 55 × 45 cm, which was equipped with an automatic water dispenser. The Gushi chickens were provided with an antimicrobial-free diet and had unrestricted access to water. The feeding environment was maintained at a temperature of 21 ± 0.6 °C, with proper ventilation, and a daily light exposure of 16 h. Before any procedures were conducted, the chickens were given a two-week acclimation period. The study protocol received approval from the Institutional Animal Care and Use Committee (IACUC) at Henan University of Science and Technology, with the approval number DK20230303.g for 10 min. The resulting plasma was transferred to 1.5 mL centrifuge tubes. All plasma samples were stored at −20 °C until further analysis.In our study, we initially weighed 32 Gushi chickens and then randomly divided them into two groups: oral (PO) and intravenous (IV). These groups consisted of equal numbers of male and female chickens. The body weights of the PO and IV groups ranged from 1.36 to 2.53 kg and 1.33 to 2.57 kg, respectively. To administer the oral dose, we prepared a 5 mg/mL solution of danofloxacin mesylate raw material using purified water. The chickens in the PO group were given a dose of 5 mg/kg BW through a gavage tube. Venous blood samples were collected from the wing veins at specific time intervals: 10, 15, 30 min, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, and 48 h after administration. For the intravenous treatment, we prepared an injection solution of 20 mg/mL danofloxacin mesylate raw material using 0.9% sodium chloride. The IV group received a dose of 5 mg/kg BW, which was administered intravenously through the left wing vein. Venous blood samples were collected from the right wing vein at specific time intervals: 5, 15, 30, and 45 min, and 1, 1.5, 2, 4, 6, 8, 12, 24, 36, and 48 h after administration. Approximately 1 mL of blood was collected at each time point and then processed by centrifugation at 4000× g for 10 min. The supernatant was carefully collected in a clean glass tube and the extraction process was repeated. All extract was then dried using a nitrogen gas flow at 50 °C. The residue was dissolved in 2 mL of the mobile phase and vortexed for 1 min. The liquid was filtered through a 0.22-μm filter into an autosampler glass vial. Finally, 20 μL of the supernatant was analyzed using the HPLC system.Danofloxacin concentrations were determined using a previous HPLC method . Brieflyv:v = 83:17), was delivered at a flow rate of 1 mL/min.The concentration of danofloxacin was determined using the Waters e2695 HPLC system, which was connected to a 2475 fluorescence detector and controlled by Empower software . The separation process utilized a Hypersil BDS C18 column . Each sample (20 μL) was injected, and the column was maintained at 30 °C for elution over a duration of 12 min. Fluorescence detection was carried out with an excitation wavelength of 280 nm and an emission wavelength of 450 nm. The mobile phase, consisting of a 0.05 mol/L phosphate buffer (adjusted to a pH of 2.6 with triethylamine) and acetonitrile (2) was further determined for the standard curve. To assess the coefficient of variation and recovery, triplicate samples containing low, medium, and high concentrations of danofloxacin  were spiked to plasma. This process was repeated three times a day for a total of three days. The limit of detection (LOD) and limit of quantification (LOQ) were determined based on signal-to-noise (S/N) ratios of ≥3 and ≥10, respectively.A standard stock solution of danofloxacin (500 μg/mL) was prepared using methanol and stored in the dark at 4 °C. This solution served as the basis for standardization and verification of the analytical method. The standard working solution was freshly prepared by diluting the standard stock solution with the mobile phase and used immediately. The standard curve was created through linear regression analysis by mixing a 450 μL aliquot of drug-free plasma obtained from healthy Gushi chickens with 50 μL of a standard working solution containing various concentrations of danofloxacin. The concentrations used for the standard curve were 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 μg/mL. The linear correlation coefficient (R1/2λz) was determined as ln2/λz [last-∞ to AUC0-∞. The mean residence time (MRT) was calculated as MRT = AUMC0-∞/AUC0-∞. For the IV route, the initial concentration (C0) was estimated using the back extrapolation method. Total body clearance (Cl) was determined as the ratio of the intravenous dose to AUC [Z) was calculated as VZ = Dose/AUC/λz, where Dose represents