Clostridium perfringens (C. perfringens) is a bacterium that commonly causes zoonotic disease. The pathogenicity of C. perfringens is a result of the combined action of α, β, and ε exotoxins. In this study, Lactobacillus crispatus (pPG-T7g10/L. crispatus) expressing the main toxoids of C. perfringens, α, ε, β1, and β2, with EGFP-labeling, was constructed, and the protective effect was estimated in chickens. The α-β2-ε-β1 toxoid was constitutively expressed for confirmation by laser confocal microscopy and western blotting, and its immunogenicity was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical assays. After booster immunization, the probiotic vaccine group showed significantly higher levels (p < 0.05) of specific secretory IgA (sIgA) and IgY antibodies in the serum and intestinal mucus. Furthermore, the levels of cytokines, including interferon (IFN)-γ, interleukin (lL)-2, IL-4, IL-10, IL-12, and IL-17, and the proliferation of spleen lymphocytes in chickens orally immunized with pPG-E-α-β2-ε-β1/L. crispatus increased significantly. Histopathological observations showed that the intestinal pathological changes in chickens immunized with pPG-E-α-β2ε-β1/L. crispatus were significantly alleviated. These data reveal that the probiotic vaccine could stimulate mucosal, cellular, and humoral immunity and provide an active defense against the toxins of C. perfringens, suggesting a promising candidate for oral vaccines against C. perfringens. Clostridium perfringens (C. perfringens) is a common zoonotic pathogen that is abundantly present in the environment. It often causes gas gangrene, necrotizing enteritis, and enterotoxemia in cattle, sheep, and chickens [C. perfringens is the main pathogenic factor that infects animals, causing rapid onset, high mortality, and serious economic losses to animal husbandry [C. perfringens results from the synergy of multiple exotoxins, of which α, β, and ε are the most common and highly toxic [C. perfringens promotes the development of necrotizing enteritis, leading to a mortality rate of up to 50% [C. perfringens infections is highly desirable. Currently, many countries use antibiotics and sulfonamides to prevent and treat diseases caused by C. perfringens infection in animals [chickens ,2. The eusbandry . The patly toxic . These tly toxic . In chicp to 50% . Therefo animals . Because animals . Therefo animals .C. perfringens mainly infects the intestinal tract of animals, developing an oral immunization vaccine is a promising approach to prevent the occurrence of this disease. Lactic acid bacteria (LAB) are Gram-positive bacteria which are widely distributed in animal intestinal tissues and play a key role in maintaining the stability of microbial ecology and inducing mucosal and systemic immunity [Lactobacillus, a member of LAB, has many advantages: it has been proven to be a food-grade safe microorganism for application in the research of food-grade gene cloning vector systems and expression vector systems; it interacts with cells and induces the production of a series of cytokines; and it has the ability to tolerate bile acids and is affected by immune adjuvants [Lactobacillus induces a mucosal immune response in the body. Some of these microorganisms pass through the microfold cells between the epithelial cells of the small intestine, whereas some pass through the dendritic cells under the epithelial cells to enter the lamina propria of the small intestine tissue, where they activate Th2 lymphocytes through the activation of plasma cells, which secrete specific IgA antibodies [Lactobacillus with better antigenic quality, whose application in production practices has far-reaching significance. Song et al. developed a stable and marker-free Lactobacillus strain, realizing expression of the alpha-toxin gene of Clostridium perfringens on the surface of bacteria [Lactobacillus casei (pPG-α/L. casei 393) for constitutively expressing the toxoid of C. perfringens α-toxin, a BLA/c mice experiment to evaluate the oral immunogenicity of pPG-α/L. casei 393, the results indicating that the strain could induce the production of mucosal, humoral, and cellular immunity, and provide a protective effect to a certain degree [As immunity . Use of djuvants . It helpdjuvants . Lactobatibodies ,14. Spectibodies ,16. Spectibodies . With cobacteria . Gao et n degree .L. crispatus, expressing the main toxoids of C. perfringens, α, β1, ε, and β2, with non-antibiotic resistance, was constructed, and the genes were modified by our laboratory for mutating the 68th histidine residue to a glycine residue in α toxin, the 234th cysteine residue to a glycine residue in β2 toxin, and the 106th histidine residue to a proline residue in ε toxin. Its immunogenicity in chickens was evaluated for its protective effect against C. perfringens infection.In this study, the recombinant strain pPG-T7g10/L. crispatus N-11 strain was isolated from the intestine of a chicken in our laboratory [C. perfringens types A (C57-1), B (CCVC-81), and C (CACC-61) were obtained from the China Institute of Veterinary Drug Control, Beijing, China. Escherichia coli strain JM109 and the engineered strain pPG-T7g10/TG1, containing the constitutively expressed plasmid were cultivated in our laboratory at 37 °C in Luria–Bertani (LB) broth with shaking. The pMD19-EGFP plasmid containing the gene encoding EGFP (Accession No. U57607) and the pMD18-T-α-β2-ε-β1 plasmid, which had been constructed to contain a fusion of the genes encoding the quadruple toxoid of C. perfringens α (H68G), β2 (C234G), ε (H106P), and β1 , were preserved in our lab [The boratory  and grow our lab . The pPG our lab ,22. The C. perfringens was acquired from the plasmid pMD19-T-α-β2-ε-β via PCR amplification and then inserted into the constitutive expression vector pPG-T7g10-PPT, generating the recombinant plasmid pPG-α-β2-ε-β1. Subsequently, the fragment encoding EGFP was inserted upstream of the fusion genes α, β2, ε, and β1 and fused with α-β2-ε-β1 by a linker, resulting in creation of the plasmid pPG-E-α-β2-ε-β1. Finally, the recombinant plasmid pPG-E-α-β2-ε-β1 was electroporated into L. crispatus N-11 competent cells and the positive strain was validated using laser confocal microscopy, flow cytometry, and western blotting.The recombinant strain was constructed using a previously described method . BrieflyL. crispatus N-11 was cultured in MRS medium at 37 °C for 16 h, centrifuged at 10,000× g for 5 min, and washed three times with sterilized phosphate-buffered saline . The precipitate was incubated with lysozyme and ultrasonicated. Then, 2 × sodium dodecyl sulfate (SDS) buffer solution was added and the sample was boiled for 10 min. After boiling, the sample was centrifuged, and the supernatant was collected. The supernatant was then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in a 10% gel, followed by western blot analysis using an anti-α toxin polyclonal and mouse anti-toxoid monoclonal antibody (generated in our laboratory) at a dilution ratio of 1:500. Subsequently, the membrane was incubated with the secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  diluted 1:5000. Immunolabeled bands were visualized using chemiluminescent substrate reagent .pPG-E-α-β2-ε-β1/L. crispatus N-11, the recombinant pPG-E-α-β2-ε-β1/L. crispatus N-11 was cultured in MRS medium at 37 °C for 16 h, centrifuged at 10,000× g for 5 min, washed three times with sterilized PBS, resuspended in sterile PBS, and then the cells were examined using laser confocal microscopy [To further analyze the protein expressed on the cell surface of pPG-T7g10-PPT/croscopy .L. crispatus N-11 grown until the optical density at 600 nm (OD600) was approximately 1.0, and then centrifuged at 5000× g for 10 min. The pelleted cells were resuspended at a concentration of 1010 colony-forming units (CFU)/mL and then washed twice with sterile PBS. The strain was then labeled with carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) and incubated for 20 min at 37 °C. Subsequently, the cells were washed and resuspended in PBS, and the labeling ratio was determined using flow cytometry. A total of 15 chickens were inoculated with CFDA-SE-labeled recombinant strain (approximately 109 CFU/mL). Intestinal segments of the jejunum, ileum, and colon were collected on days 1, 4, 7, 11, and 15 (n = 3), and the colonization capability of the recombinant strain in the intestinal tract was assessed using flow cytometry.For the colonization test, recombinant pPG-E-α-β1-ε-β2/L. crispatus N-11 as oral vaccine, 1-day-old specific pathogen-free (SPF) chickens provided by the Harbin Veterinary Institute were selected as an animal model and kept under SPF environments with free access to standard feed and water. The basal feed diet ingredients and environmental factors of all the groups were equally managed. Before oral immunization, the recombinant strain pPG-E-α-β2-ε-β1/L. crispatus N-11 was cultured to an OD600 = 1.0, washed with PBS, and resuspended to a concentration of 1 × 1010 CFU/mL in PBS. The immunization procedure was as follows. SPF chickens were divided into five groups. The recombinant strain group (n = 36) was orally immunized with 1 mL pPG-E-α-β2-ε-β1L. crispatus N-11; the empty carrier vector group (n = 36) was immunized with 1 mL of pPG-T7g10-PPT/L. crispatus N-11; the control group (n = 36) was orally inoculated with 200 μL of L. crispatus N-11; the mock control group (n = 36) received 200 μL of PBS, and this group was considered as a control without inoculation (n = 15). All chickens were immunized orally, and the immunization schedule was repeated three times at 14-day intervals. Each dose was administered (offered) once every three days. The samples were collected on days 0, 7, 14, 21, 28, 35, and 42 [The maintenance and use of the experimental animals followed the appropriate international  and national guiding principles (CNAS-CL 06:2018). In this study, to evaluate the immunogenicity of the engineered strain pPG-E-α-β2-ε-β1/, and 42 .2SO4). On day 35 post-immunization, the levels of interferon (IFN)-γ, interleukin (lL)-2, IL-4, IL-10, IL-12, and IL-17 in the samples were determined using an ELISA kit  according to the manufacturer’s guidelines.Serum and intestinal mucus samples were collected from three chickens randomly in each group on days 0, 7, 14, 21, 28, 35, and 42 after primary immunization, and specific antibody IgA and IgY levels were detected using ELISA as described earlier ,19. Brie2) were obtained from chickens immunized with pPG-E-α-β2-ε-β1/L. crispatus N-11 and the PBS control group (inoculation-free group). Collected samples were fixed with 10% neutral-buffered