Mycobacterium tuberculosis (Mtb). An effective vaccine to prevent TB is considered the most cost-effective measure for controlling this disease. Many different vaccine antigen (Ag) candidates, including well-known and newly identified Ags, have been evaluated in clinical and preclinical studies. In this study, we took advantage of a plant system of protein expression using Nicotiana benthamiana to produce N-glycosylated antigen 85A (G-Ag85A), which is one of the most well-characterized vaccine Ag candidates in the field of TB vaccines, and compared its immunogenicity and vaccine efficacy with those of nonglycosylated Ag85A (NG-Ag85A) produced with an Escherichia coli system. Notably, G-Ag85A induced a more robust IFN-γ response than NG-Ag85A, which indicated that G-Ag85A is well recognized by the host immune system during Mtb infection. We subsequently compared the vaccine potential of G-Ag85A and NG-Ag85A by evaluating their immunological features and substantial protection efficacies. Interestingly, G-Ag85A yielded moderately enhanced long-term protective efficacy, as measured in terms of bacterial burden and lung inflammation. Strikingly, G-Ag85A-immunized mice showed a more balanced proportion of multifunctional Th1-biased immune responses with sustained IFN-γ response than did NG-Ag85A-immunized mice. Collectively, plant-derived G-Ag85A could induce protective and balanced Th1 responses and confer long-term protection against a hypervirulent Mtb Beijing strain infection, which indicated that plant-produced G-Ag85A might provide an excellent example for the production of an Mtb subunit vaccine Ag and could be an effective platform for the development of anti-TB vaccines.Tuberculosis (TB) is one of the deadliest infectious diseases worldwide and is caused by  Mycobacterium tuberculosis (Mtb), remains a major infectious threat with high morbidity and mortality worldwide [Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine is the only prophylactic vaccine used, but the insufficient pulmonary protection that BCG provides against TB means that the development of effective novel vaccines is urgently needed [Tuberculosis (TB), which is caused by orldwide , and as y needed . Variousy needed ,4. Thesey needed .+ T cell responses demonstrated promising results in clinical efficacy trials [Nicotiana benthamiana and easily purified in large quantities [In 2018, two multi-Ag subunit vaccines against TB that induce Ag-specific multifunctional CD4y trials ,7. Thesey trials ,7. Therey trials . Insect y trials . Meanwhiy trials . Second,y trials ,13. Thery trials , B-cell y trials , rabies y trials , and anty trials . For vacantities ,20, and antities ,22.+ and CD8+ T cell responses are significantly increased by increasing Ag uptake by dendritic cells in a manner dependent on the carbohydrate modifications of the ovalbumin (OVA) protein [Escherichia coli expression system. In this regard, plants have the advantage of being able to produce proteins with posttranslational modifications, such as N- or O-glycosylation.Beyond the selection of Ags, the posttranslational modifications of Ags, which mimic the authentic nature of Ags, have been investigated to develop more effective vaccines against many infectious diseases. One such approach involves investigating the relationship between Ag glycosylation and vaccine effectiveness ,24. Poly protein . These sThe Ag85 complex is a 30–32 kDa family of three closely related proteins  with enzymatic mycolyl-transferase activity that are involved in the biogenesis of cord factor and in the coupling of mycolic acids to arabinogalactan in cell walls . MembersN. benthamiana and compared the protective vaccine efficacy and immunogenicity of G-Ag85A with those of nonglycosylated recombinant Ag85A (NG-Ag85A). The immunogenicity and the potential protective efficacy of G-Ag85A and NG-Ag85A were assessed by directly comparing the responses to Mtb HN878 challenge in a mouse model following the delivery of these Ags as subunit vaccines.Despite the many advantages and possibilities of plant-based expression systems, there are not many substantial studies of the efficacy of TB vaccines produced with plant systems. Therefore, we focused on the benefits of the plant expression system for the development of new effective TB vaccines and used Ag85A as a prominent target of the Mtb vaccine. In the present study, we produced N-glycosylated Ag85A (G-Ag85A) in Specific-pathogen-free 6- to 8-week-old female wild-type C57BL/6 mice were purchased from SLC Inc.  and strictly maintained under barrier conditions in a BSL-3 facility at the Avison Biomedical Research Center at Yonsei College of Medicine. The experimental protocols used in this study were reviewed and approved by the Ethics Committee and Institutional Animal Care and Use Committee (Permit Number: 2017-0264) of the Laboratory Animal Research Center at Yonsei University College of Medicine . All animal experiments were performed according to the Korean Food and Drug Administration (KFDA) guidelines and regulations.A LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit was purchased from Molecular Probes . The following Abs were used for flow cytometry analyses: phycoerythrin (PE)-conjugated monoclonal antibody (mAb) against IFN-γ, violet 450-conjugated mAb against CD44, allophycocyanin (APC)-conjugated mAb against TNF-α, brilliant violet (BV) 605-conjugated mAb against Thy1.2, BV711-conjugated mAb against CD8, and PerCP-Cy5.5-conjugated mAb against CD4; these were purchased from BD Bioscience , and Alexa Fluor 700-conjugated mAb against CD62L and PE-Cy7-conjugated mAb against IL-2 were purchased from eBioscience . The CAF01 liposome adjuvant was kindly provided by the Statens Serum Institut .The Mtb HN878 strain was collected from the Korean Tuberculosis Research Institute , and BCG (Pasteur 1173P2) was obtained from the Pasteur Institute . The mycobacterial strains used in this study were cultured as previously described . The seeE. coli expression system as previously reported [N. benthamiana was used for Ag85A expression as described below.For the expression of NG-Ag85A, we expressed the protein using the reported . To prodN. benthamiana, a recombinant construct, 1300-HCH:Ag85A, was generated as described below. The first Ag85A coding sequence was codon-optimized for efficient expression in N. benthamiana and was chemically synthesized. A DNA fragment containing Ag85A together with an enterokinase cleavage site was amplified by polymerase chain reaction (PCR) using the primers Ag85A-F and Ag85A-R  and the XmaI restriction site. PCR amplifications were conducted using Ex Taq polymerase  in 25 cycles of denaturation at 94 °C (30 s), annealing at 53 °C (30 s), and extension at 72 °C . To generate the recombinant construct 1300-HCH:Ag85A, the two PCR fragments were then inserted in the order of CBM3 and Ag85A into the sequence downstream of BiP in the pCAMBIA 1300 binary vector containing the double enhancer-containing CaMV 35S promoter (d35S), BiP leader sequence, and HSP terminator using the restriction endonucleases BamHI with XmaI and XmaI with SacI, respectively.For the expression of Ag85A in  Ag85A-R . The ford CBM3-R . The for1300-HCH:Ag85A was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation using MicroPulser  according to the manufacturer’s protocol. The gene silencing suppressor p38 construct was cotransformed to enhance the expression of fusion protein by suppressing gene silencing [1300-HCH:Ag85A and p38, two Agrobacterium each harboring 1300-HCH:Ag85A and p38 were collected by centrifugation at 3500 g for 15 min and resuspended in infiltration buffer . Two Agrobacterium suspension were mixed at a 1:1 (v/v) ratio, and the mixture was adjusted to obtain an OD600 of 1.0. The 5- to 6-week-old N. benthamiana plants were subsequently transformed with Agrobacterium mixture by vacuum infiltration [For the expression of recombinant Ag85A, the expression vector ilencing . For theltration . The infg for 30 min at 4 °C, and the supernatant was loaded into a column filled with microcrystalline cellulose  at a flow rate of 100 μL/min. The column was washed with three column volumes of washing buffer  at a flow rate of 1 mL/min. The recombinant Ag85A fusion protein-bound cellulose beads were released from the column, precipitated by centrifugation at 2000× g and 4 °C for 10 min and resuspended in 10 mL of enterokinase reaction buffer . Enterokinase  was added to the recombinant Ag85A fusion protein-bound cellulose beads and incubated at 28 °C for 4 h. The soluble fraction containing G-Ag85A was collected after centrifugation at 2000× g and 4 °C for 10 min, and enterokinase was removed by affinity chromatography using STI-Sepharose. Finally, the flow-through fraction from the affinity chromatography step that contained G-Ag85A was separately collected. Purified plant-produced Ag85A was analyzed by 10% SDS-PAGE. In addition, protein samples were saved from each fraction, including the total fraction, and subjected to western blot analysis using anti-Ag85A Ab  to check the expression of fusion protein and efficiencies of the MCC bead binding and enterokinase-mediated cleavage reactions.The harvested leaves (20 g) were frozen in liquid nitrogen and ground into powder using a mortar and pestle. Total protein extracts were prepared using five volumes (w/v) of extraction buffer . The mixture was centrifuged at 20,000× Purified plant-produced Ag85A was treated with Endo-H  or PNGase F  according to the manufacturer’s recommended protocols. Ag85A (1 μg) was added to 1 μL of 10X glycoprotein denaturing buffer , and distilled water was added to the mixture to obtain a reaction volume of 10 μL. The mixture was then incubated in boiling water for 10 min for protein denaturation and briefly cooled on ice. Subsequently, 2 μL of 10X glycobuffer 3 , 1 μL of Endo-H, and 7 μL of