the intravenous dose. The volume of distribution at steady state (VSS) was determined as VSS = MRTIV × Cl. For PO administration, the peak concentration of danofloxacin (Cmax) and the time to reach it (Tmax) were both directly read from the concentration versus time curve [PO minus MRTIV. The absorption phase rate constant (Ka) was obtained by dividing 0.693 by MAT, and the half-life of absorption (t1/2ka) was further determined as 0.693 × MAT. Bioavailability (F) was calculated as the ratio of AUCPO to the AUCIV [The concentration–time data of danofloxacin in each Gushi chicken plasma were analyzed using the non-compartment model analysis (NCA) method in Phoenix WinNonLin software  to obtain pharmacokinetic parameters. For both routes of administration, the first-order rate constant associated with the terminal phase (λz) was calculated using linear regression, from which the terminal half-life  and PO  groups, as well as Cl and VSS between IV  and IV  groups, followed a normal distribution. For these parameters, the independent sample t-test was utilized to analyze significance. Conversely, the Mann–Whitney U test was employed for other parameters that did not conform to a normal distribution. Gender effects were found to be nonsignificant in both treatment groups. Subsequently, the statistical analysis was performed on the pharmacokinetic parameters of both sexes within the two treatment groups. The normality between PO , PO , IV , and IV  groups was assessed using the Shapiro–Wilk test. Notably, t1/2λz, AUC, AUC%, AUMC, and MRT did not exhibit normal distribution. Hence, one-way ANOVA was conducted for statistical analysis, while the independent samples t-test was applied to the remaining parameters. Finally, a statistical analysis was conducted between the PO  and IV  groups. A significance level of p < 0.05 was used to determine the presence of a significant difference.The statistical analysis of pharmacokinetic parameters was conducted using SPSS software (version: 22.0). Firstly, gender effects were examined in each treatment group. The normality of each pharmacokinetic parameter between genders within each group was assessed using the Kolmogorov–Smirnov test. Results indicated that C−9 S + 0.0021 , with an R2 of 0.9999. Under these conditions, the recovery rate of danofloxacin ranged from 84.49% to 93.94%, the intra-day variation coefficient ranged from 1.85% to 4.31%, and the inter-day variation coefficient ranged from 2.44% to 3.56%. The LOD and LOQ were determined to be 0.0005 and 0.001 µg/mL, respectively.The extraction and detection methods employed in this study showcased robust specificity, high accuracy and precision, as well as excellent selectivity, in detecting danofloxacin in plasma. The retention time of danofloxacin was determined to be approximately 7.2 min, effectively avoiding interference from impurity peaks. The concentration range of danofloxacin exhibited a good linear relationship and reproducibility, ranging from 0.005 to 5 µg/mL. The regression equation for the calibration curve was C = 7 × 100-∞ after PO and IV administration was determined to be 4.72 ± 1.82 and 11.76 ± 3.25 h·µg/mL, respectively, exhibiting a significant difference. The MRT was calculated as 10.20 ± 2.47 h (PO) and 7.05 ± 1.97 h (IV), with a significant difference. The elimination half-life after PO and IV administration was 11.24 ± 3.90 and 10.17 ± 3.72 h, respectively, showing no significant difference. Following IV administration, danofloxacin demonstrated a wide distribution and slow elimination in Gushi chickens, with Vz of 6590.63 ± 2900.10 mL/kg and Cl of 446.86 ± 89.05 mL/h/kg. After PO administration, the drug concentration reached a peak of 0.53 ± 0.19 µg/mL at 4 h. The MAT was determined as 3.15 ± 2.47 h, but the bioavailability (F) was found to be only 40.12 ± 15.83%.Throughout the entire experimental period, the 32 Gushi chickens remained in good condition with no observed adverse reactions after administration. At the last plasma collection point (48 h), danofloxacin was still detectable and exceeded the LOQ. The plasma concentration of danofloxacin in Gushi chickens after PO and IV administration was graphically represented in As shown in max, Tmax, and t1/2ka. In the current study, danofloxacin reached a peak concentration of 0.53 ± 0.19 µg/mL at 4 h after PO administration, with a t1/2ka value of 2.37 ± 1.60 h. Under the same dosage (5 mg/kg) in different poultry species, the current Cmax was lower than that in Jing Hong hens (1.05 µg/mL) [max compared to all the aforementioned poultry varieties . Gushi chickens are