formalin, processed with paraffin wax, and cut to a thickness of 5 μm. Immunoglobulin (IgA) cells confined to the intestine were detected using immunohistochemistry. Immunohistochemical staining and calculations were performed as described previously [On day 42 post-immunization, intestinal samples from the duodenum, jejunum, ileum, and cecum (1 cmeviously . Polyclo6 cells/mL) was incubated in each well of a 96-well plate  at 37 °C in a 5% CO2 atmosphere for 8–12 h. Cells were then stimulated with purified recombinant α-β2-ε-β1 protein at final concentrations of 0.01, 0.1, 1, and 10 mg/mL for 60 h, and an equal volume of the medium was added to the control cells. Then, 10 µL of thiazolyl blue tetrazolium bromide (MTT) at 5 mg/mL was added to each well and incubated at 37 °C for 5 h. Finally, the absorbance was measured at 600 nm and the stimulated index was estimated as follows: SI = OD600 (sample)/OD600 (blank control).On day 42 post-immunization, splenocytes were collected from chickens in each group and used in the lymphocyte proliferation assay conducted using the respective kit from Sigma Life Science . Briefly, 100 μL of spleen cell suspension  of the natural α-β2-ε-β1 toxin combined with 108 CFU of C. perfringens type A and type B oral pathogenic bacteria via oral administration using an oral gavage needle; the respective control group was not challenged.To estimate the immune protection of chickens orally vaccinated with pPG-E-α-β2-ε-β1/The daily subsistence ratio of the experimental chickens in each group was counted post-challenge, and histopathological changes in the intestinal sections, including duodenum, jejunum, ileum, and cecum, of all the groups were observed, with special consideration given to the structure, color, and occurrences of necrosis, hemorrhage, congestion, and cell infiltration.L. crispatus pPG-E-α-β2-ε-β1/L. crispatus N-11 on the gut microbiota, we divided a total of 24 chickens into three groups: recombinant L. crispatus-, C. perfringens-, and PBS-inoculated groups (n = 8). The intestinal contents were collected on days 7 and 14 from each group. Microbial DNA was extracted using a Tiangen Stool DNA kit , according to the manufacturer’s instructions. After extraction, the concentration was measured using a NanoDrop 2000 spectrophotometer. After extracting genomic DNA from the samples, the V3-V4 region of 16S rDNA was amplified using specific PCR primers . Purified primers were mixed in equal quantities and connected to the Illumina adapter and linker sequences. A paired-end sequencing (2 × 250) was performed with equal amounts of purified amplicons on the Illumina platform, then analysed using an Illumina HiSeq2500 from GeneDenovo .To evaluate the effect of the recombinant p < 0.01 and * p < 0.05 indicated statistical significance.All data conformed to a normal distribution for analyzing with Shapiro–Wilk test, shown as mean ± standard deviation (SD). All experiments were performed in triplicate. Statistical significance was evaluated via one-way analysis of variance (ANOVA) using Tukey’s multiple comparison test and Prism 7 software. ** L. crispatus N-11 cells, but not on the pPG/L. crispatus N-11 cells  in IgA and IgY antibody levels among the groups before immunization. However, after booster immunization, particularly on day 14 post-immunization, anti–α-β2-ε-β1 toxoid specific IgA level (L. crispatus N-11 group were significantly higher (p ˂ 0.05) than those of groups “pPG/L. crispatus” L. crispatus N-11” and “PBS”. Conversely, there was no significant difference (p > 0.05) between the control groups before and after immunization. These results indicate that oral pPG-E-α-β2-β1/L. crispatus N-11 induced specific mucosal and systemic immune responses in chickens.To assay the immunity of chickens orally immunized with strain pPG-E-α-β2-ε-β1/gA level  in intesgA level  in sera p ˂ 0.01) were found in chickens immunized orally with pPG-E-α-β1-ε-β2/L. crispatus N-11 than in the pPG-T7g10-PPT/L. crispatus N-11 and PBS groups . Following taxonomic assignment, 373 operational taxonomic units (OTUs) were shared among all groups. Moreover, we found that most OTUs were present individually in different groups (L. crispatus (R. Lact-7 and R. Lact-14) was measured on days 7 and 14, and was found to be significantly different from the negative control group (PBS-7 and PBS-14) and C. perfringens groups (Cp-7 and Cp-14).We analyzed the V3–V4 regions of the bacterial 16S rRNA gene sequence and evaluated the α-diversity indices . The results showed that the gut microbiota of the control, Cp, and R. lact groups differed from each other (t groups A. The miL. crispatus N-11 (R. Lact-7 and R. Lact-14), C. perfringens (Cp-7 and Cp-14), and control (PBS-7 and PBS-14) groups were different from each other  constitutively expressing the toxoid of C. perfringens, α-toxin, and evaluated its immunogenicity. They observed a significant increase in sIgA level, with the immune protection rate being 80% [upp-deficient L. casei strain with a plasmid cross-recombination system, which expressed the α toxin in place of the upp gene. This did not necessitate antibiotic screening and provided a reference for the preparation of an oral vaccine against C. perfringens [otoxemia . The patfections . The tox poultry . It is t poultry . The toxl damage . Therefoeing 80% . Song etfringens .L. crispatus N-11, with the EGFP-marked plasmid pPG-E-α-β2-ε-β1, was effectively constructed with the constitutive expression plasmid pPG-T7g-10-PPT. Based on the different types of promoters in the Lactobacillus expression system, the system is divided into an inducible expression system and a constitutive expression system. Inducible expression is widely used in the construction of genetically engineered LAB, with lactose, xylose, pH, temperature [Lactobacillus oral vaccines. In the current study, we constructed a constitutive Lactobacillus expression system, using the pPG-T7g10-PPT carrier vector [L. crispatus to express α-β2-ε-β1 toxoids of C. perfringens. The results revealed that the fusion protein α-β2ε-β1 could be expressed by recombinant PG-E-α-β2-ε-β1/L. crispatus N-11, and the expression was sufficient for the toxoid to be recognized by an anti-toxoid monoclonal antibody. Moreover, the expression of the plasmid toxoid protein on the cell surface of the strain pPG-E-α-β2-ε-β1/L. crispatus N-11 was confirmed using laser confocal microscopy. Thus, the membrane-linked, antigen-generating, genetically engineered Lactobacillus strain, as an oral vaccine, holds potential to help the host detect antigens and efficiently stimulate antigen-specific immune responses. The ability of genetically engineered LAB to colonize and proliferate in the intestinal tract is a key factor for effective induction of an immune response in the body. Strong adhesion of LAB can effectively improve the immune response of the body [L. crispatus N-11 in the intestine. The results indicated that pPG-E-α-β2-ε-β1/L. crispatus N-11 could effectively colonize the intestine of chickens and were distributed in the jejunum, ileum, and colon, and likely among other regions. Colonization rates in the jejunum, ileum, and colon of chickens on day 1 after oral administration were high, but gradually decreased, although they remained appreciably high until the 15th day after oral administration. The good colonization capability of pPG-E-α-β2-ε-β1/L. crispatus N-11 improved its ability to effectively stimulate the immune response of the body.In the current study, as an antigen delivery vector, pPG-E-α-β2-ε-β1/perature ,30,31, aperature  being usr vector , and appthe body ,29,33. TL. casei and its complementary plasmid to express the non-toxic antigen of Porcine Epidemic Diarrhea Virus (PEDV), and the results showed that it could stimulate the production of sIgA and IgG in mice [L. crispatus N-11 in chickens, immunized orally using ELISA and immunohistochemistry. Our results revealed there was a significant increase in levels of (p < 0.01) anti-α-β1-ε-β2 toxoid sIgA-based mucosal immune responses and IgY-based systemic immune responses, indicating that the pPG-E-α-β2-ε-β1/L. crispatus N-11 constructed in this study showed better immunogenicity. Furthermore, the IgA content in the chickens immunized with the recombinant strain was significantly higher than that in the control group. The immunohistochemistry results also supported the results of the ELISA antibody detection test.An ideal mucosal vaccine must successfully induce both intestinal mucosal and systemic immune responses. sIgA is the dominant antibody on the mucosal surface and plays a significant role in host defense against infections ,24. Avia in mice . TherefoL. crispatus N-11 can also induce cellular immune responses. The ratio of T helper 1 (Th1)/(Th2) cells determines the direction of the immune response. According to a previous study [Lactobacillus can stimulate the progression of innate immune responses and trigger Th1-mediated immune reactions. IFN-γ is mainly produced by Th1 cells and can improve the activity of Th1 cells in promoting cellular immune activity [p ˂ 0.01) in chickens immunized orally with pPG-E-α-β1-ε-β2/L. crispatus N-11 than in the empty vector PPG-T7g10-PPT/L. crispatus, non-recombinant L. crispatus N-11, and PBS groups. Additionally, it is worth noting that the concentrations of these cytokines in the serum of chickens in the PPG-T7g10-PPT/L. crispatus and L. crispatus N-11 groups were higher than those in the PBS group, indicating that the non-engineered probiotics themselves can directly improve the nonspecific immune response. The experimental results show that oral immunization with recombinant L. crispatus not only induces a systemic humoral immune response in the body of the animal, but also stimulates the body to produce a cellular immune response. To verify the immune-protective effect of orally administered recombinant L. crispatus, we conducted a post-immunization challenge test. The toxin used was a mixture of natural toxins of C. perfringens types A and B. The experiment showed that the immunity induced by oral administration of recombinant L. crispatus in the vaccine group was able to resist the effect of the natural toxin of C. perfringens, with all four chickens tested showing protection, whereas all of the pPG-T7g10-PPT/L. crispatus N-11 and PBS control chickens died. Our histopathological observations show that the intestines of the vulnerable groups showed obvious pathological changes compared with that of the chickens orally immunized with recombinant L. crispatus. These results indicate that recombinant pPG-E-α-β2-ε-β1/L. crispatus N-11 is immunogenic. We also examined the fecal microbiome to explore the effects of oral recombinant L. crispatus on the gut microbiota. We analyzed the V3-V4 regions of the bacterial 16S rRNA gene sequence and found a significant difference in the microbial diversity in intestinal feces of the chickens orally administered recombinant L. crispatus N-11, compared with the control groups. The relative abundance of Bacillus increased significantly, whereas that of Clostridium decreased compared to that in the control group.We found that there was a positive association between cell immunity and lymphocyte proliferation rate. Subsequently, we were able to identify increased spleen lymphocyte proliferation in immunized chickens. Our results indicated that recombinant pPG-E-α-β1-ε-β2/us study , Lactobaactivity . The Th2activity . In the L. crispatus constitutively expressing the α, β1, ε, and β2 toxoids of C. perfringens. Its immunogenicity as an oral probiotic vaccine was explored, and our results showed that pPG-E-α-β2-ε-β1/L. crispatus N-11 could effectively induce mucosal, humoral, and cellular immune responses and provide protective effects against a C. perfringens challenge. These results demonstrate that the engineered strain is a promising candidate vaccine against C. perfringens infections.In conclusion, this study constructed EGFP-marked, genetically engineered