distilled water were added, and the reaction mixture was incubated at 37 °C for 1 h. For PNGase F treatment, Ag85A (1 μg) was added to 1 μL of 10X glycoprotein denaturing buffer, and distilled water was added to the mixture to obtain a reaction volume of 10 μL. The Ag85A protein was denatured by incubation in boiling water for 10 min, and the sample was then chilled on ice. The reaction volume was increased to 20 μL through the addition of 2 μL of 10X glycobuffer 2 , 2 μL of 10% NP-40, 5 μL of distilled water, and 1 μL of PNGase F, and the resulting mixture was incubated at 37 °C for 1 h. Both samples were analyzed by western blotting using anti-Ag85A polyclonal Ab .5 CFUs of BCG Pasteur 1173P2. For the subunit vaccine immunization, mice were immunized three times at 2-week intervals via subcutaneous injection. Each immunization contained 2 μg of NG-Ag85A or G-Ag85A protein adjuvanted with CAF01 liposomes. The mice in the control group were immunized with only CAF01. Four weeks after the last immunization, spleen and lung cells were prepared and analyzed in terms of their immunogenicity (n = 4–6 mice/group). Six weeks after the final immunization, the adjuvant control group and the vaccinated groups  were infected with the Mtb Beijing strain HN878 at approximately 70 CFUs per mouse via aerosol . Specifically, the mice were exposed to the HN878 strain for 60 min in the inhalation chamber, which had been previously calibrated to deliver a predetermined dose . One day after infection, the infected mice were sacrificed to confirm the initial bacterial burden (n = 3). Four weeks and twelve weeks after challenge, mice were sacrificed to measure the efficacy of vaccination.C57BL/6J female mice were subcutaneously immunized once with 2 × 102 and 1 mM MgCl2) for 30 min at 37 °C. Splenocytes and lung cells were filtered via a 40 μm cell strainer  in RPMI medium supplemented with 2% fetal bovine serum  using a sterile 10-mL syringe. The erythrocytes were lysed using red blood cell lysis buffer  for 3 min at room temperature, and then single cells were washed twice with RPMI medium supplemented with 2% FBS.Single-cell suspensions from the lungs and spleen were prepared as follows. The spleens and lungs from mice in each group were minced into 2–4 mm pieces using scissors. The lung tissue was incubated in 3 mL of cellular dissociation buffer  containing 0.1% collagenase type IV  and 1 mM CaClSingle cells from the lungs and spleens of Mtb-infected or immunized mice were stimulated with Ag proteins (NG-Ag85A or G-Ag85A) for 12 h at 37 °C. The levels of secreted IFN-γ in the culture supernatant were measured using a commercial ELISA kit according to the manufacturer’s instructions .The levels of NG-Ag85A- or G-Ag85A-specific total immunoglobulin G (IgG), IgG1, and IgG2c in serum were evaluated as Ag-specific type 1 or type 2 immune responses. Briefly, 96-well plates were coated with 1 μg/mL NG- or G-Ag85A. ESAT-6 was used for negative control. After incubation of the diluted serum, horseradish peroxidase (HRP)-conjugated Ab against total IgG, IgG1  or IgG2c  was used as a secondary Ab. The optical densities (ODs) were determined at 450 nm.TM Fixable Dead Cell Kit was used for 30 min at 4 °C. The cells were then washed with PBS, fixed, and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 30 min at 4 °C. The permeabilized cells were washed twice with Perm/Wash (BD Biosciences) and stained with PE-conjugated anti-IFN-γ, APC-conjugated anti-TNF-α, and PE-Cy7-conjugated anti-IL-2 Abs for 30 min at 4 °C. The cells were washed twice with Perm/Wash and fixed with IC fixation buffer  for flow cytometry analysis.Individual lung and spleen cells were prepared from immunized and Mtb-infected mice and stimulated with 2.5 µg/mL NG- or G-Ag85A at 37 °C for 12 h in the presence of GolgiPlug and GolgiStop . First, the cells were washed with 1X PBS (pH 7.4), and the Fc receptor was blocked with an anti-CD16/32-blocking Ab at 4 °C for 15 min. Surface molecules were stained with fluorochrome-conjugated Abs against Thy1.2, CD4, CD8, CD44, and CD62L, and the LIVE/DEADThe protective efficacy of the various vaccination strategies was determined through analysis of the histopathology and bacterial growth in the lung and spleen. The mice from every vaccinated group were sacrificed at 4 and 12 weeks after Mtb infection to evaluate the protective efficacies of each vaccination. For the lung histopathology analysis, the right superior lobes were preserved overnight in 10% formalin and embedded in paraffin. The lung was sectioned at 4–5 μm, stained with H&E, and evaluated by light microscopy. For the bacterial growth analysis, the lung and spleen were homogenized, and serially diluted samples were plated onto Middlebrook 7H11 agar plates  supplemented with 10% OADC , 2 μg/mL 2-thiophenecarboxylic acid hydrazide , and amphotericin B (Sigma–Aldrich). After incubation at 37 °C for 3–4 weeks, the bacterial colonies were counted.t-test. One-way ANOVA followed by Tukey’s multiple comparison tests was used to analyze data from more than two groups. All the values are expressed as the means . Statistical significance was determined at * p < 0.05, ** p < 0.01 or *** p < 0.001.The statistical analyses were conducted using GraphPad Prism V5.0 . The differences between two groups were analyzed using an unpaired Student’s N. benthamiana as the host for the production of Ag85A in plants, and we compared the Ag85A produced in N. benthamiana with that expressed in E. coli. Accordingly, Ag85A was codon-optimized to N. benthamiana. Specifically, for the production of recombinant Ag85A in plants, we designed a recombinant Ag85A fusion construct, 1300-HCH:Ag85A  at position 203 of mature Ag85A and has been reported to be N-glycosylated when expressed in the ER of mammalian cells . To inveo Endo-H . Thus, w+ T cell and is important for protecting the host against Mtb [+ and CD8+ T cells compared with that induced by NG-Ag85A-stimulated BMDCs. These results indicated that Ag85A is well recognized by the host immune system at the early and late infection phases and that G-Ag85A induces different immunogenic properties compared with those induced by NG-Ag85A.To determine the potential of an Ag as a vaccine target, the IFN-γ response could be used as a marker for Ag recognition because IFN-γ is a key Th1 CD4inst Mtb . Thus, wTo evaluate the efficacy of G-Ag85A and NG-Ag85A, we vaccinated mice with BCG, G- or NG-Ag85A. CAF01 was used as an adjuvant for G- or NG-Ag85A vaccination. Four weeks after the final immunization, the CAF01-, BCG-, CAF01/NG-Ag85A-, and CAF01/G-Ag85A-immunized groups were sacrificed for analysis a. The lu+ and CD8+ T cells is associated with a protective immune response of the host at the time of Mtb infection [+ T cells is a crucial component of the protective response against Mtb infection [+ and CD8+ multifunctional T cells in the lungs and spleens that secrete IFN-γ, TNF-α, and IL-2. Live individual T cells were gated on CD4+ or CD8+, and triple-positive (IFN-γ+TNF-α+IL-2+), double-positive , and single-positive  T cells were analyzed among the CD62LloCD44hi T cells  staining of lung sections from all groups were analyzed a. Four w+ T cells and TNF-α+IL-2+ double-positive cytokine-secreting CD4+ T cells in the lung  and 4 times as many IFN-γ+ CD4+ T cells  as the CAF01/G-Ag85A-immunized group. However, the percentages of every combination of cytokine-secreting CD4+ T cells were balanced in the CAF01/G-Ag85A-immunized group but not in the CAF01/NG-Ag85A-immunized group, and the overall frequency of triple-positive and double-positive CD4+ T cells was also higher in the CAF01/G-Ag85A-immunized group than in the CAF01/NG-Ag85A-immunized group  and a higher overall portion of polyfunctional T cells than did the NG-Ag85A-immunized group . Because E. coli-produced Ag85A had no modifications, such as glycosylation or lipidation, plant-produced G-Ag85A might induce immune responses that are different from those induced by E. coli-produced Ag85A [During the production of Mtb Ags in plants, one of the most crucial steps is an easy and efficient purification, unless the aim is the oral administration of plants expressing Ags. In fact, in most cases of recombinant protein production, the purification process is the rate-limiting step. Thus, the development of a cost-effective purification system is important. For easy purification, two important components are an affinity tag and a low-cost matrix for the binding of the tag. CBM3 is a cellulose-binding domain of  for MCC ,47. In t for MCC . The 35- for MCC ; however for MCC . These d for MCC . Althouged Ag85A .N. benthamiana induced a greater Ag-specific IFN-γ response in the lung cells of infected mice than that induced by NG-Ag85A expressed in E. coli  following immunization with G-Ag85A or NG-Ag85A. Consequently, the percentage of polyfunctional CD4+ T cells that secreted more than one cytokine was higher and more balanced in the G-Ag85A-immunized group than in the NG-Ag85A-immunized group at 4 weeks postinfection  rather than earlier time points. In addition, the CD8+ T cell response induced by G-Ag85A immunization at 12 weeks postinfection involved a higher multifunctional CD8+ T cell response than that induced by NG-Ag85A immunization, which correlated with reductions in the bacterial load and lung inflammation. Notably, the synergy between CD4+ and CD8+ T cells suggests that a vaccine that induces both T cell subsets has the best opportunity to prevent TB. Recently, Moguche et al. demonstrated differential responses of CD4+ T cells to two leading TB vaccine Ags, Ag85B and ESAT-6, in a mouse model [+ T cells with a substantial proportion of KLRG1+ cells restricts the protective ability of CD4+ T cells to recognize ESAT-6, whereas Ag85B-specific T cells exhibit a limited ability to control Mtb infection by reducing Ag expression