usually raised in a free-range environment, which allows them to roam freely. This exercise habit may have caused its Tmax to be delayed more than the other birds. In this study, Gushi chickens were initially raised in a free-range environment before being placed in cages for the experiment. They underwent a 14-day acclimatization period in the new environment.This study is the first to examine the pharmacokinetics of danofloxacin in Gushi chickens, and danofloxacin was given at 5 mg/kg via PO and IV routes. In a pharmacokinetic study, important parameters for assessing drug absorption rate include C5 µg/mL) , Muscovy5 µg/mL) , and Bil5 µg/mL) . However5 µg/mL) , and Arb5 µg/mL) , followi1/2λz) after PO and IV administration was 11.24 ± 3.90 and 10.17 ± 3.72 h, respectively, with no significant difference. While the current PO and IV t1/2λz values in Gushi chickens were longer than those in Lohmann broiler chickens  [1/2λz values. This phenomenon is not only observed in poultry but also in livestock. For example, in donkeys, the t1/2λz after IV and intramuscular (IM) administration was 7.25 and 6.36 h, respectively [1/2λz after IV and subcutaneous (SC) administration was 3.27 and 3.07 h, respectively [The elimination of danofloxacin in Gushi chickens occurred at a slow pace. The half-life (tctively)  and turkctively) . Howeverectively , withoutSS) of 3035.00 ± 428.84 mL/kg, which was lower than those observed in Lohmann broiler chickens  [SS values. The wider distribution of danofloxacin compared to other fluoroquinolones may be attributed to its higher lipid solubility and intracellular permeability [After IV administration, Gushi chickens exhibited a volume of distribution at steady state  . On the 0 mL/kg)  and enro0 mL/kg) , demonsteability . Additio1/2ka was further calculated as 2.37 ± 1.60 h, which was longer than those in Japanese quails (0.98 h) [After PO administration, the MRT value was 10.20 ± 2.47 h, longer than that of IV administration (7.05 ± 1.97 h), and the difference between both was calculated to be the MAT (3.15 ± 2.47 h). The PO t(0.98 h) , but sho0-∞ in Gushi chickens was found to be 11.76 h·µg/mL. This is comparable to the AUC0-∞ observed in Jing Hong hens (10.41 h·µg/mL) [0-∞ was only 4.72 h·µg/mL. The current result is consistent with the values observed in most birds  [After administering 5 mg/kg of danofloxacin through IV administration, the AUCh·µg/mL)  and turkh·µg/mL) . Howeverh·µg/mL)  and Lohmh·µg/mL)  which reh·µg/mL) ,21,28,29 hens 10.1 h·µg/mLh·µg/mL) , Bilgorah·µg/mL) , Lohmannh·µg/mL) . The lowUpon comparing all the pharmacokinetic data, we observed that the gender of Gushi chickens had minimal impact on the pharmacokinetics of danofloxacin. Despite the larger body weight of males in comparison to females , the pharmacokinetic parameters displayed only slight variations between the two sexes. However, these differences were not statistically significant when comparing males and females under the same administration route. It is worth mentioning that previous studies exploring the pharmacokinetics of danofloxacin in other birds did not separate the data based on gender ,22. ThisIn our experiments, we overlooked the possible effects of danofloxacin on chicken blood parameters. However, according to a report by FAO, female dogs that were orally administered danofloxacin at a dose of 50 mg/kg BW per day for two weeks showed increased levels of serum blood urea nitrogen and creatinine . This suEscherichia coli [Escherichia coli [Pasteurella multocida [Escherichia coli and Pasteurella multocida in Gushi chickens are consistent with the findings of previous studies, the current oral dosage of 5 mg/kg would yield an AUC/MIC ratio of 18.88 h and 94.4 h against Escherichia coli and Pasteurella multocida, respectively—both ratios surpassing 6.73 h. Since the same IV dose resulted in a significantly higher AUC (Escherichia coli or Pasteurella multocida.A previous study conducted on turkeys found that an AUC/MIC ratio greater than 6.73 h could effectively eradicate hia coli . In addihia coli  and an Multocida . Assumingher AUC , the theEscherichia coli or Pasteurella multocida.The current study demonstrated excellent pharmacokinetic profiles for danofloxacin, including quick absorption, wide distribution, and slow elimination in Gushi chickens. However, incomplete oral absorption was also observed in Gushi chickens with absolute bioavailability at 40.12 ± 15.83%. Based on the ratio of AUC/MIC determined in the current study, an intravenous or oral dose of 5 mg/kg danofloxacin would be expected to successfully treat Gushi chickens infected by