The metabolism and pharmacokinetics of Ptb are still sparse, probably due to the lack of tools that allow following the Ptb destiny both in living cells and in vivo. In this contest, we propose two Ptb fluorescent derivatives where Ptb has been functionalised by benzofurazan and rhodamine-B-isothiocyanate, compounds 1 and 2, respectively. Here, we report the synthesis, the optical and structural characterisation of 1 and 2, and, their putative cytotoxicity in two different cell lines.Several epidemiological studies suggest that a diet rich in fruit and vegetables reduces the incidence of neurodegenerative diseases. Resveratrol (Res) and its dimethylated metabolite, pterostibene (Ptb), have been largely studied for their neuroprotective action. The clinical use of Res is limited because of its rapid metabolism and its poor bioavailability. Ptb with two methoxy groups and one hydroxyl group has a good membrane permeability, metabolic stability and higher  It has been hypothesised that, in 2030, the incidence of only AD, in the world population, will be around 100 million individuals, with a very high emotional and economic cost,Unfortunately, the treatments available against these pathologies are not effective and, in most cases cannot prevent the onset of the diseaseTherefore, it appears very important to focus the attention on the research to prevent or to slow down the initial steps of the neurodegeneration process. The discovery of promising agents against neurodegeneration with little or no toxicity represents an important goal for all people suffering from this debilitating disease and their caregivers.,,in vitro and in vivo studiesRecently, several studies have confirmed that plant derivate natural substances, whether of terrestrial or marine origin can reduce the incidence of different pathologies including neurodegenerative,However, the clinical use of Res is limited because of its rapid metabolism and its poor bioavailability, encouraging researchers to derivatize this stilbene to overcome its limit,Studies show that, following oral administration, Ptb has an approximately 4-fold greater (about 80% versus 20%) bioavailability and 7.5-fold (105 min versus 14 min) longer half-life if compared to Res,via the subtype α of oestrogen receptor (ER-α) and may be a new neuroprotective agent for nerve injury treatment and preventionRecent studies have demonstrated that Ptb has anti-inflammatory, anti-carcinogenic and anti-diabetic and neuroprotective properties together with a very low toxicity in animal and human modelsin vivoHowever, the metabolism and pharmacokinetics of Ptb are still scarce probably due to the lack of tools that allow following the Ptb destiny both in living cells and 1 and 2  as fluorescent moiety while compound 2 is functionalised with rhodamine-B-isothiocyanate (Rhd B-Itc)  . The use2.2.1.1 and 2 were synthesised as reported in Compounds 1 and 2 were synthesised via amine derivative 6 that was obtained from the reaction of Ptb with N-Boc-bromoethylamine, and subsequent deprotection of the Boc group in acidic conditions. After, the conjugation of compound 6 with benzofurazan  or rhodamine-B-isothiocyanate (Rhd B-Itc) afforded the desired fluorescent pterostilbene derivatives 1 and 2.In detail, compounds 1, the NBD was chosen as fluorescent probe skeleton because the NBD fluorescent derivatives possess long excitation and emission wavelengths  that avoid interference due to biomatrices as reported in the literature,2 was functionalised with Rhd B-Itc; a moiety present in numerous fluorescent compounds used to perform biological testsFor compound 2.2.1 and 2 compounds. For all samples, absorption spectra were collected in DMSO solutions prepared at three different concentrations, and the respective calibration lines were obtained by applying a linear fitting procedure (origin included), as to calculate the molar extinction coefficient (ε). Maximum absorption wavelengths and ε values are summarised in UV–visible absorption and emission characterisations were carried out on NBD, Res, λexc), emission wavelength (λemis), and emission intensity (Iemis) values are summarised in Figures S1–S5), as well as absorption wavelengths, absorbance values, emission wavelengths, and emission intensity values for each measured solution (Tables S1–S5).Emission spectra were also collected by exciting the four samples DMSO solutions at the maximum absorption wavelength. Excitation wavelength . NEXAFS spectra are reported in The proposed molecular structure of samples 1 and at 287.0 eV in 2 are assigned to C 1 s → π* C = N/O resonance. In spectrum 1, the features at 286.2 eV is due to C 1 s(C-R) → π* C = C transition. In spectrum 2 the feature at 288.0 eV is associated to C 1 s → π* C = S transitionC K-edge spectra  present ar-N bond. The previous peaks, at 400 eV in 1 and at 398.6 eV in 2, are assigned to N 1 s → π* C = N transition of the oxadiazole ring and to S-C = N 1 s → π* transitionIn N K-edge spectra , the fea,In the O K-edge spectra  there is−1 are mainly related to aromatic and olefinic stretching vibrations. Similar features can be detected in the spectra of samples 1 and 2 (lines b and a) (According to literatureb and a) .−1, related to asymmetric and symmetric stretching of the nitro group; the same peaks can be evidenced in the spectrum of sample 1, evidencing successful coupling of Ptb to 7-nitrobenzofurazan.The IR spectrum of 7-nitrobenzofurazan (line c) shows two main features at 1500 × 1360 cm1 and 2.In synthesis, NEXAFS and FTIR results confirm the molecular structures proposed in 2.3.1, 2) modify cell vitality or promote PARP-1 cleavage confirming the very low toxicity of this compound as already reported,The putative cytotoxic effects of Ptb fluorescent derivatives was evaluated by the Propidium iodide (PI) assay, which is membrane impermeant and generally excluded from viable cells. This assay is commonly used for identifying dead cells in a population. In particular, the breast cancer (i.e. MCF-7) and the neuroblastoma (i.e. SH-SY5Y) cell lines have been used as experimental models and results were compared with that of Res 1 µM, which shares many biosynthetic pathways with pterostilbene. Notably, none of tested concentrations (i.e. 0.1–10.0 µM) of Ptb or its fluorescent derivatives modified cell morphology (data not shown) or their vitality . The com1, 2) raised the question if these compounds still maintain some of neuro-protective effects ascribed to their precursor,1 maintained the same efficacy than Res, with a value higher with respect to its precursor (Ptb), whereas the compound 2 reduced NGB levels under the control value. As a whole, these data, for the first time, indicate that Ptb neuroprotective effects, like Res, could be mediated by the accumulation of NGB suggesting that both polyphenols could share the same signalling pathways. Moreover, only Ptb functionalised on phenolic ring with benzofurazan (compound 1) maintained the same efficacy of its precursor in enhancing NGB levels, while Ptb derivative containing rhodamine B-isothiocyanate (compound 2) significantly reduced NGB levels under the control values, The lack of any toxics effects of Ptb fluorescent derivatives  and from Fluorochem , and were used without further purification. Chemical reactions were monitored by thin layer chromatography (TLC) using aluminium plates pre-coated with silica gel and containing a fluorescent indicator (Merck Silica Gel 60 F254). The spots on TLC were visualised by a UV lamp (254 nm). NaLeica Galen III microscope. 1H and 13 C NMR spectra were recorded in the opportune solvent with a Bruker Ultrashield™ 400 MHz spectrometer , at 25 °C. Chemical shifts (δ) are given in ppm and coupling constants (J) are reported in Hz. Signals in NMR spectra are indicated by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartette, m = multiplet, bs = broad singlet. Mass spectra (ESI-MS) were recorded with a high-resolution Q Exactive plus Orbitrap spectrometer , resolution 140,000 at m/z 200.Flash chromatographic purifications were performed in glass columns using silica gel 230–400 mesh Sigma-Aldrich . Compounds were characterised by the determination of melting points and of NMR and Mass Spectrometry spectra. Melting points (m.p.) were measured with a 3.1.1.3  and K2CO3  in anhydrous DMF (2 ml) and was added dropwise, under nitrogen, a solution of N-Boc-ethylamine bromide 4  in anhydrous DMF. The reaction mixture was stirred at 50 °C for 8 h and then left to r.t. for 36 h monitoring by TLC. Then H2O was added and the aqueous solution was extracted with CHCl3. The organic phase was washed twice with H2O and ice, dehydrated with Na2SO4 and finally evaporated under reduced pressure in rotating evaporator. Compound 5 was obtained as a transparent oil (95% yield). 1H NMR  δ: 8.01 ; 7.44–7.41 ; 7.04–7.00 ; 6.91–6.87 ; 6.89–6.83 ; 6.64–6.63 ; 6.37–6.36 ; 4.04–4.01 ; 3.82 ; 3.54–3.52 ; 1.45 .To a solution of 4-phenol 3.1.2.5  in DCM (10 ml), was added dropwise TFA . The reaction mixture was left stirring overnight at r.t. The reaction mixture was then neutralised with saturated solution of NaHCO3 to pH ∼ 8. The aqueous solution was extracted with DCM and the organic phase was washed twice with H2O. The organic phase was dried with Na2SO4 and evaporated under reduced pressure. To the pale-yellow oil obtained was added a solution of Et2O • HCl, dropwise at 0 °C, to get the hydrochloride salt and then solution was evaporated. The crude product was purified by trituration in Et2O to afford compound 6 as a pink solid (61% yield). 