during persistent Mtb infection. These results suggest that different vaccination strategies will be required to achieve optimal protection mediated by the recognition of Ags expressed at distinct stages of Mtb infection by T cells. Similar to Ag85B, Ag85A is exclusively expressed during the early infection phase, but its expression is reduced during the late phase of Mtb infection. Interestingly, the maintenance of multifunctional Th1 T cell responses and the switching of CD4+ T cells to CD8+ T cells is associated with the long-term protective efficacy of G-Ag85A immunization in mice. Although we are unable to define how this switching occurred in the current study, our data have important implications for the rational design of TB vaccines tailored to optimize the protection conferred by specific CD4+ T cells that recognize Ags expressed at distinct stages of Mtb infection.In our study, the effects of G-Ag85A and NG-Ag85A immunization did not exhibit any differences at 4 weeks postinfection, but G-Ag85A immunization, unlike NG-Ag85A immunization, exhibited protective effects in the lung and spleen at 12 weeks postinfection . Howeverse model . They reCompared with NG-Ag85A, G-Ag85A induced a strong IFN-γ response in the lung cells of Mtb-challenged mice, and G-Ag85A-matured BMDCs resulted in enhanced T cell proliferation with IFN-γ secretion compared to NG-Ag85A-matured BMDCs. In addition, vaccination with G-Ag85A induced robust, durable, Ag-specific, and balanced Th1 T cell responses, and ultimately conferred enhanced protection against Mtb HN878. However, the association between Ag glycosylation and the immunological responses induced by G-Ag85A and NG-Ag85A has not yet been clearly established. Therefore, further investigations with glycosylation of other Mtb Ags are necessary to confirm this relationship. Collectively, our results suggest that post-translationally modified Ag85A, which might mimic the native form of Ag85A produced in a plant system, has high potential as a vaccine Ag by generating balanced Th1 T cell responses with multifunctional capacities. In addition, vaccination through the combination of G-Ag85A with other Ags or its application as a BCG booster could be an effective strategy to increase vaccine efficacy, which might provide new opportunities for the future development of anti-TB vaccines.


BIM on apoptosis of GC were investigated. Finally, the pathways of two phosphorylation sites were investigated by kinase inhibitors. The results revealed that p-BimEL-S65 levels were higher in GC from H than SA and A, whereas p-BimEL-T112 was reversed. The prosurvival factors like FSH and IGF-1 upregulated the level of p-BimEL-S65, while the proapoptotic factor, heat stress, increased the level of p-BimEL-T112 in cultured GC. Compared with the overexpression of wild BimEL, the apoptotic rate of the GC overexpressed BimEL-S65A (replace Ser65 with Ala) mutant was significantly higher, but the apoptotic rate of the cells overexpressing BimEL-T112A did not differ. In addition, inhibition of the ERK1/2 or JNK pathway by specific inhibitors reduced the levels of p-BimEL-S65 and p-BimEL-T112. In conclusion, the levels of p-BimEL-S65 and p-BimEL-T112 were reversed during follicular atresia. Prosurvival factors promote p-BimEL-S65 levels via ERK1/2 to inhibit GC apoptosis, whereas proapoptotic factor upregulates the level of p-BimEL-T112 via JNK to induce GC apoptosis.BimEL protein is involved in follicular atresia by regulating granulosa cell apoptosis, but the dynamic changes of BimEL phosphorylation during follicular atresia are poorly understood. The aim of this study was to explore the changes of key BimEL phosphorylation sites and their upstream regulatory pathways. First, the levels of BimEL-Ser65 and BimEL-Thr112 phosphorylation  in granulosa cells (GC) from healthy (H), slightly-atretic (SA), and atretic (A) follicles and in cultured GC after different treatments were detected by Western blotting. Next, the effects of the corresponding site mutations of  DKK3, GADD45A, CAMTA2, CCDC80, DAPK2, ECSIT, MSMB, NUPR1, RUNX2, SAMD4A, and ZNF628 having a fold-change higher than five between healthy and atretic follicle granulosa cells could likely serve as markers of pig follicular atresia [Although follicle development is an ongoing process, atresia prevents follicles from ovulating to affect reproductive activity. In mammals, more than 99% of developing follicles undergo atresia ,2,3. It  atresia . The letThe Bcl-2 protein family plays irreplaceable roles during apoptosis, and one of the most important proteins is the BH3-only protein, Bim. Bim binds with high affinity to antiapoptotic Bcl-2 family members and regulates apoptotic signaling through Bax and Bak . The genDuring the process of follicular atresia, the level of BimEL protein in porcine granulosa cells is elevated , but theThe ovaries from gilts aged about 5 months old were collected at a local abattoir and transported to the laboratory in a vacuum flask (30–35 °C) containing sterile physiological saline within 2 h. Ovaries were washed twice with sterile physiological saline 37 °C) containing 100 IU/L penicillin and 50 mg/L streptomycin. Healthy, slightly atretic, and atretic follicles were classified according to previously established morphological criteria  °C conta,23,24. B2 for 24 h. The cells were passaged upon reaching confluence. Granulosa cells from about 10 ovaries can be selected and cultured in 10 dishes with a diameter of 35 mm as primary granulosa cells, and the same amounts of cells cultured in 4 dishes with a diameter of 100 mm as adherent granulosa cells.Porcine granulosa cells were cultured as previously described ,13,23. B2 for 12 h. Both levels of p-BimEL S65 and p-BimEL T112 were compared among different treatments. Experiment III: The effects of phosphorylation at Ser65 and Thr112 were observed during apoptosis in porcine granulosa cells. BimEL and BimEL mutation vectors were constructed and transfected into porcine granulosa cells. The levels of recombinant Bim proteins and morphological changes of granulosa cells were compared among the cells transfected by different vectors. The apoptotic rates were examined by flow cytometry. Experiment IV: The potential pathways were suggested to induce the levels of p-BimEL S65 and p-BimEL T112 by the prosurvival or proapoptotic factors.The PCR-amplified full-length porcine BimEL cDNA was constructed into pEGFP-N1 (Clontech). The reading frame of BimEL cDNA was connected with enhanced GFP to obtain a recombinant pEGFP-N1-BimEL plasmid. The plasmid was propagated using Trans1-T1  as the host strain and purified with a Plasmid MiniPrep Kit , followed by sequencing to confirm the open reading frame. Mutations of BimEL phosphorylation sites were performed by a Fast Mutagenesis System , and primers for mutations were designed according to the manufacturer’s instructions. Primers used in this experiment are shown in TM Kit for Primary Mammalian Fibroblasts  were used for plasmid transfection. The transfection protocol followed the manufacturer’s instruction. Briefly, monolayer adherent granulosa cells were harvested after reaching confluence and divided into 6 equal parts (about 2 × 106 cells each) named as CN (with no plasmid but treated the same with other groups), vector (pEGFP-N1 transfection), BimEL (pEGFP-N1-BimEL transfection), BimEL-S65A (pEGFP-N1-BimEL-S65A transfection), BimEL-T112A (pEGFP-N1-BimEL-T112A transfection), and BimEL-S55A65A73A (pEGFP-N1-BimEL-S55A65A73A transfection). A total of 100 µL Nucleofector™ solution and 1.5 µg plasmid DNA were added to each group except for the CN; then, the cuvette with cell/DNA suspension was inserted into the Nucleofector™ Cuvette Holder to carry out the Nucleofector™ Program V-024 individually. After that, a total of 600 µL of DMEM media were added into each group, and the samples were transferred immediately into six-well plate . Finally, the cells were cultured for 24 h or 48 h in humid air with 5% CO2 at 37 °C for further analysis.NucleofectorTM 2b Device  and Basic NucleofectorThe granulosa cells were first transfected with BimEL, BimEL-S65A, BimEL-T112A, and BimEL-S55A65A73A vectors and cultured in cell slides with or without QVD-OPH  treatments, respectively. After that, the cells were fixed with 4% paraformaldehyde, and the nuclei were stained with Hoechst 33342. The samples were analyzed for morphological changes in cells and nuclei by a laser confocal scanning microscope .v/v) protease inhibitor Cocktail (HY-K0010) and 1% (v/v) phosphatase inhibitors  purchased from MCE . Western blotting was performed as described previously [The cells were harvested and washed once in PBS, then lysed on ice for 30 min with RIPA buffer  and supplemented with 1% (eviously . ProteinPorcine ovaries (2–5 mm in diameter) were fixed overnight in 4% phosphate-buffered formaldehyde at 4 °C for 7 days and then embedded in paraffin. Randomly selected sections (5 μm each) were used for subsequent Hematoxylin–eosin, TUNEL, and immunohistochemical analyses. Hematoxylin and eosin were purchased from Solarbio .2O2 in PBS for 10 min (to block endogenous peroxidase). They were then incubated in a humidified chamber with equilibration buffer for 5 min and finally incubated with terminal deoxynucleotidyl transferase for 1 h at 37 °C. Negative control slides were incubated as described above, without the final addition of terminal deoxynucleotidyl transferase. After TUNEL reactions were complete, slides were washed in PBS, sealed under coverslips with nail varnish and examined under fluorescence microscopy.Apoptosis was analyzed by TUNEL  according to the manufacturer’s protocol. Briefly, after deparaffinization and rehydration, sections were incubated with proteinase-K (20 g/mL) for 15 min at room temperature and quenched in 3% HImmunohistochemical analysis was performed as previously