S. typhimurium TA1535. In contrast, MNNG and NNK mutagenicity detected in S. typhimurium YG7108, a strain lacking O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of beer, NABs, or beer components, suggesting that they may mediate its antimutagenic effect by enhancing DNA damage repair. Phosphorylation of Akt and STAT3, with or without epidermal growth factor stimulation, in lung epithelial-like A549 cells were significantly decreased following beer, NABs, GB and PU. They targeted both the initiation and growth/progression steps of carcinogenesis, specifically via antimutagenesis, stimulation of alkyl DNA-adduct repair, and suppression of Akt- and STAT3- mediated growth signaling. GB and PU may contribute, in part, to the biological effects of beer and NABs via the suppression of Akt and STAT3 phosphorylation.We investigated the chemopreventive effects of beer, nonalcoholic beers (NABs), and beer-components (glycine betaine (GB)) on NNK-induced lung tumorigenesis in A/J mice, and the possible mechanisms underlying the antitumorigenic effects of beer, NABs, and beer-components. Beer, NABs, and GB reduced NNK-induced lung tumorigenesis. We investigated the antimutagenicity of beer, NABs and beer-components (GB and pseudouridine (PU)) toward the mutagenicity of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Beer, NABs, and beer components were antimutagenic toward MNNG and NNK in the Ames test using  Lung cancer is the most common cause of cancer-related deaths . The deaBeer is one of the most consumed alcoholic beverages in the world and is rich in nutrients such as carbohydrates, amino acids, minerals, vitamins, and other compounds such as polyphenols. Hops are one of the raw materials for beer, which serves as an important source of phenolic compounds . ApproxiRecently, nonalcoholic beers (NABs) have been developed, and the size of this market is increasing because of the increasing adoption of healthier lifestyles and awareness of the benefits of alcohol-free beverages. Nonalcoholic beer can be produced in various ways, including the removal of alcohol from beer after production or the use of special yeast to avoid the production of alcohol during brewing . DependiIn our previous study, we identified pseudouridine (PU) and glycine betaine (GB) as active components in beer responsible for antimutagenicity , 16. GB In the present study, we investigated the antimutagenic activity of NABs and beer components (GB and PU) toward the alkylating agent 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and NNK, and its mechanisms. We also investigated the chemopreventive effects of beer, NABs, and GB on NNK-induced lung tumorigenesis in A/J mice, and the molecular mechanism of the antitumor effect and suppression of tumor growth in the presence of beer and beer components.Commercial beer, low alcoholic beer , and nonalcoholic beer  were purchased from local stores in Okayama City. The alcohol content of the beer sample was 5%, that of LAB was 0.5%, and those of NABs were 0%. All the samples except NAB(B) were produced in Japan, and NAB(B) was imported from Germany. For the Ames test, beer, LAB, and NABs were sterilized by filtration. For further experiments, the beer was freeze-dried and the alcohol was removed. NAB(A) and NAB(B) were freeze-dried. The residues were dissolved in water to one-tenth the original volume (hereafter referred to as the 10-fold concentrate). The 10-fold concentrates were stored at -20 °C and diluted just before use. GB and PU were purchased from FUJIFILM Wako Pure Chemical Co. . NNK were purchased from Toronto Research Chemicals , MNNG was from Nacalai Tesque , and 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) was from Dojindo Laboratories . For metabolic activation, the supernatant fraction of rat liver homogenate (S9) prepared using phenobarbital and 5,6-benzoflavone was obtained from FUJIFILM Wako Pure Chemical Co. Akt (pan) (C67E7) rabbit mAb (#4691), phospho-Akt (Ser473) (D9E) rabbit mAb (#4060), STAT3 (79D7) rabbit mAb (#4904), phospho-STAT3 (Tyr705) (D3A7) XP rabbit mAb (#9145), LY294002 (#9901) monoclonal antibodies (mAbs) was purchased from CST, Japan , and Stattic (ab120952) from Abcam . All the other reagents were purchased from commercial sources.Salmonella enterica subspecies I, serovar Typhimurium  strain TA1535 [hisG46 ΔuvrB gal bio chl1005 rfa1001] was a gift from Dr. B. N. Ames of the University of California, Berkeley [S. typhimurium YG7108 [hisG46 ΔuvrB gal bio chl1005 rfa1001 Δadast::Kmr Δogtst::Cmr], a strain lacking O6-methylguanine