1H NMR  δ: 7.04–7.02 ; 6.64–6.59 ; 6.53–6.51 ; 6.51–6.47 ; 6.20–6.19 ; 5.89–5.88 ; 3.77–3.74 ; 3.31 ; 2.89–2.87 .To a solution of compound 3.1.3.6  and commercially available 4-chloro-7-nitrobenzofurazan 7  were dissolved in a H2O/THF solution. To the resulting solution was added LiOH  and then the mixture was stirred at r.t. overnight, protected from light. The brown solid precipitate was washed with H2O and filtered under vacuum. The crude product was purified by flash chromatography (n-hexane/EtOAc in ratio 1.5:1) and then triturated in n-hexane. Compound 1 was obtained as a brown solid (93% yield). M.p.: 166–168 °C. 1H NMR  δ: 9.61 ; 8.56–8.54 ; 7.53–7.51 ; 7.22–7.18 ; 7.03–6.99 ; 6.97–6.95 ; 6.73–6.72 ; 6.58–6.55 ; 6.38–6.37 ; 4.33–4.31 ; 3.90 ; 3.76 . 13 C NMR  δ: 159.8 ; 157.9 ; 145.3, 144.4 ; 139.3; 137.9 ; 129.9 ; 128.4, 127.8 ; 126.3 ; 121.1; 114.7; 104.1; 99.7 ; 65.4 ; 55.1 ; 43.0 . m/z ESI-MS: [M-H]− 461.15.Compound 3.1.4.6 , as hydrochloride salt, was precipitated in 10 ml of NaOH 0.1 M. The resulting precipitate was solubilised in 5 ml of DMF, and then Rhd B-Itc 8  was added to the solution. The reaction mixture was left stirring 48 h at r.t. and protected from light. The reaction was monitored by TLC. The aqueous solution was extracted with EtOAc and then the organic layer was washed with H2O. The organic phase was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography  to afford compound 2 as a red solid (12% yield). M.p. 140–142 °C. 1H NMR  δ: 7.96 ; 7.82–7.80 ; 7.46–7.44 ; 7.36–7.33 ; 7.18–7.16 ; 7.07–7.03 ; 6.99–6.88 ; 6.98–6.95 ; 6.92–6.88 ; 6.64–6.63 ; 6.36–6.35 ; 4.28–4.25 ; 4.06–4.03 ; 3.79 ; 3.66–3.61 ; 1.30–1.26 . 13 C NMR  δ: 183.0 ; 163.4 ; 162.7 ; 160.2 ; 159.6, 157.0, 141.3, 133.5, 132.0, 131.4, 130.0 ; 129.1 ; 128.0, 116.2, 115.4, 115.0, 105.5, 100.7, 97.2 ; 67.5 ; 56.0 ; 46.9 ; 45.4 ; 33.3, 30.9, 23.9; 14.6, 13.0 . m/z ESI-MS: [M + H]+ 799.35.Compound 3.2.UV–visible absorption measurements were carried out in the range 200–800 nm, using DMSO solution in quartz cells, by Shimadzu 2401 PC UV–vis spectrophotometer−1 range with a Bruker Vector 22 equipped with a DTGS detector. Thin films of samples 1 and 2, deposited from DMSO solution onto gold substrates, were analysed in reflectance mode with a Specac P/N 19650 series monolayer/grazing angle accessory. KBr pellets of 7-nitrobenzofurazan samples were prepared to record the FTIR spectrum.Fourier transform infra-red spectroscopy (FT-IR) spectra have been recorded in the 4000–400 cmar-N bond and the resonance at 531.5 eV, assigned to the C = O carbonyl 1 s − π* transition, respectively. The raw C, N, and O K-edge NEXAFS spectra were normalised to the incident photon flux by dividing the sample spectrum by the spectrum collected on a freshly sputtered gold surface. Spectra were then normalised subtracting a straight line that fits the part of the spectrum below the edge and assessing to 1 the value at 330.00, 430.00, and 560.00 eV for carbon, nitrogen, and oxygen, respectively.Near-edge X-ray absorption fine structure (NEXAFS) spectra were acquired at the BEAR beamline (bending magnet for emission absorption and reflectivity) at the ELETTRA storage ring. BEAR is installed at the left exit of the 8.1 bending magnet exit. The apparatus is based on a bending magnet as a source and beamline optics delivering photons from 5 eV up to about 1600 eV with selectable degree of ellipticity. The UHV end station is equipped with a movable hemispherical electron analyser and a set of photodiodes to collect angle-resolved photoemission spectra, optical reflectivity, and fluorescence yield. In these experiments, we used ammeters to measure drain current from the sample. C, N, and O K-edge spectra were collected at grazing (20°) incidence angles of the linearly polarised photon beam with respect to the sample surface. In addition, our carbon, nitrogen and oxygen K-edge spectra have been further calibrated using the resonance at 285.00 eV, assigned to the C = C aromatic 1 s − π* transition, the resonance at 401.00 eV, assigned to the 1 s − π* transition of the C3.3.3.3.1.2 in modified, phenol red‐free, Dulbecco’s Modified Eagle’s Medium (DMEM) medium. Ten % (vol/vol) of charcoal‐stripped foetal calf serum, l‐glutamine (2 mM), gentamicin (0.1 mg/mL), and penicillin (100 U/mL) were added to the media before use. Cells were used at passage 13–17. The cell line authentication was periodically performed by amplification of multiple short tandem repeat loci by BMR genomics S.r.l . Cells were treated for 48 h with either vehicle  or resveratrol  or with different concentration of Pterostilbene and its fluorescent derivatives 1 and 2.Human breast cancer cells, MCF‐7, and human neuroblastoma cells, SH-SY5Y  were grown in air containing 5% CO3.3.2.Briefly, after the treatments, cells were lysed and protein were solubilised in the YY buffer  containing 0.70% (wt/vol) sodium dodecyl sulphate (SDS). Total proteins were quantified using the Bradford protein assay . Solubilised proteins (20 µg) were resolved by 7% or 15% SDS‐polyacrylamide gel electrophoresis at 100 V for 1 h at 24.0 °C and then transferred to nitrocellulose with the Trans‐Blot Turbo Transfer System  for 10 min. The nitrocellulose was treated with 5% (wt/vol) bovine serum albumin in 138.0 mM NaCl, 25.0 mM Tris, pH 8.0, at 24.0 °C for 1 h and then probed overnight at 4.0 °C with anti‐PARP‐1  or with anti-NGB  antibodies. Moreover, anti‐α‐tubulin  antibody was used to normalise the protein loaded. The antibody reaction was visualised with the chemiluminescence western blot analysis detection reagent . The densitometric analyses were performed by the ImageJ software for Microsoft Windows .3.3.3.After grown up to 80% confluence in 96‐well plate, cells were stimulated as reported above. Fixation and permeabilization were performed through ice cold EtOH 70% for 15 min at −20 °C. After EtOH solution removal, the cells were incubated with PI buffer for 30 min in the dark. Again, solution removal was performed, and the cells were rinsed with PBS solution. The fluorescence was revealed  with TECAN Spark 20 M multimode microplate reader .3.3.4.t-test to compare two sets of data or by ANOVA followed by Bonferroni test to compare different group of data by INSTAT software system for Windows. In all cases, p < 0.05 was considered significant.The statistical analyses were performed by Student’s Click here for additional data file.


Lysinibacillus sp. was isolated, numbered strain GG242. The degradation rate of 100 mg·L−1 17β-estradiol (E2)) > 95% in one week, and compared with extracellular enzymes, intracellular enzymes have stronger degradation ability. Strain GG242 can maintain a stable E2 degradation ability under different conditions . Under appropriate conditions , the degradation rate increased by 32.32% in one week. Based on the analysis of transformation products, inferred E2 was converted via two distinct routes. Together, this research indicates the degradation potential of strain GG242 and provides new insights into the biotransformation of E2.Environmental estrogen pollution has long been a concern due to adverse effects on organisms and ecosystems. Biodegradation is a vital way to remove estrogen, a strain of  Natural estrogens include estrone E1), 17β-estradiol E2), and estriol E3). The main sources of natural estrogen are human and animal excrement, so estrogen inevitably enters the environment, and estrogen is difficult to degrade in nature, resulting in its gradual accumulation. Estrogen can cause many adverse effects, such as the feminization of male fish, and when exposed to E2 concentrations of 550–630 ng·L, 17β-est, and est. The mai−1 E2 within 144 h [−1 of E2 within five days [Natural estrogen E2 is noted for its high content and strong activity, many E2-degrading strains have been isolated, for example, five E2-degrading bacteria were isolated from activated sludge, which achieved in vitro a 99% removal rate of 1 mg·Live days . The stuLysinibacillus sp. was isolated from cattle farm sewage . (1) Explore the ability to degrade E2 and the E2-degrading capacity of intracellular and extracellular enzymes. (2) Explore the effects of culture conditions  on E2 degradation. (3) Explore the changes in degradation ability under suitable conditions. (4) The E2 transformation products were identified by liquid chromatography tandem electrospray ionization mass spectrometry (LC-ESI-MS) and infer possible transformation routes.Here, to degrade E2, a bacterium of 2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4, and 1.0 g (NH4)2SO4 in 1 L ultrapure water. Luria Bertani medium (LB): 10.0 g tryptone, 10.0 g NaCl, 5.0 g yeast extract in 1 L ultrapure water, solid plates were made by adding 1.5% agar to a liquid medium.E2 and E1  were purchased from Solarbio . High-performance liquid chromatography (HPLC)-grade acetonitrile  and methanol  were purchased from Thermo Fisher Scientific . Ultrapure water was produced using an ultrapure water device . The basal salt medium (BSM) was comprised of 1.0 g Kv/v), sampling at specific time intervals, and detected with the OD600 . Sterilized LB was used as a control. The growth curve was determined by plotting the average value for each time point. The culture was centrifuged (3000 revolutions per minute (rpm), 10 min, 4 °C), the supernatant was discarded, 2.5% glutaraldehyde solution (electron microscope fixative) was added to the pellets and placed at 4 °C for 12 h. After fixing, the mixture was centrifuged , the supernatant was discarded, the pellets were washed 3 times with phosphate-buffered saline (PBS) solution, then centrifuged. Thereafter, the bacteria were dehydrated by sequential exposure to 10, 30, 50, 70, and 90% ethanol, 5 min at each concentration, and then centrifuged . Finally, the bacteria were exposed to 100% ethanol. Scanning electron microscopy was done by the Gechuang Testing Technology Center .To observe bacterial colony morphology, the enriched strain was streaked on LB plates, a single colony was obtained by repeated streaking, and the single colony was picked for gram staining and microscopic examination . Furthermore, bacteria were inoculated into the LB at 1%  was extracted from the cultured strain, and DNA was used as a template for 16S rDNA gene sequence amplification via polymerase chain reaction (PCR). 