described . BrieflyQVD-OPH (20 μM) was added immediately after transfection. After 24 h, QVD-OPH was withdrawn by changing the culture media, and another 24 h later, the cells were harvested and stained with PE-conjugated Annexin V and 7-AAD in a binding buffer for 15 min as described previously ,27. GFP-p < 0.05.All data are presented as the mean ± SD and were analyzed using a one-way ANOVA with SPSS 22  for Windows. The ANOVA followed by a post hoc Dunnett’s test was used to determine significant differences between different groups. Differences were considered statistically significant at To confirm the follicular status identified by the morphology criteria, HE staining was performed in healthy, slightly-atretic, and atretic follicles. The results showed that all granulosa cells tightly lined the basement membrane in healthy follicles, but cells began to diffuse away from the basement membrane in slightly atretic follicles. More seriously, the cells drifted to the follicular antrum in the atretic follicles A. FurtheProapoptotic factors like heat stress in summer may induce granulosa cell apoptosis and cause follicular atresia, while prosurvival factors like FSH and IGF-1 can promote granulosa cell survival and proliferation. Therefore, the effects of these factors on the levels of p-BimEL S65 and p-BimEL T112 in granulosa cells were investigated. Porcine primary granulosa cells were cultured in vitro and treated with proapoptotic (heat stress) or survival factors , and the levels of p-BimEL S65, p-BimEL T112, and other related proteins were detected A. The reA65A73A were transfected into porcine granulosa cells with about 30% efficiency. The results demonstrated that BimEL-GFP and BimL-GFP in the four transfections were all expressed, while endogenous Bim expression levels did not change compared with the pEGFP-N1 vector and transfection control (CN)  A and B-1rol (CN) B-2.A65A73A plasmids compared to those in the pEGFP-N1 vector. Moreover, the positive cells were almost wrinkled with fragmented or shrunk nuclei  to regulate its activity . Previounchioles . Exposurnchioles . Herein,Based on our results, a hypothetical model of BimEL phosphorylation participating in animal follicular atresia is presented in


Introduction: Severe Combined Immunodeficiency (SCID) is a life-threatening immunodeficiency caused by several pathogenic genetic variants, and it is characterized by profound defects in T-cell numbers and immune function. First performed in the late 1960's, hematopoietic stem cell transplantation remains the standard treatment for most cases of SCID. There is a growing number of post-transplant SCID patients, and it is imperative to assess the long-term outcomes of these patients. We have reported here the longest follow-up of a post-transplant SCID patient who, to our knowledge, bears the first gamma chain (γc) variant to show intact IL-21 signaling.Case Presentation: The patient presented at 5 months of age with recurrent thrush and Pneumocystis jiroveci pneumonia. In 1971, at the age of 11 months, he received an unconditioned, matched, related donor transplant comprising whole, unprocessed bone marrow. He is now 48 years old without significant illness and has never required immunoglobulin replacement. He exhibits T-dependent vaccine responses. He does suffer from chronic warts and bacterial infections that have worsened in recent years. We confirmed a known pathogenic variant in the IL2RG gene showing a hemizygous variant NM_000206.2:c.675C>A, resulting in p.Ser225Arg. His chimerism studies revealed donor T cells, host B cells, host myeloid cells, and mixed NK cells. Lymphocyte enumeration revealed normal numbers and distribution of B cells. The host B cells carry the pathogenic variant in IL2RG, but, when stimulated with IL-21, they demonstrated intact, γc-dependent signaling.Conclusions: Even with host B cells, reconstitution with donor T cells can be sufficient to allow over four decades of survival when B-cell function is intact. Our case demonstrates that satisfactory B-cell function can arise as a consequence of both intact IL-21 signaling due to a hypomorphic γc variant, and close HLA matching with the donor to allow for effective T-cell help. Severe Combined Immunodeficiency (SCID) is a collection of life-threatening diseases characterized by an absence of functional T cells and requiring treatment with hematopoietic stem cell transplantation (HSCT), gene therapy, or enzyme replacement therapy. At least 70 SCID patients born yearly in the United States and Canada undergo definitive treatment , 2. OverIL2RG) gene variants causing X-linked SCID (X-SCID) (The most common genetic defects resulting in SCID are IL-2 receptor gamma ((X-SCID) . B cells(X-SCID) , 5.The survival rate of patients with SCID who receive HLA-identical transplants is ~90% , 6. LongPneumocystis jiroveci pneumonia. He later developed Candida esophagitis and failure to thrive. The patient received an unconditioned transplant at 11 months of age in 1971 that comprised whole, unprocessed bone marrow  replacement or nutritional support. He had recurrent dental infections, and one skin carcinoma had been removed. He developed cutaneous, oropharyngeal, and genital warts that relapsed and remitted but had worsened in recent years . In the IL2RG gene was sequenced from patient's buccal mucosal cells, and this revealed a hemizygous variant on Chromosome X at location g.70329160 (based on build GRCh37/hg19), NM_000206.2:c.675C>A, resulting in p.Ser225Arg. Cases of X-SCID have been reported due to missense variants of nearby amino acids  (−B+NK− SCID phenotype (The d L230P) . This vad L230P)  and has henotype . Thus, w+ and CD8+ T cells .Lymphocyte enumeration showed normal numbers of T and B cells but low numbers of NK cells. A predominantly memory phenotype was seen for both CD4 T cells . TRECs w+ , 22.5% of his T cells were Vβ1+ , and 5.7% of his T cells were Vβ16+ , while proportions of 15 other Vβ regions were lower than normal. Thus, while T-cell numbers were normal, the patient's T cells had a diminished repertoire.The diversity of the patients' T cells was examined by assessing the frequency of usage of 24 TCR Vβ regions in T cells by flow cytometry. We found 31.1% of his T cells were Vβ3In vitro assays revealed that NK-cell-dependent cytotoxicity was impaired in the patient. We examined killing after normalizing for numbers of NK cells and offered a range of erythroblastoid target cell concentrations, but killing was <2% and was not accentuated by pre-incubation with the γc cytokines IL-2 or IL-15. This result suggested that his NK cells were poorly functional.IL2RG, while 100% of the monocytes and B cells showed the pathogenic variant of IL2RG. Thus, a somatic reversion mutation was not responsible for the resiliency of the patients' B cells nor were any of the B cells derived from the donor.HLA typing was performed by sequencing DNA isolates from the patient's buccal mucosal cells, confirming a 12-out-of-12 match with the donor. To assess engraftment and the origins of specific hematopoietic lineages, we performed chimerism studies on sorted cells by short-tandem repeats. As expected, myeloid cells were entirely from the recipient, and T cells were entirely from the donor. However, B cells were 93% from the recipient and 7% from the donor, and the degree of uncertainty in this test did not preclude the possibility that 100% of the B cells are from the recipient. NK cells were 69% from recipient. To further understand the origins of these cells, we rigorously purified T cells, B cells, and monocytes by flow sorting and analyzed each population with whole exome sequencing. None of the T cells showed rare variants in Streptococcus pneumoniae, polio, or HiB. He had absent titers to EBV and HSV-2. Boosting with polysaccharide pneumococcal vaccine resulted in poor responses  AF647 (clone 4/P-Stat3), anti-pStat6 (Tyr641) AF647 (clone 18/P-Stat6), anti-pStat5 (Tyr694) AF488 [clone 47/Stat5(pY694)], and Mouse Isotype Control AF488 or AF647 (clone MOPC-173) from BD Biosciences; anti-CD4 AF488 or BV421 (clone OKT4), anti-CD14 BV421 (clone HCD14), anti-CD19 BV421 (clone HIB19), and Human TruStain FcX from Biolegend. All cytokines were from Peprotech: IL-2 (Cat# 200-02), IL-4 (Cat# 200-04), and IL-21 (Cat# 200-21).Stimulation: 20 μL of 10X cytokines in PBS (or PBS only control) were added to 180 μL of heparinized whole blood in a 5 mL FACS tube to achieve these final concentrations: IL-2 (10 ng/mL), IL-4 (100 ng/mL), or IL-21 (50 ng/mL). Tubes were incubated at 37°C for 20 min, at which point 4 mL of pre-warmed 1X BD Lyse/Fix buffer (Cat# 558049) was added. Cells were fixed for 10 min at 37 °C, centrifuged and washed twice with FACS buffer (1X PBS supplemented with 2% FBS and 1 mM EDTA). Staining and permeabilization: Fc receptors were blocked for 5 min at RT, followed by a 20 min stain on ice with anti-CD4 and anti-CD19. Cells were washed with FACS buffer and permeabilized for 30 min on ice with 1 mL BD Phosflow Perm Buffer III (Cat# 558050) that had been pre-cooled to −20°C. After permeabilization, 2 mL FACS buffer was added and the samples were centrifuged. After three additional washes, the cells were stained with anti-pStat antibodies or an isotype control for 30 min at RT. Samples were washed three times and data were collected on a Cytek DxP10 flow cytometer. Data were analyzed with FlowJo software.All data for this paper are available in this article/supplementary materials.The studies involving human participants were reviewed and approved by UCLA Office of the Human Research Protection Program. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CD, MG-L, and MB provided care. CD wrote the manuscript. All authors edited the manuscript. MB and ES designed the experiments. TT performed the experiments.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.