DNA methyltransferases, was gifted from Dr. M. Yamada of the National Institute of Health Sciences [Berkeley , and S. Sciences . Human lQuantitative analysis of GB and PU in NAB was outsourced to the Saitama Prefectural Industrial Technology Center. Analyses were performed using Agilent Technologies 1260 Infinity II HPLC and 6120 single quadrupole mass spectrometers . The GB content was 92 µM in NAB(A) and 290 µM in NAB(B). The PU contents were 5.2 µM in NAB(A) and 22 µM in NAB(B).Mice  were purchased from Japan SLC, Inc. . Five mice were housed in animal compartments for each cage and randomly divided into treatment groups at least one week before the start of the experiment. Murine chow  was mixed with water or an experimental solution to produce feed-lumps. The mice had free access to feed-lumps and water, and were maintained with optimal air exchange and a 12-h light/12-h dark cycle at a constant room temperature of 20 °C. All the experiments were conducted in accordance with the Guidelines for Animal Experiments of the Okayama University Advanced Science Research Center  based on the Act on Welfare and Management of Animals  and Standards Relating to the Care, Management, and Alleviation of Pain and Distress of Laboratory Animals (Notice of the Ministry of the Environment No. 88 of 2006).S. typhimurium TA1535 in the absence of rat liver homogenate (hereafter referred to as -S9) as previously described [S. typhimurium YG7108 (-S9) were also investigated. The amounts of beer, LAB, and NAB used for the assay calculated as microliter equivalents of the original volume (µL eq.) of the original beer, LAB, and NABs, respectively. The effects of beer, NAB(A), NAB(B), PU and GB on NNK-induced mutagenicity were also investigated using the Ames test with S. typhimurium TA1535 and YG7108 in the presence of rat liver homogenate for metabolic activation (hereafter referred to as + S9). Other procedures were as described above. The experiments were performed in triplicates. All plates were examined with a stereo microscope whether background lawn was impaired.The effects of beer, LAB, NABs, ethanol, PU and GB on MNNG-induced mutagenicity were investigated using the Ames test  with S. escribed . Beer, LThe mutagenic activity (%) was derived as follows:NNK-induced tumorigenesis experiments were conducted according to previous studies . In expeIn experiment 2, mice  were divided into 5 groups (groups VII-XII) of 10–15 animals each. Prior to the experiment, the two nonalcoholic beers (NAB (A) and NAB (B)) were freeze-dried and dissolved in water to half the volume of the original NAB (hereinafter referred to as 200% NAB (A) and 200% NAB (B)). The feed-lumps were produced by mixing murine-chow powder with water, 200% NAB(A) or 200% NAB(B) , or NAB(B) diets, respectively). At 8 weeks of age, mice in groups VII, VIII, and IX were injected with 100 mM NNK in 0.1 ml saline, while mice in groups X, XI, and XII were given a single i.p. injection of 0.1 mL saline. The mice in groups VII and X received a water diet throughout the experiment. Mice in groups VIII and XI received the NAB(A) diet, and mice in groups IX and XII received the NAB(B) diet from 4 weeks of age until the end of the experiments. The mice were euthanized at the age of 30 weeks. In both experiments, the number of surface lung nodules in the left lung lobe was counted using a loupe and a Digital Caliper . Counted nodules were with a diameter of > 1 mm. The left lung lobes were fixed, the sections were stained with hematoxylin and eosin (HE), and pathological analysis was performed.4 cells, 90 μL/well) were seeded in 96-well plates. After 24 h incubation , 10 μL of beer , NAB , PU  or GB  was added to each well. After incubation for 24 h, MTT solution (10 μL/well) was added and incubated for 2 h. The MTT-containing medium was removed, and for dissolving the formazan complexes, dimethyl sulfoxide (DMSO) was added to the wells. The optical density of the cells was measured, and optical density of the control (untreated) cells was set at 100% viability.Cell viability was measured using the MTT assay to determine the concentration of beer, NAB, PU and GB for the studies of phosphorylation effect as previously described . BrieflyThe effects of phosphorylation of STAT3 and Akt in A549 cells was determined as previously described . BrieflyP < 0.05. Statistical analyses were performed by KaleidaGraph  and Excel statistics .For each data point as indicated in each figure, data are expressed as means ± standard deviation. Statistical significance was set at S. typhimurium TA1535 without metabolic