16S rDNA was connected to the T vector to construct a recombinant plasmid, and the vector universal primer was used as the sequencing primer for DNA sequencing. After removing the vector sequence, the gene sequence was obtained. The obtained gene sequences were aligned to other 16S rDNA sequences in the NCBI database, and the sequences with high scores in the aligned result were selected to construct the phylogenetic tree (MEGA7).−1, respectively. First, a single colony was transferred to 5 mL LB and cultured in a shaker  at 30 °C and 135 rpm·min−1 for 12 h, then 1 mL culture to 100 mL LB, grown to the logarithmic growth phase and centrifuged , supernatant was removed and the pellets were washed twice with PBS solution, centrifuged  after each washing. Finally, the pellets were suspended in PBS solution, and the OD600 was adjusted to 2.0 (4.3·108 CFU·mL−1). The obtained bacterial suspension was added to BSM for the E2 degradation experiment, the degradation period was 7 days, with daily sampling to detect the OD600 and residual E2. To explore the ability of intracellular and extracellular enzymes to degrade E2, bacteria were grown in 100 mL LB to log phase, intracellular and extracellular enzymes were obtained based on the method described in. In brief, the bacterial culture was centrifuged , the supernatant was used as an extracellular enzyme, the remaining bacteria were resuspended in PBS, and the bacteria were disrupted in ice water bath to obtain intracellular enzymes. The ultrasound cell crusher  was used to crush bacteria, the instrument parameter settings were: Power was 150 W, total time was 5 min, working time and interval time were 5 and 7 s, the whole process was repeated 3 times. After the preparation, the enzyme solution was filtered , and finally placed at 4 °C. The 5% (v/v) intracellular enzymes and extracellular enzymes were added to the BSM to degrade E2, respectively, the degradation period was 5 days, and the degradation rate of E2 was used to evaluate the degradation ability.To explore the E2-degrading ability of strain GG242, the initial concentration of E2 was set to 10, 50, 100, and 200 mg·L600 was adjusted to 2.0. To explore the effect of different culture conditions on E2 degradation, the following experiments were set up: Group 1: The pH was set to 5–11, group 2: The temperature was set to 20–40 °C, Group 3: The NaCl was set to 0–40 g·L−1, only one condition was changed in each group, the rest were the same. The degradation period was 5 days, with daily sampling to detect E2. Thereafter, according to the above experiments, the appropriate conditions were selected, and E2 was degraded under appropriate conditions.As described above, the suspension was prepared, and the OD−1 (to ensure sufficient products), the degradation period was set to 5 days, and samples were taken on days 1 and 4 for LC-MS detection. Before detection, 400 μL methanol (−20 °C) was added to 100 μL sample, fully mixed and centrifuged . The supernatant was concentrated and dried under vacuum. After drying, 150 μL 2-chlorophenylalanine (4 ppm) 80% methanol solution was added to redissolve it, and the supernatant was filtered to obtain the sample to be detected. Chromatographic conditions: ACQUITY UPLC® HSS T3 column , column oven temperature 40 °C, autosampler temperature 8 °C, injection volume 2 μL, mobile phase for gradient washing was positive ion 0.1% formic acid water (C), 0.1% formic acid acetonitrile (D), negative ion 5 mM ammonium formate water (A), acetonitrile (B), and the flow rate was 25 mL·min−1. The setting of the gradient elution program was 0–1 min, 2% B/D; 1–9 min, 2–50% B/D; 9–12 min, 50–98% B/D; 12–13.5 min, 98%B/D; 13.5–14 min, 98–2% B/D; 14–20 min, 2% D positive mode . MS conditions: Electrospray ion source (ESI), positive and negative ion ionization mode. The positive ion spray voltage and the negative ion spray voltage were 3.5 kV and 2.5 kV, respectively, sheath gas 30 arb, and auxiliary gas 10 arb. The capillary temperature was 325 °C, the full scan was performed with a resolution of 70,000, the m/z scanning range was 81–1000, data-dependent acquisition (DDA) MS/MS experiments were performed with HCD scan, and the collision voltage was 30 eV. Unnecessary MS/MS information was removed with dynamic exclusion .To detect the transformation products of E2, the concentration of E2 in the BSM was set to 100 mg·L−1 and 30 °C, bacteria suspension was inoculated with 1% (v/v), in triplicate, and without bacteria as control. Using Excel (version 2016) to process data and calculate results, statistical analysis of data with SPSS software . Graphs were prepared using GraphPad Prism (version 7.00). Detection of E2 by HPLC, HPLC setting conditions: the instrument was Shimadzu LC-2030 Plus . The chromatographic column was Agilent ZORBAX SB-C18 . The mobile phase was acetonitrile: Water = 55:45 (v/v) with a flow rate of 1.0 mL·min−1, column oven temperature 30 °C, injection volume 25 μL, detection wavelength 280 nm.Unless otherwise specified, the above degradation experiments were performed in 100 mL BSM at 135 rpm·minLysinibacillus sp.7B-842 (KF441703.1), and the sequence similarity >99%  on the 7th day. Furthermore, as E2 was degraded, E1 was detected and accumulated over time  was significantly lower than other groups (p < 0.01). At pH 6–10, strain GG242 can maintain a stable degradation capacity, the remaining E2 was <3.6 mg·L−1 and the degradation rate was >88%. At pH 8, strain GG242 has the strongest degradation ability with 0.3 mg·L−1 remaining E2 and 98.9% degradation rate. At different temperatures . This shows that 30 °C is the preferred temperature for strain GG242. When the temperature increased from 30 to 40 °C, the degradation ability gradually decreased at 35 °C and 40 °C, the remaining E2 was 6.7 mg·L−1 and 24.9 mg·L−1, with 77.5% and 16.9% (p < 0.01) degradation rates. Under different salinity, the degradation rate elevated as the salinity increased . Under appropriate degradation conditions, strain GG242 can degrade 200 mg·L−1 E2 to about 54 mg·L−1 in seven days, and the degradation rate reaches 72.7% (p < 0.01) compared to before (40.3%), therefore, in practical applications, optimizing controllable factors will undoubtedly enhance degradation capacity.The E2 degradation results at different pH are shown in eratures c,d, as tncreased e,f. Whenes 72.7% . The degm/z), etc., are shown in E2 converted to 2-methoxyestradiol, next, the rings except for the benzene ring on the 2-methoxyestradiol were cleaved and converted to trans-Ferulic acid. On route II, E2 converted to E1, and E1 underwent conversion and cleavage. On route II-a, the saturated ring cleavage, formed 4-hydroxycinnamic acid, 4-hydroxycinnamic acid continued to cleavage and became hydroquinone, finally, hydroquinone entered the tricarboxylic acid cycle. On route II-b, the benzene ring on E1 underwent an addition reaction, converted to dehydroepiandrosterone, and further converted to ursodeoxycholic acid, ursodeoxycholic acid was subsequently dehydroxylated to form lithocholic acid. On route II-c, the benzene ring of E1 was hydroxylated to form 4-hydroxyestrone, as on routes I and II-a, the saturated ring of 4-hydroxyestrone was cleaved to 3,4-dihydroxymandelic acid, and further cleaved into catechol, then catechol entered the tricarboxylic acid cycle. This study deduces several possible E2 transformation routes, which enriched the fate of E2 in the degradation process and provided new ideas for the biotransformation of E2. Subsequent work will focus on validating these routes and mining potential genes and enzymes involved in the reaction.Eight transformation products were identified by LC-ESI-MS, identification information, such as retention time and mass–to–charge ratio  [Pseudomonas sp. and Lysinibacillus sp. can form biofilms on the surface of plastic debris, which contributes to its degradation [−1 E2, the degradation rate is more than 96%, and the first five days were the main phase of degradation, and the degradation capacity was weakened on days 6 and 7, this may be because the bacteria entered a lag phase. At 200 mg·L−1 E2, GG242 degraded 46.5 mg·L−1 E2, and the degradation rate reached 23.2% on the 1st day, but as the degradation progressed, the degradation rate increased less, on the 7th day, the degraded E2 was 70.8 mg·L−1, and the degradation rate only increased to 35.3%. This may be due to two reasons, the first reason may be the high E2 concentration inhibits the degradation ability, and the other may be that the gradually produced E2 metabolite affects the bacteria. With the initial E2 increases, the biomass increases, resulting in a higher degradation rate, this may be due to higher biomass leading to the higher efficiency of biochemical reactions [−1. Although GG242 can use E2 as energy for growth, as E2 concentration gradually increases, E2 may adversely affect bacterial growth. From the analysis of biomass changes, E2 metabolites may influence bacteria, such as at 100 mg·L−1 E2, the biomass on the 1st day was comparable to 50 mg·L−1 E2, but as degradation proceeded, it showed a sharp drop, which could be that the high concentration of E2 produced more metabolites and affected the growth of bacteria. Compared to other strains, strain GG242 can degrade higher concentrations of E2, such as a Novosphingobium strain can degrade about 70% of 100 mg·L−1 E2 within 7 days [Nubsella strain, which can achieve 99% removal of 2 mg·L−1 E2 in 7 days. The difference may be due to the different E2 concentrations selected when screening bacteria, strains screened at low E2 concentrations may have higher-affinity enzyme systems, whereas these strains may not have strong estrogen tolerance. Therefore, more studies should be carried out to better evaluate the performance of the two types of bacteria. Both intracellular and extracellular enzymes can degrade E2, and the degradation ability of intracellular enzymes is stronger than extracellular enzymes, this may be due to the mechanism of repairing the activity of endogenous enzymes and proteins [Gordonia sp. strain R9, E2 degradation decreased from 30 to 40 °C [−1 NaCl, this indicates that GG242 can adapt to changing environments. Probing the effect of different factors on degradation ability not only shows the potential of bacteria in applications, but also reveals the suitable environment for bacteria.In vitro,  mg·L−1) . In addiradation . Howevereactions . Howevern 7 days . Zhou etproteins . When thproteins ,33, and proteins . The effto 40 °C . The reato 40 °C . The effto 40 °C , low conto 40 °C . This isTwo possible routes were deduced based on the analysis of the products, one is the cleavage of E2, and the other is the cleavage and transformation of E1. On routes I and II-a, trans-Ferulic acid and 4-hydroxycinnamic acid have a similar chemical structure, this further illustrates the result of cleavage. Trans-Ferulic acid and 2-methoxyestradiol have the same benzene ring structure, so inferred the two substances are on the same route, but the key compound 2-methoxyestradiol was not identified, which requires further identification. On route II-a, 4-Hydroxycinnamic acid has the same benzene ring structure as E1 and E2. Since E1 is the major transformation product of E2, it is inferred that 4-Hydroxycinnamic acid is the cleavage product of E1. On route II-b, dehydroepiandrosterone can be used to synthesize ursodeoxycholic acid , ursodeoLysinibacillus sp. Strain GG242 has a robust degradation ability, strong and compared with extracellular enzymes, intracellular enzymes have stronger degradation ability. The strain has cultural adaptability, but the elevated temperature has a significant impact on degradation, degradation capacity is improved under suitable conditions . Two possible E2 transformation routes were deduced based on the analysis of the product. This study provides an option for estrogen-degrading microorganisms and proposes 2 possible E2 transformation route.An E2-degrading strain GG242 was isolated from cattle farm sewage and identified as


Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity. Obesity is a serious risk factor for cardiovascular diseases, particularly for myocardial infarction . It aggrOxysterols also play a physiopathological role during ischemia-reperfusion, as they can induce apoptosis . They acUsing ob/ob mice, we showed, in the present study, that chronic exercise significantly reduced 7βOH myocardial content, and this was accompanied by an increase in Akt phosphorylation, thus showing an activation of the cardioprotective RISK pathway as a pre-emptive mechanism before any ischemic insult. Conversely, cardiomyocytes exposed to 7βOH elicited reduced Akt phosphorylation. Finally, as a translational attempt, we provide results showing a concomitant increase in 7βOH concentration in the serum of obese patients, as compared to healthy volunteers. Concomitantly, Akt phosphorylation in the PBMC issued from their blood was reduced, respectively.We previously reported that regular exercise was able to induce cardioprotection in both lean and obese mice . We, theTo investigate the link between the cytosol and mitochondria, we examined the subcellular localization of PKCε, following regular exercise . The expNumerous studies suggest that certain oxysterols could inactivate the pro-survival kinases of the RISK pathway. We, therefore, decided to investigate the effect of 7βOH on cardiomyocytes in WT mice specifically. The phosphorylated Akt/total Akt ratio was significantly reduced in the 7βOH-treated mice cardiomyocytes . This sup < 0.05). Exercise significantly reduced the 7βOH cytosolic concentrations in both the Ex-WT and Ex-ob/ob mice , compared to sedentary wild-type mice (Sed-WT mice) . Mean body mass index was 22.7 ± 0.4 kg/m² for healthy volunteers vs. 42.9 ± 2.2 kg/m² for obese patients (p < 0.05). None of the patients had history of cardiac disease.To investigate whether the oxysterols could be considered candidate as biomarker in patients, nine healthy volunteers  and seven obese patients  were included in the study and experienced oxysterol assays in plasma. Mean body weight was 60 ± 2 kg for healthy volunteers vs. 122 ± 7 kg for obese patients  Animal Ethics Committee . Male 8- to 10-week-old wild-type C57BL/6J (WT) and obese (ob/ob) mice were used . Mice were housed in an air-conditioned room with a 12-h light-dark cycle and received standard rodent chow and drinking water ad libitum. n = 55) and ob/ob (n = 52) mice were randomly subjected to treadmill exercise (Ex-WT and Ex-ob/ob) or sedentary conditions (Sed-WT and Sed-ob/ob). Exercised animals ran 5 consecutive days per week for 4 weeks. The first week was a 30-min adaptation period with gradual speeds . For the three other weeks, mice ran during one hour per day . To take into account the differences in body weight [Both WT  and competent health authorities. All subjects included provided written informed consent before participating in the study. Blood samples were collected on lithium heparinate and then rapidly centrifuged, and the plasma was frozen at −80 °C until the oxysterol assays. Peripheral blood mononuclear cells (PBMC) were freshly isolated from whole blood collected on lithium heparinate by Ficoll density gradient. Cell recovery and viability were assessed using a hemacytometer and trypan blue.2 in a humidified incubator. This concentration of 7βOH has been chosen on the basis of preliminary experiments guided by previous reports [Adult mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (90 mg/kg) and heparin (500 IU). The heart was quickly excised, placed in an ice-cold calcium-free perfusion buffer, and first perfused using a Langendorff apparatus with the perfusion buffer at 37 °C. Then, a digestion buffer containing calcium, liberase, and trypsin was used. Once enzyme digestion of the heart was completed, the left ventricle was dissected. It was then minced with scissors and homogenised with a pipette in a stopping buffer containing calcium and 10% bovine calf serum. The cellular pellet was washed 6 times with a perfusion buffer containing 5% bovine calf serum and increasing concentrations of calcium. If the final viability was greater than 65%, these adult mouse cardiomyocytes were then incubated with 25 µM of 7β-hydroxycholesterol (7βOH) solubilized in ethanol during 24 h at 37 °C and 5% CO reports ,19.g for 10 min at 4 °C. Cell pellet was resuspended in a homogenization buffer containing 220 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EGTA and supplemented with protease and phosphatase inhibitors (pH 7.4 at 4 °C). Two cycles freezing-thawing at −80 °C, followed by 5 sonication cycles, were then used to break the cell membranes. A final centrifugation at 16,100× g for 1 h at 4 °C allowed for collecting the cytosol.Adult mouse cardiomyocytes and human PBMC, when available, were collected by centrifugation at 1000× g for 5 min. Supernatants were centrifuged at 10,000× g for 10 min at 4 °C. The final mitochondrial pellet was then resuspended in 150 µL of a homogenization buffer containing 220 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EGTA, and supplemented with protease and phosphatase inhibitors (pH 7.4 at 4 °C). The supernatants were centrifuged at 100,000× g for 60 min at 4 °C. The mitochondrial and cytosolic protein concentrations were determined by the BCA protein assay kit .Cytosol and mitochondria were extracted from fresh left ventricular tissue. The tissues were scissor minced, homogenized on ice using a Teflon Potter homogenizer, and centrifuged at 1000× To measure mitochondrial calcium retention as an index of the resistance to calcium-induced mPTP opening, cardiac mitochondria (0.8 mg/mL), incubated with 3.3 µM calcium Green-5N fluorescent probe  were loaded by the addition of successive 2.5 µM calcium pulses. When maximal calcium-loading threshold was reached, mitochondria underwent a fast process of calcium release due to mPTP opening. The concentration of calcium in the extra-mitochondrial medium was monitored by means of a Perkin Elmer LS 50B fluorescence spectrometer at excitation and emission wavelengths of 506 and 532 nm, respectively.Western blot analyses were performed to investigate phosphorylated Akt, total Akt, PTEN, and PKCε. To extract proteins, myocardial tissues were placed in medium containing 220 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EGTA, 5µL/mL of protease inhibitor cocktail, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 1 mM sodium Na2β-glycerol phosphate, and pH 7.4 at 4 °C. Extracted protein samples were denatured at 95 °C for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with primary antibodies for β-actin , phospho-Akt , total Akt (1/2000), PTEN (1/1000) , and PKCε . Blots were washed and then incubated with anti-rabbit IgG secondary antibody . Bands of interest were scanned and quantified in a blinded manner using gel analysis software ImageJ-1.37 .v/v. The organic solvents were evaporated under a gentle stream of argon and converted into trimethylsilyl ethers [Oxysterol measurements were performed on cytosol extracts from mouse fresh cardiac tissue and plasma samples from human subjects. Concentrations of 7β-hydroxycholesterol (7βOH) were measured by gas chromatography-mass spectrometry (GC-MS) ,40. D7-7l ethers .p < 0.05. Non-parametric tests were used, with respect of the low number of animals in each experiment.Statistical analysis was performed using Kruskal–Wallis for overall analysis followed by individual comparisons using Mann–Whitney tests, with significance at


Bacillus sp. GHS8, Pseudoalteromonas sp. GHS5 and Psychrobacter sp. GHF10, which were originated from natural bioflocculant of Ruditapes philippinarum conglutination mud (RPM), respectively. The EPS had similar function groups C-H, N-H, C-O, and C = O. The EPS were composed of different monosaccharides . The molecular weights (Mw) of EPS were ordered as 51.4 kDa (EPS-S5) > 9.15 kDa (EPS-S8) > 4.41 kDa (EPS-F10). Three types of EPS all showed higher peak flocculation activities than the reported crude EPS from the RPM. Besides, the EPS also exhibited efficient decoloration and antioxidation activities, especially for EPS-S8, which might be due to the low Mw and specific monosaccharide composition.Three novel types of exopolysaccharides (EPS) EPS-S8, EPS-S5, and EPS-F10 were extracted and purified from bacterial isolates  In the niformis , Bacillus cereus , Edwardsla tarda , Sphingoae BBL01 , Acinetotobacter , Pseudoaeromonas , Bacillusis AP-4 . The chesis AP-4 . HoweverRuditapes philippinarum, a very popular mudflat shellfish, is widely distributed in the coast and received much attention (Ruditapes philippinarum conglutination mud (RPM), and two complex heteropolysaccharides  were screened with similar monosaccharides composition except glucose content  purify novel EPS and analyze their structural characterization, including functional groups and monosaccharides components; (2) study the diverse biological functions of the different EPS, including flocculation, decolorization and antioxidation.2.2.1.Psychrobacter sp. GHF10, Pseudoalteromonas sp. GHS5, and Bacillus sp. GHS8 isolated from the RPM, were identified by the 16S rDNA sequences analysis with the NCBI accession Numbers of KX702266, KX702256, and KX702261, respectively. Each bacterial strain was inoculated in a 10 L fermentation tank  with culture medium. The compositions of medium were presented as followings (g L−1): glucose, 20; (NH4)2SO4, 0.2; urea, 0.5; yeast extract, 0.5; MgSO4·7H2O, 0.2; KH2PO4, 2.0; K2HPO4, 5.0. The components were dissolved in synthetic seawater, which was composed of the followings substances (g L−1): MgCl2·6H2O, 9.68; KCl, 0.61; Na2SO4, 3.47; NaCl, 30.0; Na2HPO4, 0.014; NaHCO3, 0.17; CaCl2·2H2O, 1.36; KBr, 0.10; SrCl2·6H2O, 0.04; H3BO3, 0.03. After culture for 48 h in the condition of 25°C and 180 rpm, 6 L of fermentation broth was obtained and centrifuged at 9,000 g for 20 min, and the supernatant was preserved and concentrated. The concentrated solution was packed into a dialysis bag with a Mw cut-off of 3,500 Da. The dialysis bag was placed in deionized water and dialyzed for 2 days at 4°C to remove salts and other small molecular compounds. The deionized water was replaced every 120 min. Then, the dialysate was concentrated by rotary evaporator (RE-2000). Finally, the concentrated solution was mixed with threefold of cold ethanol at 4°C for 1 day, and the precipitation was crude EPS. All the materials were used under aseptic conditions.Three bacterial strains −1) were used as the eluent to elute the anion exchange column gradually with a flow rate of 1 ml min−1. Thus, three types of pure EPS were obtained and named as EPS-F10 (Psychrobacter sp. GHF10), EPS-S5 , and EPS-S8 (Bacillus sp. GHS8), respectively.The Sevag method was utilized to remove protein in the crude EPS . The tot2.2.