Lactococcus lactis MG1363 FnBPA+ strains carrying plasmids encoding IL-4 and IL-10 in the streptozotocin- (STZ-) induced diabetes model and in nonobese diabetic (NOD) mice. The STZ-induced mice that were treated with combined bacterial strains carrying plasmids encoding IL-4 and IL-10 showed lower incidence of diabetes and more preserved pancreatic islets than the mice that received the individual bacterial strains. Combined administration of L. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  resulted in protection against diabetes in NOD mice. It was shown that the combined treatment with recombinant bacterial by oral route prevented hyperglycemia and reduced the pancreatic islets-destruction in NOD mice. In addition, increased levels of IL-4 and IL-10 in serum and pancreatic tissue revealed a systemic effect of the treatment and also favored an anti-inflammatory microenvironment. Reduced concentrations of IL-12 in pancreas were essential to the regulation of inflammation, resulting in no incidence of diabetes in treated NOD mice. Normal levels of intestinal sIgA after long-term treatment with the L. lactis strains carrying plasmids encoding IL-4 and IL-10 indicate the development of oral tolerance and corroborate the use of this potent tool of mucosal delivery. For the first time, L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10 are tested in STZ-induced and NOD mouse models. Therefore, our study demonstrates this innovative strategy provides immunomodulatory potential for further investigations in T1D and other autoimmune diseases.Type 1 diabetes (T1D) is an autoimmune disease that culminates in beta cell destruction in the pancreas and, subsequently, deficiency in insulin production. Cytokines play a crucial role in the development of diabetes, orchestrating the recruitment and action of immune cells, to not only destroy insulin-producing cells but also preserve them. Therefore, the aim of this study was to investigate the effect of orally administered  Trafficking of autoreactive T lymphocytes to the pancreatic islets and the resulting inflammatory environment culminates in disease establishment in at-risk individuals [Type 1 diabetes (T1D) is a chronic immune-mediated disease characterized by destruction of ividuals . After dTo study the pathogenesis and immunophysiology of T1D, some murine models have been developed over the years, each one sharing different mechanisms of the human disease. Chemical induction of T1D through multiple doses of streptozotocin (STZ) is an immune-mediated T1D model discovered by Like and Rossini –5. The SCytokines play a key role in connecting the risk factors that contribute to T1D development and trigger the destructive action of T cells in pancreatic islets , 15. In Lactococcus lactis as the vehicle for mucosal delivery of plasmids encoding IL-4 and IL-10. This lactic acid bacteria also have been handled as a genetic tool due to some advantages, as do not colonize the tract gastrointestinal, present safe use in humans, and be able to induce tolerance successfully [L. lactis bacteria have been engineered to express a variety of molecules, including an invasin called fibronectin-binding protein A (FnBPA) (L. lactis MG1363 FnBPA+ strain) to more efficiently deliver the eukaryotic expression plasmids encoding IL-4 and IL-10 [L. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  allows an increased rate of internalization and plasmid transference to eukaryotic cells, which will be able to express IL-4 and IL-10. Systematic administration of these cytokines was shown to be a successful approach in studies involving T1D [The recurrent problem of biocompatibility and an undesirable immune response during the delivery process encouraged us to use essfully , 22. L. nd IL-10 , 24. Thuving T1D –27. Moreving T1D , 29. ThuL. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  in the two most extensively used animal models of T1D, the STZ-induced model, and NOD mice. The combined treatment of these bacterial strains for the first time is an interesting approach, since individual administration has already been shown to be able to reduce inflammation in intestinal inflammation models by our research group [L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression plasmids encoding IL-4 and IL-10 could result in beneficial effects on T1D.In the present study, we examined the effect of oral administration of ch group –32. TherL. lactis MG1363 FnBPA+, L. lactis MG1363 FnBPA+ , and L. lactis MG1363 FnBPA+  were grown in M17 medium (Sigma-Aldrich) supplemented with 0.5% glucose with or without chloramphenicol (10 μg/mL) (Sigma-Aldrich) and erythromycin (5 μg/mL) (Sigma-Aldrich) at 30°C.The strains used in this work are listed in Male C57BL/6 mice, aged 8-9 weeks, were obtained from Biotério Central of Federal University of Minas Gerais . Female NOD mice were kindly donated by Dr. Ana Maria Caetano de Faria from UFMG. The animals were kept in microisolators on ventilated shelves with air filtration under a 12 h light-dark cycle with free access to standard mouse chow and water. All animal procedures were approved by the Ethics Committee on Animal Use  of UFMG/Brazil.μL of the corresponding L. lactis strain at a final dose of 1 × 109 colony forming units (CFU) in 0.9% saline.C57BL/6 mice were subjected to a 4 h fast and were then injected intraperitoneally (i.p.) with 50 mg/kg/day STZ (Sigma-Aldrich) for 5 consecutive days (days 0-4) . STZ wasμL of bacterial suspension at a final dose of 1 × 109 CFU in 0.9% saline 5 times/week for 15 consecutive weeks , L. lactis MG1363 FnBPA+  (F-IL-4-STZ group), L. lactis MG1363 FnBPA+  (F-IL-10-STZ group), L. lactis MG1363 FnBPA+ , and L. lactis MG1363 FnBPA+  (F-IL-4/IL-10-STZ group).C57BL/6 mice were allocated into groups that received 0.9% saline i.g., and by intraperitoneal route, the animals received 0.9% saline  or sodium citrate buffer (vehicle control (VC) group) and groups that received the STZ injections i.p. and, by gavage, administration of 0.9% saline , L. lactis MG1363 FnBPA+ , and L. lactis MG1363 FnBPA+  (F-IL-4/IL-10-NOD group).NOD mice were distributed into two groups, according to the constitution of dose i.g.: 0.9% saline  and μL Tween 20; 5 g BSA; 34 mg PMSF; 1 mL DMSO; 44.6 mg BC; 372 mg Na2EDTA; and 40 μL aprotinin (10 mg/mL) (Sigma-Aldrich); PBS 1× q.s.p. 1 L) per 100 mg of tissue. Then, the samples were centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant was stored at -80°C until use.The blood, the pancreas, and the colon were harvested immediately after euthanasia. Blood samples were incubated at 37°C for 40 minutes and then centrifuged at 5,000 rpm for 10 minutes at 4°C. The serum was collected and stored at -20°C until use. Pancreatic and colon tissues were homogenized in 1 mL buffer  in pancreatic and colon homogenates were determined using BD OptEIA (BD) and R&D DuoSet (R&D Systems) ELISA kits, according to the manufacturers' instructions.The concentrations of IL-4 and IL-10 were measured in serum, and the levels of IL-2, IL-4, IL-10, IL-12, and transforming growth factor α chain specific) horseradish peroxidase (HRP) conjugate (Southern Biotech), as recommended by the manufacturer.The concentration of secretory immunoglobulin A (sIgA) in intestinal lavage fluid was measured by capture ELISA using goat anti-mouse Ig, human ads-UNLB (Southern Biotech), and goat anti-mouse IgA  and L. lactis MG1363 FnBPA+ ) at a final dose of 1 × 109 CFU in 0.9% saline by oral gavage. The spleen, liver, and mesenteric lymph nodes (MLNs) were collected under strict aseptic conditions 2 h, 4 h, 6 h, 12 h, and 24 h after bacterial administration. The tissues were macerated in 500 μL of 0.9% saline, and 100 μL of this homogenate was plated in M17 medium (Sigma-Aldrich) supplemented with agar, 0.5% glucose, chloramphenicol (10 μg/mL) (Sigma-Aldrich), and erythromycin (5 μg/mL) (Sigma-Aldrich). The plates were incubated at 30°C for 24 h.C57BL/6 mice received 100 μm sections were stained with hematoxylin and eosin (HE), and at least 15 islets per pancreatic sample were evaluated and scored for infiltration progression (by an independent investigator in blinded fashion), as follows: no insulitis: no visible signs of infiltration; peri-insulitis: few infiltrating elements with patchy distribution primarily at the periphery of the islet; mild insulitis: mild infiltration (peri- and intraislet) with less than 50% of the islet infiltrated by the immune elements; and severe insulitis: extensive inflammation with more than 50% of the islet infiltrated.After euthanasia, the pancreas was removed, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. Five-χ2 test was used. p values less than 0.05 (p < 0.05) were considered statistically significant.The GraphPad Prism software, version 5.0 (GraphPad), was used for statistical analysis, and the results are presented as the mean ± standard deviation (SD). One-way ANOVA followed by Tukey's post hoc test was used for the analysis of variance. For diabetes incidence, the Glycemic levels were analyzed, and oral bacterial treatment of STZ-induced groups  did not alter the increases in glycemic levels at any time after T1D induction; blood glucose levels in these groups were similar to that in the saline-STZ group . A progrL. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  (F-IL-4/IL-10-NOD group) showed normoglycemia (glycemic levels less than 200 mg/dL) until the end of the experimental period  and L. lactis MG1363 FnBPA+  exhibited significantly higher serum production of IL-4  and L. lactis MG1363 FnBPA+  than in the saline-NOD group  in the colon homogenates from STZ-induced and NOD mouse models were measured and did not show significant differences among the groups (data not shown). However, there was decreased production of sIgA in all C57BL/6 mice that received bacterial strains  in comparison to that of groups that received only oral saline (negative and VC groups)  . InteresL. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+ ) was not detected in the spleen (data not shown) at 24 h after gavage. However, we observed a minute quantity of bacterial translocation in the liver at 2 h and 4 h after oral administration of the bacteria  and L. lactis MG1363 FnBPA+  in MLNs, primarily at 6 h after oral gavage  and L. lactis MG1363 FnBPA+ ; (F-IL-4/IL-10-STZ group)) in STZ-induced mouse model preserved more pancreatic islets without visible signs of inflammation than when these strains were administered individually (F-IL-4-STZ and F-IL-10-STZ groups)  . As showβ cells are destroyed by autoreactive T cells. After diagnosis, patients have to adapt their lives in an attempt to reach wellness mainly because the available treatment, insulin therapy, is laborious and requires constant care and attention. Therefore, more studies investigating T1D development and improved treatments are absolutely needed.T1D is a chronic condition in which pancreatic L. lactis, since it is considered an ideal tool to mucosal delivery and oral tolerance induction [L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10  to treat the two most extensively used animal models of T1D is reported here for the first time.Early studies using multiple doses of STZ and NOD mice have shown that IL-4 and IL-10 have the potential to change the natural course of T1D by impairing insulitis and maintaining normal levels of glycemia , 34, 35.nduction . Oral adIn the STZ-induced T1D model, we decided to verify the effects of the bacterial strains carrying the plasmids encoding IL-4 and IL-10 individually and combined. Despite all groups that received STZ injections became diabetic, it was noteworthy that the F-IL-4/IL-10-STZ group presented the highest number of mice protected against diabetes incidence.Moreover, STZ-injected mice orally administered both bacterial strains encoding IL-4 and IL-10 showed pancreatic IL-2 levels similar to those of the negative and vehicle control groups and were significantly higher than the saline-STZ group. Previous studies reported that IL-2 plays a key role in the balance between immunoregulation and immunosuppression, since this cytokine is able to expand and activate regulatory T cells (Tregs), which subsequently provide active suppression of the effector response , 37. ParL. lactis by oral route [The evaluation of sIgA levels is important because it is already well established that this antibody exhibits immunomodulatory properties and is involved in not only responding to pathogens but also maintaining homeostasis between the gastrointestinal tract and commensal and ingested microorganisms –42. In aal route .L. lactis in the STZ-induced T1D model, these results were important and led to the investigation of the effects of the combined administration of L. lactis MG1363 FnBPA+ strains carrying the IL-4 and IL-10-coding plasmids in the NOD mouse model.Corroborating the others analysis, histological findings confirmed that combined treatment with the bacterial strains encoding IL-4 and IL-10 was more successful in attenuate insulitis progression than the individual administration of these bacterial strains. Results indicating more pancreatic islets with no signs of inflammatory infiltrates suggest a beneficial effect resulting from the combined bacterial strains. Some studies have shown that IL-4 and IL-10 induce a synergistic effect to suppress cellular immunity , 45. AltL. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  are able to regulate glycemia levels effectively throughout the observational period. Therefore, the oral treatment with these genetically modified bacteria during 15 weeks resulted in the absence of T1D incidence in NOD mice.It is commonly known that the high and uncontrolled levels of glycemia are associated with a rapid progression of disease and more severe complications in patients with T1D. Using the NOD mouse model, we demonstrated that oral administered L. lactis MG1363 FnBPA+ encoding IL-4 and IL-10 by oral route presented significantly increased levels of IL-10 in serum and pancreatic tissue. IL-10 is a major anti-inflammatory cytokine that can modulate immune processes, inhibiting the synthesis of proinflammatory cytokines and favoring a tolerant status [L. lactis-based therapy in the NOD mouse model [L. lactis encoding IL-4 and IL-10 was at least initially due to the bacteria administration and subsequent expression of these cytokines. We suggest this result was crucial for regulation of inflammation observed, probably through Treg cells, and consequent control of glycemia and no incidence of T1D.In the present study, we demonstrated that oral administration of the bacterial strains led to systemic effects. NOD mice treated with t status . IL-10 ht status . IL-10 ct status . Moreovese model . T1D is L. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  showed promising results indicating increased levels of IL-4 in serum and pancreas of NOD mice, further corroborating the beneficial role of the strategy tested here for the prevention of T1D.IL-4 is a pleiotropic immunomodulatory cytokine produced and secreted primarily by Th2 cells. Approaches favoring Th2 activity and administration of cytokines that downregulate Th1 activities, such as IL-4 and IL-10, have shown success in suppress insulitis and prevent diabetes , 18, 26.Additionally, both IL-4 and IL-10 appear to suppress the production of the proinflammatory cytokine IL-12 by antigen-presenting cells (APCs), thereby downregulating the differentiation of Th1 cells , 53. AccL. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  was obtained by the histological analysis. Corroborating the normoglycemia presented by NOD mice that received oral treatment with both bacterial strains, the majority of pancreatic islets was preserved, without signs of insulitis. Collectively, our data indicate the preservation of pancreatic islets is a result of the reduced autoimmune destruction, triggered by the increased levels of IL-4 and IL-10 and the decreased levels of IL-12. Consequently, this alteration in cytokine production in pancreatic environment possibly altered the T cells subsets, skewing to Treg cell proliferation, which may have contributed to the disease protection.More clear evidence of the protective effect of the combined administration of L. lactis have been used as a versatile tool for immunotherapy in several inflammatory conditions, such as T1D [L. lactis encoding IL-4 and IL-10 were detected mostly in MLNs, which are known to be the key site for oral tolerance. The induced Treg cells proliferate in MLNs and are subsequently released in circulation to modulate pancreatic beta cell autoimmune destruction in a IL-10-dependent manner [L. lactis as a delivery tool of antigens and bioactive compounds revealed the induction of oral tolerance and proliferation of Treg cells were able to revert new-onset diabetes in NOD mice [L. lactis MG1363 FnBPA+  and L. lactis MG1363 FnBPA+  was able to protect against beta-cell destruction due to expansion of Treg cells producing IL-4 and IL-10 in pancreas. Additionally, neither genetically modified bacteria was detected in the MLNs, spleen, and liver from 12 h after oral administration, indicating that these strains are indeed transient, which is a relevant attribute in the context of biosafety practices.Genetically modified h as T1D . Since th as T1D –60. Genet manner , 61, 62.t manner . PromisiNOD mice , 64. ThuL. lactis MG1363 FnBPA+ strains carrying pValac::dts::IL-4 and pValac::IL-10 verified in NOD mice was not observed in the STZ-induced model. This difference between the two main T1D models has already been reported in other studies [Altogether, the protective effect against T1D triggered by the oral administration of  studies –67, and  studies –70. Sinc studies , 72. In  studies .L. lactis MG1363 