activation (-S9). The results are shown in Fig. + revertants per plate found in the absence of beer, LAB, NABs, GB, or PU was 2,620 ± 178 for 2.5 nmol of MNNG  (positive control). The number of spontaneous revertants/plate (negative control) for S. typhimurium TA1535 (-S9) was 6.3 ± 1.3. The numbers of His + revertants per plate found for 100 µL of samples were 10.8 ± 0.96 for beer, 8.5 ± 2.5 for NAB(A), 11.5 ± 2.6 for NAB(B), 12.5 ± 5.0 for NAB(D), 9.3 ± 3.3 for NAB(E), 8.3 ± 4.5 for NAB(F), and 10.5 ± 4.4 for LAB assayed with TA1535 (-S9). Beer, LAB, and NABs did not show mutagenic activity toward S. typhimurium TA1535 (-S9). We examined all plates with a microscope and confirmed background lawn was not impaired and no cytotoxicity was observed. The mutagenicity of MNNG detected using TA1535 was significantly reduced in the presence of beer  of MNNG mutagenicity detected with TA1535 is summarized in Table 50 of NAB(B) and NAB(A) were the lowest compared with those of NAB(D-F) and LAB, we selected NAB(A) and NAB(B) for further investigation.The antimutagenic properties of beer, LAB, and various NABs against MNNG were investigated using the Ames test with O6-alkylguanine DNA adducts. If these adducts are not removed, they mispair with the wrong base during DNA replication, resulting in mutation. O6-methylguanine DNA methyltransferases play a critical role in MNNG mutations. Therefore, we investigated the anti-mutagenic activities of samples detected with the strain deficient in O6-methylguanine DNA methyltransferase, YG7108, compared to those of the proficient strain TA1535. The number of His+ revertants per plate found in the absence of samples was 1,732 ± 230 for 0.25 nmol of MNNG . The number of spontaneous revertants/plate (negative control) of S. typhimurium YG7108 (-S9) was 8.3 ± 5.0. The numbers of His+ revertants per plate found for 100 µL of samples were 16.5 ± 6.4 for beer, 9.3 ± 1.9 for NAB(A), 12.5 ± 5.0 for NAB(B) assayed with YG7108 (-S9). The numbers of His+ revertants per plate found for 250 µmol of GB was 2.5 ± 0.7  and 25.5 ± 4.0 ; and for 5 µmol of PU was 5.0 ± 1.4  and 10.7 ± 2.5 . Beer, NAB(A), NAB(B), GB and PU did not show mutagenic activity toward S. typhimurium YG7108 (-S9). In contrast to the results obtained using TA1535  for GB, and 0.50 µmole/plate (i.e. 0.122 mg/plate) for PU.The constituents in beer, GB, and PU, inhibited the mutagenicity of MNNG, as detected using TA1535  (positive control). The number of spontaneous revertants/plate (negative control) of S. typhimurium TA1535 (+ S9) was 7.3 ± 0.5, and that of S. typhimurium YG7108 (+ S9) was 14.3 ± 1.7. The numbers of His+ revertants per plate found for 100 µL of samples assayed with TA1535 (+ S9) were 11.3 ± 7.7 for beer, 10.7 ± 5.2 for NAB(A), 7.3 ± 2.1 for NAB(B) and those assayed with YG7108 (+ S9) were 10.0 ± 0.8 for beer, 7.7 ± 3.9 for NAB(A), 9.0 ± 2.9 for NAB(B). The numbers of His+ revertants per plate found for 250 µmol of GB was 5.6 ± 1.9  and 8.0 ± 2.1 ; and for 5 µmol of PU was 10.6 ± 2.4  and 9.0 ± 1.4 . Beer, NAB(A), NAB(B), GB and PU did not show mutagenic activity toward S. typhimurium TA1535 (+ S9) and YG7108 (+ S9). We investigated the anti-mutagenic activities of samples detected with YG7108, compared to those of TA1535. The number of His+ revertants per plate found in the absence of beer, LAB, NABs, GB, or PU was 2583 ± 38 for 100 nmol of NNK  (positive control). In contrast to the results obtained using TA1535 . No significant differences in food intake were observed among groups I–XII.P < 0.05)  of mice in groups I–VI at 30 weeks of age are shown in Table 5) Table . The num5) Table . The num5) Table . No miceP < 0.05)  are associated with tumor development . We inveThe phosphorylation of STAT3 in A549 cells treated with 40%beer, 70%NABs, 1 mM PU, and 250 mM GB was examined. Without EGF or IL-6 stimulation, the phosphorylation of STAT3 was inhibited in the presence of beer  Fig. . WithoutBeer has been reported to exert various biological effects, including cancer chemoprevention . As awarS. typhimurium TA1535. We investigated the differences in antimutagenicity between beer, LAB, and five NABs. The ID50 for the antimutagenicity of beer (0.5 µL/plate) was lowest and those of NABs and LAB toward MNNG ranged from 1.5 µL (NAB(B)) to 15.8 µL (NAB(F))  was nearly 10 times fewer than ID50 of NAB(F). These differences in the antimutagenic effects on MNNG may be due to the variety of raw materials, ingredients, and components in each drink  was not suppressed by beer, NABs, GB, or PU  on MNNG mutagenicity (3.1 µL/plate) was calculated