−1, respectively. A satisfactory standard (r2 > 0.999) was made by different Mw (2.7–133.8 kDa) of dextran standards. Then, the Mw of the samples were calculated using the GPC software.The Mw of the purified EPS was determined by high performance gel permeation chromatography (HP-GPC) coupled with a TSK gel G3000PWXL column  and the refractive index detector . The det−1; injection volume, 20 μl; detector wavelength, 245 nm. Twelve kinds of monosaccharide standards were used for drawing standard curves, including mannose (Man), galactose , xylose (Xyl), glucosamine (GlcN), ribose (Rib), rhamnose (Rha), arabinose (Ara), galacturonic acid (GlaUA), galactosamine , glucose (Glc), fucose (Fuc), and glucuronic acid (GlcUA).The hydrolysis of purified EPS was the same as previous study . Then, t−1 and speed of 1 cm−1.The FT-IR analysis was utilized for ascertaining the function groups of purified EPS. The mixture of dried EPS (1 mg) and KBr (0.1 g) were pressed and then scanned in the frequency range of 4,000–400 cm2.2.1.−1) were mixed with kaolin suspension (4 g L−1) and CaCl2 solution (10 g L−1) at the rotation speed of 180 r min−1 for 1 min and then mixed at 30 r min−1 for 2 min. The supernatant was collected after 10 min precipitation and measured at wavelength of 550 nm by an ultraviolet spectrophotometer. The calculation of the flocculation rate is shown in Flocculation activity of the purified EPS were determined using the modified method. Briefly, various concentrations of EPS  was used for determining the decolorization activity of purified EPS. The mixture of dyes solution was stirred with 0–1 mg ml−1 EPS at 30 rpm min−1 for 1 min. After precipitation and centrifuge, the supernatant was measured at wavelength of 620 nm. The calculation formula of decolorization activity is shown in Crystal violet  and 0.5 ml FeSO4 solution (9 mM), and then bathed in a water bath at 25°C for 10 min. Finally, 1 ml of 9 mmol L−1 salicylic acid was added in a water bath at 37°C for 1 h. The control group was distilled water. The calculation of the scavenging rate of hydroxyl radicals of polysaccharide samples is shown in OH radical scavenging assay was also conducted. The various concentrations of purified EPS (0.5 ml) were mixed with 1 ml HROH, the OH clearance rate of the sample; A1, the absorbance value of the sample at wavelength of 510 nm; A0, the absorbance value of the blank group.Where, 3.3.1.Psychrobacter sp. GHF10, Pseudoalteromonas sp. GHS5, and Bacillus sp. GHS8) had the percentages of total polysaccharides of 87.02, 92.16, and 78.2%, respectively. The isolated EPS were scanned under the ultraviolet spectrum (200–400 nm) and there were no sharp absorption peaks of protein or nucleic acid. The EPS were further purified via DEAE-52 Cellulose Ion Exchange Chromatography  from the three strains  > 9.15 kDa (EPS-S8) > 4.41 kDa (EPS-F10) . Figure spectrum . The broH or N-H . The samectively . Besidespyranose . Besidesnose (−1 . Results3.3.The monosaccharide compositions of EPS are presented in 3.4.−1) and CaCl2 solution (10 g L−1) (−1. The peak flocculation rates of the EPS occurred at 0.6 mg mL−1 (EPS-F10), 0.6 mg mL−1 (EPS-S5), and 0.8 mg mL−1 (EPS-S8), respectively. Besides, the EPS-S5 had the highest peak flocculation rates (80.1%).The flocculation activity of the different EPS was determined in kaolin clay (4 g L0 g L−1) . The flo−1 (EPS-F10), 2.0 mg mL−1 (EPS-S5), and 1.5 mg mL−1 (EPS-S8), respectively. Obviously, the EPS-S8 exhibited better performance of decoloration activity.In terms of decoloration activity, similar phenomenon of first rise and then fall trend were found in the three types of EPS . The sam−1, the scavenging ability of the two free radicals gradually became stronger, but much lower than those of ascorbic acid (Vc). The DPPH radical scavenging ability reached 61.9% (EPS-F10), 40.8% (EPS-S5), and 61.6% (EPS-S8) at an EPS concentration of 1 mg mL−1, and the EC50 values of scavenging ability of DPPH radicals were 0.23 mg mL−1, 0.35 mg mL−1, and 0.24 mg mL−1, respectively , 24.5% (EPS-S5), and 47.0% (EPS-S8), respectively  and function groups . To our knowledge, these EPS molecules showed no similarity with reported microbial bioflocculants for their specific molecular weights and complicated composition  have been extracted from RPM . The Mw Leuconostoc mesenteroides strain XR1  is presented in Pseudoalteromonas and Bacillus, but this study firstly reported the EPS from genera Psychrobacter. Besides, there is still little information about the function of flocculation, decoloration or antioxidation from the both genera Pseudoalteromonas and Psychrobacter. In our previous study, the crude EPS of 14 strains isolated from the Zhoushan RPM were proved to have efficient bioflocculation . EPS-S5 with the highest Mw exhibited the lowest DPPH scavenging ability in this study. The low Mw polysaccharide were reported to exhibit stronger antioxidant activity than high Mw of polysaccharide , which w5.Bacillus sp. GHS8, Pseudoalteromonas sp. GHS5 and Psychrobacter sp. GHF10 isolated from the RPM, respectively. Similar function groups , the similar magnitude (4.41–51.4 kDa) of Mw, and neutral sugars were all found in the three types of EPS. However, EPS-S8 from Bacillus had low Mw, specific monosaccharide composition, which exhibited the better performance of decoloration activity and scavenging ability on both DPPH as well as hydroxyl radicals.Three novel heteropolysaccharides with flocculation, decoloration, and antioxidation activities were generated and purified from The original contributions presented in the study are included in the article/LF: conceptualization, methodology, software, data curation, visualization, writing – original draft preparation, and writing – reviewing and editing. TQ: data curation, visualization, methodology, software, and visualization. GY: visualization, methodology, software, and writing – reviewing and editing. JM: conceptualization, resources, and writing – reviewing and editing. All authors contributed to the article and approved the submitted version.The authors gratefully acknowledge the financial support by the Scientific Research Foundation of Hainan Tropical Ocean University (No. RHDRC202118).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1068922/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


In this experiment, mouse secondary follicles were cultured in the three-dimensional culture system of alginate gel with different concentrations of IGF-1. According to the results of follicle growth, oocyte maturation, the levels of hormone  and the expressions of genes related to hormone secretion, oocyte-secreted factors, apoptosis and gonadotropin receptor, the optimal concentration of IGF-1 was determined to be 10 ng/mL in the culture medium. Moreover, the intraperitoneal injection of IGF-1 before superovulation in mice could increase the number of ovulated oocytes and reduce their degraded rates.p < 0.05). However, G50 significantly inhibited follicular growth (p < 0.05), while G10 showed a higher oocyte maturation rate. Thus, the 10 ng/mL IGF-1 was used in subsequent experiments. IGF-1 enhanced the function of granulosa cells (GCs) by upregulating expressions of Star, Cyp19a1, Hsd3b1, Fshr, and Lhcgr. Oocyte secretory function was promoted by upregulating expressions of Bmp-15, Gdf-9, and Fgf-8. Addition of IGF-1 showed anti-apoptotic effect. However, G10 did not improve fertilization rate of MII oocytes compared to G0. In an intraperitoneal injection experiment in mice, IGF-1 significantly increased the number of ovulated oocytes (p < 0.05). In conclusion, 10 ng/mL IGF-1 can promote the production of mature oocytes in the 3D culture medium and injection of IGF-1 before superovulation increases the number of ovulated oocytes.Insulin-like growth factor-1 (IGF-1) plays a crucial role during folliculogenesis, which has been demonstrated by previous research. However, the optimal IGF-1 dosage in the three-dimensional (3D) culture system is unknown. Mouse secondary follicles (140–150 µm) were cultured for 6 days within an alginate bead in a medium supplemented with 0 (G0), 5 ng/mL (G5), 10 ng/mL (G10), or 50 ng/mL IGF-1 (G50). Secretions of 17β-estradiol and progesterone were significantly increased in G10 and G50 ( Igf-1 was expressed in the ovaries of different species [Igf-1 knockout mice were infertile, and the size of the ovaries of Igf-1−/− female mice was less than half of the normal. Furthermore, most follicles were mostly arrested in the preantral stage, while a few follicles developed into the antral stage without maturation and ovulation [Igf-1 are low during the primary follicle stage and reach a high level in the late preantral and early antral stages in mice [Follicular development is gonadotropin-independent at the earliest growth stage  when it  species ,6, and I species . Both mavulation . The tra in mice . In addi in mice . These rFshr in preantral follicles of IGF-1−/− mice remained at a low level, while the IGF-I can significantly increase Fshr expression in murine granulosa cells in vivo [In rats, IGF-1 and IGFR are located in oocytes during the preantral follicle stage and the intensity of IGFR is stronger than IGF-1 based on immunohistochemistry analysis . The ooc in vivo . The effExcessive concentrations of IGF-1 can upregulate apoptosis-regulating genes, increase the vulnerability of GCs and lead to apoptosis . Yang et2+) can be added to complete polymerization [Maintaining follicular structure during the culture period facilitates the gap junction between the oocyte and the GCs, which is essential for oocyte growth and meiotic competence . The disrization . This prrization . In the rization . Optimizrization .In this study, we determined the optimal concentration of IGF-1 in a 3D in vitro culture system. The function of IGF-1 was analyzed by the changes in hormone secretion and gene expression and the synergistic effect of IGF-1 and gonadotropin was demonstrated in vivo during superovulation.