FnBPA+ strains carrying the eukaryotic expression plasmids encoding IL-4 and IL-10 in the NOD mouse model.Regarding the limitations of our study, we could not provide all the desirable experimental control groups with NOD mice, since the acquisition and maintenance of these animals are costly. However, the assessments performed with all groups in the STZ-induced model showed that we should focus efforts on the combined administration of bacterial strains carrying the cytokine-coding plasmids. Additionally, these limitations do not invalidate our valuable findings on combined administration of L. lactis MG1363 FnBPA+ strains carrying the eukaryotic expression plasmids pValac::dts::IL-4 and pValac::IL-10 attenuated the initial progression of insulitis in the STZ-induced model by increasing levels of IL-2 in pancreatic tissue and protected NOD mice against T1D in the observational period. Our findings suggest the dynamic cytokine signaling triggered by the oral treatment with the genetically modified bacteria plays a crucial role in the immunomodulation of the disease in the NOD mouse model. Our results showed the oral treatment with L. lactis MG1363 FnBPA+ strains encoding IL-4 and IL-10 can effectively reduce the incidence of T1D and control the glycemia levels in NOD mice. Additionally, we demonstrated this combined treatment was able to prevent insulitis progression, primarily by increasing the levels of IL-4 and IL-10, known by suppressing the proinflammatory cytokine milieu, in pancreas and serum and decreasing the levels of the proinflammatory cytokine IL-12 in pancreas. This is the first study to administer orally the combined genetically modified L. lactis strains carrying eukaryotic expression plasmids encoding IL-4 and IL-10 in the STZ-induced and in the NOD mouse models. In summary, our study preliminarily provides novel and relevant observations, enhancing the potential of this innovative approach as part of an alternative therapy to T1D.In conclusion, the results of our study demonstrate that the combined administration of


Nature Communications 10.1038/s41467-019-14119-9, published online 15 January 2020.Correction to: In the original version of this Article, the Y axis label in Fig. 6, panel E was incorrectly labelled as CD4+ CAR+T cells, instead of CD8+ CAR+T cells. This error has been corrected in both the PDF and HTML versions of the Article.


Dendropanax morbifera is a versatile plant that has been used as a herbal medicine due to its various useful medicinal effects. To protect its active component from biological stress and increase its drug efficacy as well as drug bioavailability, nanoemulsion was prepared. Dendropanax morbifera zinc oxide nanoparticles (DM-ZnO NPs) were synthesized using the plant extract via the co-precipitation method and loaded with active indole-3-carbinol for nanoemulsion formulation using the ultrasonication process. Field emission transmission electron microscope revealed the flower shape of the Dendropanax morbifera indole-3-carbinol zinc oxide nanoemulsion (DM-ZnO-I3C-NE). In contrast, DM-ZnO NPs showed a spheroid shape that coincides agreeably with field emission electron scanning microscope. The hydrodynamic sizes by dynamic light scattering are about 65 ± 3 nm and 239.6 ± 6 nm and the crystallite sizes from X-ray diffraction are 11.52 nm and 16.07 nm for DM-ZnO NPs and DM-ZnO-I3C-NE, respectively. In vitro analysis revealed the cytotoxicity of DM-ZnO-I3C-NE against a human lung cancer cell line (A549) at 12.5 µg/mL as well as reactive oxygen species (ROS) production. The DM-ZnO-I3C-NE-induced ROS generation level was higher than that of DM-ZnO NPs and free indole-3-carbinol. The synergistic effect of DM-ZnO and indole-3-carbinol indicates DM-ZnO-I3C-NE as a potential candidate for future lung cancer drug and could be scope for functional food. Cancer shows the highest mortality rate worldwide, which is due in part to mutations that enable cancer cells to survive and spread to other organs . ApproxiDendropanax morbifera (DM) for synthesizing zinc oxide nanoparticles and further entrapped with them with indole-3-carbinol (I3C) using the oil-in-water (O/W) nanoemulsion technique. Dendropanax is a genus of flowering plants in the family Araliaceae that consists of almost 92 species of evergreen trees and shrubs found at the seashore and islands in the southern part of South Korea. Dendro means “tree” and Panax means “Panacea,” and DM falls under the subtropical evergreen panacea family. The leaf, stem, and root of this oriental medicinal plant provides significant remedy for infectious diseases, skin, migraine, and various anti-inflammatory activities [DM show anti-complementary activity [DM, it has been applied as functional food materials with anti-diabetic, anti-cancer, and anti-atherogenic activities. DM extract contains triterpenoids, polyacetylene, and phenolic substances that have been shown to exhibit anti-inflammatory and anti-cancer properties. The information and composition of the extracts still not much explored. However, the biological applications of these substances are under investigation. These plant extracts with nanoformulation have become more prevalent in the past decades, due to the valuable components and nutraceutical properties. Oleifolioside B, a component in DM, exhibits anti-cancer activity by activating autophagy and inducing apoptosis through caspase activation [In our recent approach, we used the traditional tivities . A recentivation .Nano ZnO has gained recent attention in food additives as a GRAS  substance by the US Food and Drug Administration (FDA) . Nano ZnDM and Zn, synthesizing DM-ZnO NPs and nanoemulsion (NE) has been explored for food supplements and cancer treatment. To increase the efficacy of DM-ZnO NPs, I3C, another green source and anti-cancer substance, was loaded using non-ionic surfactant and olive oil in an O/W nano emulsification system. We prepared the oil-in-water nanoemulsion by an ultrasonication method for enhancing the solubility of the active component inside DM and I3C by trapping the hydrophobic part inside of the hydrophilic part by creating ultrasonic cavitation. Moreover, the lipophilic part solubility will improve in the water form of nanoemulsion that sequentially increases the bioavailability and medicinal properties of the drug.Considering the advantages of I3C is found in cruciferous vegetables such as broccoli, cabbages, and cauliflower and shows excellent tumor-blocking initiation capabilities . I3C alsDM-ZnO NPs were successfully synthesized from DM extract, and I3C was entrapped using the oil-in-water nanoemulsion system in which ZnO NPs water solution was used as the aqueous phase and olive oil was used as the oil phase with Tween 80 as a surfactant. The effect of the synthesized nanoformulation was investigated using the A549 lung cancer cell line and the HaCaT human keratinocyte cell line. Furthermore, the anti-cancer activity was checked via measurement of ROS level.In this article, we carefully synthesized biologically active nanoemulsion and performed structural identification. Morphology was determined using dynamic light scattering (DLS), Fourier transforms infrared analysis (FTIR), field emission transmission electron microscopy (FE-TEM), ultraviolet-visible (UV-Vis) spectrophotometry, and FE-SEM, which aided in the elucidation of the morphological and chemical natures of the NE. Dendropanax morbifera sample was collected from of Wando-gun city of South Korea from a 100-year-old tree in the September 2019. A total of 10 kg of plant were collected and extracted.The Zinc nitrate hexahydrate (>98.0%), indole-3-Carbinol , sodium hydroxide (>98.0%). All media was supplied from Difco, MB Cell . Absolute alcohol was supplied by Samchun Pure Chemical Co. Ltd. . Olive oil and Tween 80 were obtained from Samchun Pure Chemical Co. Ltd. The human keratinocyte cell line (HaCaT) and lung cancer cell line (A549) used in this study were obtained from the Korean cell line bank . RPMI 1640  culture medium was purchased from GenDEPOT Inc. . Dulbecco’s modified eagle’s medium (DMEM)  with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (p/s)  was also used for experiments.The sample was washed to remove any debris and ground into fine particles. Plant powder (10 g) was placed in conical flasks with 100 mL of DW (Distilled water) and autoclaved for 60 min at 100 °C under high pressure to obtain an aqueous extract. The autoclaved extract was filtered with filter paper (Whatman-No.1) and centrifuged at 5000 rpm for 15 min to remove debris. The collected supernatant was kept at 4 °C until experiments.DM-ZnO NPs were prepared using the co-precipitation method with minor modifications [DM extract (w/v) was mixed with 80 mL of distilled water under stirring, and 0.1 mM (10 mL) of zinc nitrate salt was added to the light brown solution with continuous stirring (500 rpm). Then, the