to be 285 pmol/plate, which is 0.13% of the ID50, and the amount of GB in the ID50 of NAB(B) (1.6 µL/plate) was calculated to be 464 pmol/plate, which is 0.21% of the ID50. ID50 of PU on MNNG mutagenicity was 0.50 µmol/plate, whereas the amount of PU in ID50 volume of NAB(A) on MNNG mutagenicity (3.1 µL/plate) was calculated to be 16.1 pmol/plate, that is 3.2% of ID50, and the amount of PU in ID50 of NAB(B) (1.6 µL/plate) was calculated to be 35.2 pmol/plate, that is 7.0% of ID50. The contribution ratios of GB and PU to the antimutagenicity of NABs were comparable to those in beer , as previously described [The GB content was 92 µM and 290 µM in NAB (A) and NAB(B), respectively, and the PU content was 5.2 µM and 22 µM in NAB (A) and NAB(B), respectively. As GB contents in beer was reported as 7.00 mg/100 mL (0.600 mM) , and PU escribed , 16. TheWe investigated the antitumorigenic activity of beer, NAB(A), NAB(B), and GB on NNK-induced lung tumorigenesis in vivo. Unfortunately, PU is too expensive for in vivo experiments. NNK-induced lung nodule formation was significantly reduced Table . More thDuring multistep carcinogenesis, the suppression of cancer cell growth is an important target for anti-tumorigenesis. These samples may suppress the growth of cancer cells to form countable nodules. Tumor nodules > 1 mm in diameter were counted, and the number of nodules > 1 mm in diameter was significantly decreased in mice that received beer, NABs, or GB Table . We inveThe concentration of GB in the 40%beer, 70%NAB(A) and 70%NAB(B) was calculated as 239 µM, 64.4 µM and 203 µM, respectively, and the concentration of PU in the 40%beer, 70%NAB(A) and 70%NAB(B) was calculated as 7.1 µM, 3.64 µM and 15.4 µM, respectively. STAT3 and Akt phosphorylation experiments were performed using 1 mM PU or 250 mM GB. Therefore, the amounts of PU and GB in beer and NABs may be insufficient to suppress Akt and STAT3 phosphorylation. Other components of beer and NABs, along with GB and PU, may also suppress the phosphorylation of Akt and STAT3 in A549 cells.The raw materials of beer and NABs are malts, hops and other ingredients Table , and comIn conclusion, beer, NABs, and GB reduced the NNK-induced lung tumorigenesis. They target both the initiation and growth/progression steps of carcinogenesis, specifically via antimutagenesis, stimulation of alkyl DNA adduct repair, and suppression of Akt- and STAT3- mediated growth signaling. GB and PU may contribute, in part, to the biological effects of beer and NABs via antimutagenicity and the suppression of Akt and STAT3 phosphorylation.


N6-Methyladenosine (m6A) modification plays a vital role in lung disorders. However, the potential of m6A in neonatal Bronchopulmonary Dysplasia (BPD) has not been reported. This study aimed to investigate the roles of METTL3 in BPD.BPD models were established by hyperoxia in vivo and in vitro. Histological analysis was determined using HE staining. Gene expression was determined using Western blotting, qRT-PCR, and immunofluorescence. The release of IL-1β and IL-18 was detected using ELISA. The m6A sites of ATG8 were predicted by SCRAPM and verified by MeRIP assay. The location of GSDMD and ATG8 was determined by FISH assay. The interaction between ATG8 and GSDMD was detected using Coimmunoprecipitation (Co-IP). Cell pyroptosis was determined using flow cytometry and TUNEL assays.METTL3 was overexpressed in BPD, which was accompanied by an increase in m6A levels. Interestingly, METTL3 suppressed hyperoxia-mediated damage and pyroptosis in BEAS-2B cells and promoted cell autophagy. METTL3-mediated m6A modification of ATG8 suppressed its expression and disrupted the interaction between ATG8 and GSDMD. However, autophagy inhibition induced pyroptosis in BEAS-2B cells. In vivo assays showed that METTL3-mediated autophagy inhibition induced a decrease in the radial alveolar count and an increase in the mean linear intercept and promoted cell pyroptosis.In conclusion, METTL3-mediated cell pyroptosis promotes BPD by regulating the m6A modification of ATG8. This may provide new insight into the development of BPD. Bronchopulmonary Dysplasia (BPD) is a common complication of prematurity and is characterized by damage to the alveolar structure. N6-Methyladenosine (m6A) modification is deemed the most common mRNA modification in eukaryotic cells. A pyroptosis is a form of programmed cell death characterized by the activation of inflammasomes, such as NLRPs and NLRCs. ,17 The aBlood samples were collected from 30 premature infants with BPD and 30 non-BPD age-matched controls hospitalized in