™, Ham’s F-12 Nutrient Mixture, Leibovitz’s L-15, Collagenase IV, and fetal bovine serum (FBS) were purchased from Gibco . Other chemicals were obtained from Sigma-Aldrich  unless otherwise specifically indicated.MEM α with GlutaMAXAll animals were purchased from SPF  Biotechnology Co., Ltd. All mice were housed in polypropylene cages and provided food and water ad libitum. Animals were kept on a 12 h light/dark cycle (8:00 a.m.–8:00 p.m.) at 22–25 °C with 30–50% relative humidity.w/v) alginate bead. Beads were incubated in half MEM α and half F-12 of the mixture containing 10% FBS at 37 °C, 5% for 30 min. Then, beads were plated (one follicle/well) in 96-well plates in 100 µL of culture media composed of MEM α, 3 mg/mL of BSA, 1 mg/mL bovine fetuin, 5 µg/mL of insulin, 5 µg/mL of transferrin, and 5 ng/mL of selenium. Encapsulated follicles were cultured at 37 °C in 5% CO2 for 6 days. Every other day, half of the media (50 µL) was exchanged and stored at −80 °C. Follicle survival and diameter were assessed using an inverted microscope (Olympus IX-70) with transmitted light and phase objectives. Follicles were designated dead if the oocyte was no longer surrounded by a granulosa cell layer or if the GCs had become dark and fragmented.Ovaries isolated from 16 days old ICR-1 female mice were divided into two pieces and placed in enzymatic media containing L-15 with 0.2% Collagenase IV and 0.2% DNase I for 40 min at 37 °C before follicle isolation. Secondary follicles (140–150 µm) with theca cells were then mechanically isolated using insulin-gauge needles in L-15 media with 1.0% FBS and encapsulated into a sterile 0.5% , 10 ng/mL epidermal growth factor (EGF), and 10 mIU/mL porcine FSH  for 16 h at 37 °C, 5% CO2. Twenty metaphase II oocytes were placed in 50 μL mHTF medium drops containing 106 sperm/mL and incubated under mineral oil for 6 h at 37 °C, 5% CO2. Oocytes were then washed 3 times in fresh G1-PLUSTM  medium to remove all bound sperm and cultured in G1-PLUSTM medium at 37 °C, 5% CO2. Fertilized oocytes were identified by the presence of 2 pronuclei (2PN).Sperm was collected from the cauda epididymis of 9-week-old ICR-1 male mice. One hour before IVF, motile sperm was incubated in 100 μL mHTF medium  under mineral oil at 37 °C, 5% COFor immunostaining, oocytes were fixed in 4% paraformaldehyde at 4 °C for 60 min. The oocytes were washed three times in PBS containing 0.1% PVA and transferred to 3% bovine serum albumin (BSA) at 37 °C for 60 min. Blocked oocytes were incubated at 37 °C with CoraLite594-Conjugated Alpha Tubulin Monoclonal Antibody  for 60 min in the dark. After washing three times with PBS containing 0.1% PVA, chromosomes were labeled using Hoechst 33342 for 5 min and oocytes were mounted in 2.5 μL Antifade Mounting Medium drops on glass bottomed dishes. Samples were examined under a confocal laser scanning microscope.The hormone determination work was completed by the Beijing JinHaiKeYu Biological Technology Development Co. Ltd. . The 17β-estradiol and progesterone in conditioned media were measured using a radioimmunoassay. AMH in conditioned media was determined by ELISA. The sensitivities for the 17β-estradiol, progesterone and AMH assays were 5 pg/mL, 30 pg/mL, and 10 pg/mL. Intraassay and interassay coefficients of variation were determined to be 10 and 15%, respectively. To obtain sufficient media for each assay, media collected from follicles in identical alginate conditions were pooled for each time point (10 samples pooled per measurement). Five independent measurements for each hormone at each time point were performed.TM H Minus cDNA Synthesis Master Mix  and the first-strand cDNAs were used for Q-PCR analysis with GoTaq® qPCR Master Mix . The primer sequences used are listed in Total RNAs from the follicles were extracted and purified using the RNeasy Micro Kit . The total RNAs were reverse transcribed by MaximaReal-time quantitative polymerase chain reaction (qPCR) was performed using the qTOWER 2.0/2.2 sequence detection system . For each reaction, 2 μL cDNA, 0.4 μL primers, 10 μL 2×Master SYBR Green mix (Promega), and 7.2 μL nuclease-free water were added to a final volume of 20 μL. PCR cycling conditions were 95 °C for 15 min, followed by 39 amplification cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 15 s. After testing, the efficiency of qPCR is between 95–98%. To maximize accuracy, each sample was run in triplicate with a negative control of the reaction mixture with no cDNA added.Sixty ICR-1 8-week-old female mice were randomly divided into two groups. There were 3 repetitions per group and 10 mice per repetition. The mice were injected intraperitoneally with IGF-1 (2 μg/g body weight per mouse) 24 h before equine chorionic gonadotropin (eCG) (7.5 IU per mouse) injection, followed by injection with hCG (7.5 IU per mouse) 48 h after eCG injection in the IGF-1 treatment group. Mature metaphase II oocytes were collected from the oviducts 16 h after hCG injection a. The cot-test. The percentages of normal and degraded oocytes were analyzed by chi-square analysis. Data were reported as mean ± SD. p < 0.05 * was considered statistically significant. All statistical calculations were performed using SAS (version 9.0) software .Experimental data were obtained from three or more independent repeated experiments. Percentage data were arcsine transformed. Differences in follicular diameter, survival rate, maturation rate, degeneration rate, hormonal production, and the number of ovulated oocytes were analyzed by one-way ANOVA . The expression level of different genes was analyzed using Student’s p < 0.05) (p < 0.05) (p > 0.05)  d,e. Mean < 0.05) c. Howeve > 0.05) c.p < 0.05) while G50 was significantly decreased (p < 0.05) (p > 0.05) (p > 0.05) (p < 0.05)  f. Meanwh > 0.05) f. There  > 0.05) g. Howeve < 0.05) g.p < 0.05), and the level of 17β-estradiol was positively correlated with the dosage of IGF-1 (p < 0.05) (p > 0.05), but still significantly increased the amount of 17β-estradiol secretion compared with the G0 (p < 0.05) (p < 0.05) (p > 0.05) and was maintained until the end of the culture (p > 0.05) (p < 0.05) (p < 0.05) (p < 0.05) (The 17β-estradiol significantly increased from days 2 to 4 of culture a. On dayof IGF-1 a. On day < 0.05) a. Howeve < 0.05) a. Proges < 0.05) b. On day culture b. The pr > 0.05) b. Howeve < 0.05) b. AMH, s < 0.05) c. The G1 < 0.05) c. The am < 0.05) c.Star, Cyp19a1, and Hsd3b1) were increased with the development of follicles, and IGF-1 treatment significantly increased the expression level (p < 0.05) in all stages of in vitro culture of follicles  , Gdf-9      d. Gdf-9,at day 6 e–g. On d94-fold) e, Bmp-1586-fold) f, and Fg63-fold) g. The go < 0.05) h,i. On d.45-fold h, and by.49-fold i.Bcl2 increased with the development of follicles (Bcl2 in the treatment group (p < 0.05) (Bcl2 compared with the G0 at day 6 (p < 0.05)  (Bcl2/Bax ratio after 4 days of culture (p < 0.05) (Bcl2/Bax ratio at day 6 (p < 0.05)  (Caspase3 compared with the G0 at each stage (p < 0.05) (The expression of ollicles a. The re < 0.05) a. Additi < 0.05) a. Bax inelopment a. The ad < 0.05) a. Furthe < 0.05) b. Meanwh < 0.05) b. The ex < 0.05) a. This t < 0.05) a. Finall < 0.05) a.p > 0.05)  c. The oo > 0.05) b.(p < 0.05) (p < 0.05), and the IGF-1 treated group had a significantly decreased percentage of degraded oocytes .Mice in the control and IGF-1 treated groups exhibited significant differences. Both the number of ovulated oocytes and normal oocytes were significantly higher in the IGF-1 treated group than in the control group  < 0.05) a. The nu < 0.05) a. Figurep < 0.05) (p < 0.05)  f, while  < 0.05) a,c. Ther < 0.05) a,b. SimiAmh until the early follicle stages in mice [In hormone production, our results showed that IGF-1 had a promotional effect on follicular steroidogenesis. Estradiol synthesis is primarily regulated by gonadotropins and IGF-1 can positively or negatively alter the concentration of estradiol produced by promoting differentiation and proliferation of GCs . With th in mice . These oStar, Cyp19a1, and Hsd3b1) at each stage. These results indicated that the synthesis increased steroid hormones by upregulating the expression of genes involved in steroid synthesis. The expression of Star, Hsd3b1, and Cyp19a1 in GCs is regulated by FSH stimulation, while IGF-1 alone can increase the expression of Star, Hsd3b1, and Cyp19a1 [Amh increased in the early stage of culture and decreased in the late stage of culture. In research on rhesus macaques, the trend of Amh was similar to our result [Considering follicular development, oocyte maturation efficiency and hormone secretion level, the optimal concentration of IGF-1 in the 3D culture of mouse follicles was 10 ng/mL. With the proliferation of GCs, the steroid secretion activity in follicles increased during the culture period. IGF-1 significantly promoted the expression of the steroidogenesis-related genes  in mouse follicles from day 4 to day 6, cultured in vitro . We suggest that IGF-1 increased the function of GCs and steroidogenesis, and upregulated the expression of gonadotropin receptors. IGF-I mediates the effect of FSH on the ovary and improves follicular development.Previous reports indicated that IGF-1 can act synergistically with FSH to enhance follicular development . During The results of the current research have proven that designing modern and powerful approaches to the murine ex vivo 3D model of IGF-1-assisted growth and maturation of ovarian secondary preantral follicles in order to obtain satisfactory outcomes of IVM might be reliable and feasible for the purposes of achieving high efficiency rates in such novel ARTs as IVF/ICSI in humans and other mammalian species ,66,67,68