Huai'an Maternity and Child Healthcare Hospital. All the procedures were performed in accordance with STARD guidelines. This study was approved by the Ethical Committee of Huai'an Maternity and Child Healthcare Hospital. This study was approved by the parents of the enrolled patients.2. Cells were treated with 2 mM 3-methyladenine (3-MA) for 24 h. Cells were then divided into normoxia and hyperoxia (95% O2) for 24 h. METTL3 #1‒2 and its Negative Control (NC), METTL3 Overexpression plasmids (OE) and empty vector, ATG8 OE and empty vector, and GSDMD OE and empty vector were provided by GeneChem, China. Cells were transfected with Lipofectamine 3000 (Invitrogen). Five microliters of Lipofectamine and 20 pM shRNA and/or ATG8/GSDMD OE were mixed for 6 h at 37 °C. Then, transfection was performed for 48 h.BEAS-2B cells were purchased from ATCC. Cells were cultured in an incubator containing 12% FBS and 1% penicillin/streptomycin (Gibco) at 37 °C in COThe levels of IL-1β and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA).The levels of LDH were measured by an LDH kit .−ΔΔCq method. The primers used in this study were as follows: METTL3 F: 5’-TTGTCTCCAACCTTCCGTAGT-3’ and R: 5’-CCAGATCAGAGAGGTGGTGTAG-3’.RNA was extracted from cells. QuantiTect Rev. Transcription Kit (QIAGEN) was used for cDNA synthesis. PCR was conducted using a QuantiNova SYBR Green PCR Kit (QIAGEN). The RNA levels were normalized to GAPDH and calculated using the 2After transfection, cells were collected and lysed. Then, total protein was collected and concentrated using a BCA kit. Protein (30 µg) was isolated using 10% SDS-PAGE at 120v. The protein was transferred onto PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies such as antiLC3I/II , antiATG8 , antiNLRP3 , antiASC , anti-Caspase1 , GSDMD , antiβ-actin  and goat-anti-rabbit antibody . Finally, the bands were captured by ECL reagents and analyzed using ImageJ (V.2.3.0).Cells were seeded into a 24-well plate. After mounting on 4% paraformaldehyde, the cells were hybridized with 2 µM Cy3-labeled GSDMD and FITC-labeled ATG8 RNA probes. Then, the cells were counterstained with DAPI. Finally, images were captured using confocal microscopy .Cells were collected, mounted on 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Afterward, cells were incubated with primary antibodies against LC3 puncta  and Alexa Fluor fragment of goat anti-rabbit IgG . The cells were counterstained with DAPI. Subsequently, the cells were visualized using a fluorescence microscope .Total RNA was collected and purified to remove ribosomal RNA and contaminated DNA. Afterward, RNA was sheared into fragments and denatured. Then, RNA was incubated with protein A/G magnetic beads conjugated with anti-m6A antibody. After elution, RNA was analyzed by qRT-PCR.After transfection, cell lysates were collected using a lysis buffer. Then, the lysates were incubated with anti-GSDMD, anti-ATG8 and anti-IgG primary antibodies at room temperature for 2h and secondary antibodies at 4°C overnight. Then, the complexes were mixed with anti-IgG magnetic beads, rinsed in IP buffer, and analyzed using a western blot assay.Cells were harvested, mounted on 4% paraformaldehyde, and permeabilized in 0.25% Triton-X100. Afterward, the cells were stained using an in-situ Cell Death Detection Kit. The images were visualized by fluorescence microscopy . Cell death rates = TUNEL-positive cells/total cells ×100%.−/−), and the respective Wild-Type (WT) control mice were purchased from the Experimental Animal Center of Nanjing Medical University. Mice were divided into three groups: 1) C57BL/6J mice were exposed to 21% oxygen  for 14 days; 2) C57BL/6J, METTL3−/− and WT control mice were exposed to 80% O2 oxygen  for 28 days; 3) Mice were injected with 15 µg of 3-MA 30 min before hyperoxia exposure. The procedures were performed in accordance with ARRIVE guidelines.Neonatal C57BL/6J, B6.129S4-Ccr2tm1Ifc/J , gene expression determination (ELISA and immunofluorescence), and m6A level determination (MeRIP).http://www.cuilab.cn/sramp/).The m6A modification sites and RNA secondary structure were predicted by the online database SCRAMP .The data generated in the present study may be requested from the corresponding author.This research was funded by grants fromJiangsu Provincial Health and Family Planning CommissionLili Xu: Formal analysis, Data curation, Writing – original draft. Zhan Shi: Formal analysis, Data curation, Writing – original draft. Zhaojun Pan: Data curation, Writing – review & editing. Rong Wu: Conceptualization.The authors declare no conflicts of interest.