solution was heated up to 65 to 70 °C. Then, 0.2 M (15 mL) aqueous solution of NaOH was added dropwise into the hot solution with continuous stirring over 120 min. During this process, the precipitate begins to accumulate, thicken, and form a uniform mixture. The mixture was allowed to cool down without stirring, after which it was centrifuged at 8000 rpm at 15 min to remove the unreacted substances. A cold-water wash (5 °C) was performed. Solid particles were kept at 60 °C for 4–5 h in the oven for the complete conversion of Zn(OH)2 to ZnO NPs as a white DM-ZnO-NP powder.The ications . Zinc niIn this approach, we adapted a higher energy ultrasonication technique for the development of O/W nanoemulsion using the ultrasonication method . This teThe synthesized nanoemulsion and nanoparticles were characterized via various analytical instruments for measuring size, morphology, and stability.DM-ZnO NPs and DM-ZnO-I3C NE.The green synthesized NP solutions were observed between 200 and 700 nm with a UV-Vis spectrophotometer (Ultrospec TM-2100 pro) for the confirmation of the −1. The spectral studies were plotted as transmittance (%) versus wavenumber (cm−1).The surface capping materials of the NPs and NE were determined by FTIR analysis  between 4000 and 450 cmDM-ZnO NPs and DM-ZnO-I3C-NE in a 2θ range of 20°–80° angle with an operating voltage of 40 kV as well current of 40 mA &Cu-Kα radiation of 1.54 Å.XRD was analyzed using D8 Advance  that confirmed the crystallinity and size of DLS analysis data were obtained at 25 °C with a zeta potential and particle size using the zeta analyzer and ELSZ-2000 series , which was used to define the size distribution.The morphology of the purified metallic NPs and nanoemulsion was examined using a multifunctional 200 kV-operated JEM-2100 F . The samples were prepared using a copper grid on which a small amount of the samples was dropped.In this study, we used an FE-SEM to obtain topographical and elemental information ; gun: thermal field emission type; resolution: 1.0 nm @15 kV 1.7 nm @1 kV 4.0 nm @0.1 kV; magnification: 12× to 900,000×). FE-SEM provides more transparent and higher regulation than typical SEM.2 atmosphere.A549 cells were cultured in RPMI 1640 with 10% FBS, 1% penicillin. HaCaT cells were cultured in DMEM with 10% FBS and 1% p/s. Both cell lines were cultured at 37 °C in a humidified incubator with 5% CO4 cell/well. Cells were treated as indicated with various concentrations  and incubated for 24 h. Then, cells were treated with 20 µL of 3--2, 5-diphenyl tetrazolium bromide solution  for 4 h at 37 °C. Viable cells convert the MTT solution to purple-colored formazan; the insoluble formazan was dissolved by 100 µL DMSO in each well. Readings were taken at 570 nm with an Enzyme-Linked Immunosorbent Assay (ELISA) reader .Cells were seeded in X-well plates at 1 × 104 cells per well in X-well plates overnight. Cells were treated with various concentrations of DM-ZnO-I3C-NE for 24 h. Next, 25 μM of the DCFH-DA (2′-7′dichlorofluorescin diacetate) solution was added into each well and samples were incubated for 30 min. The supernatant was discarded, and cells were washed twice with 1 × dPBS . A multi-model plate reader was used for measurement of the fluorescence intensity by an excitation wavelength of 485 nm and an emission wavelength of 520 nm. ROS generation assays were also performed using DM-ZnO-I3C-NE with DM-ZnO NPs and I3C at the same concentrations.ROS levels were determined using 2′,7′-dichlorofluorescein (DCF-DA). A549 cells were seeded at 1.0–1.2 × 10To ensure the stability of the nanoemulsion, we synthesized three different concentrations: S1, S2, and S3. All three contained different compositions of the oil and water ratio, as shown in DM-ZnO-I3C-NE contains 80% aqueous part containing 37.5 mg of DM-ZnO NPs and olive oil (8%) as well as a surfactant (7%) and was loaded with 5% I3C (12.5 mg).Sample S2 was centrifuged at 3000 rpm for 30 min, and we did not observe any precipitation during the observation time. To evaluate temperature stability, the formulation was tested at different temperatures  for 15 days of observation. S2 showed stability as shown by observing the zeta potential and PDI (Poly dispersity index) value. The low polydispersity value of NE (less than 0.2) indicated efficient formulation of the system as well homogeneity, which may help attain excellent stability . Based oDM-ZnO nanoemulsion, and I3C alone. Results indicated that DM successfully synthesized ZnO NPs using the co-precipitation method. DM-ZnO NPs showed a sharp surface at plasmon resonance wavelength (λspr) at approximately 363 nm, demonstrating the formation of ZnO NPs. The DM extract showed a sharp absorption peak around 280–320 nm due to the presence of polyphenol [DM extract functionalized with ZnO NPs and ZnO-I3C-NE both showed the absorption spectra at 250–380 nm [DM extract in the formulation  and secondary amine (–NH) group due to the polyphenol from Dendropanax extract and secondary amine (–NH) of I3C [−1 and 1383 cm−1 represents the functional group of medium (–CH) stretch and (–C=O) stretching. The DM-ZnO NPs and DM extract exhibit functional groups at 3382–3271 cm−1, also representing the phenolic (–OH) group from the polyphenol compound of the plant extract. The DM-ZnO NPs also demonstrated an absorption peak at 1598 cm−1 observed in plant extract, which may be due to the presence of (–OH), (–CH) groups. I3C showed a narrow peak at 3379 cm−1 due to the presence of the (–NH) secondary amine bond and also absorption bands at 2800–3000 cm−1 and 1450–1554 cm−1 representing C–H stretching and C=C stretching bond for alkyne molecules. Finally, the DM-ZnO NPs and DM-ZnO-I3C-NE both contain an IR transmission peak at (450–500) cm−1, indicating the presence of metal ZnO NPs and confirming the successful formation of DM-ZnO-I3C-NE from plant extract mediated with DM-ZnO NPs and I3C [FTIR spectra of ) of I3C ,25. Abso and I3C ,27.The crystalline structure and crystallite size insight into the nanomaterial are provided through the XRD analyzer. The XRD pattern of the synthesized DM-ZnO NPs and DM-ZnO-I3C-NE is shown in D is denoted as the crystalline size of the particles, k is the Scherrer constant, which is equal to the shape factor 0.9, the wavelength of light diffraction is λ where (λ = 1.54 Å), β is the FWHM , and θ is the Bragg angle of refraction.The average crystalline size of the nanoparticles and nanoemulsion were calculated using Scherrer Equation (1), which is also shown in DM-ZnO NPs and DM-ZnO-I3C-NE, and the nanoemulsion is 11.52 nm and 16.07 nm, which are respectively shown in DM-ZnO NPs and DM-ZnO-I3C-NE [The crystalline sizes were calculated for both O-I3C-NE . The XRDDM-ZnO NPs loaded with I3C using the oil-in-water system, the morphology changed to a flower shape with systematic orientation, and the entrapment of I3C was confirmed as successful with DM-ZnO in the 500 nm range  for 24 h. Cytotoxicity analysis was determined using MTT assay  as well as the production of ROS. DM-ZnO-I3C-NE induced higher ROS levels compared with free DM-ZnO and I3C on human lung carcinoma cells. The results of ROS generation validated with cell morphological studies as the higher concentration of the DM-ZnO-I3C-NE was found to be effective for apoptosis against the free I3C and DM-ZnO NP. These results suggest the efficacy of the compounds at the target site and open a door for detailed investigation of the compound mechanisms.DM-ZnO-I3C-NE showed lower toxicity in the HaCaT human keratinocyte cell line. The increased ROS level and toxicity on human carcinoma lung cancer cells by DM-ZnO-I3C-NE indicates it may be a potential source of future nano-drugs for lung cancer treatment. This approach also provides a novel nanoformulation for functional food, which is a proven safe and efficient approach for commercial applications.


MYC amplification (MYCamp-G3-MB) has been shown to be highly aggressive and exhibited worst prognosis, indicating the need for novel effective therapy most urgently. A few epigenetic targeted therapeutic strategies have recently been proven to effectively treat preclinical models of MYCamp-G3-MB, including BET inhibition, HDAC inhibition and SETD8 inhibition, unveiling a promising direction for further investigation. In this study, we carried out systemic bioinformatic analyses of public-available MB datasets as well as functional genomic screening datasets of primary MYCamp-G3-MB lines to search for other potential therapeutic targets within epigenetic modulators. We identified SSRP1, a subunit of histone-chaperone FACT complex, to be the top drug target candidate as it is highly cancer-dependent in whole-genome CRISPR-Cas9 screening across multiple MYCamp-G3-MB lines; significantly upregulated in MYCamp-G3-MB compared to normal cerebellum and most of the rest MB subtypes; its higher expression is correlated with worse prognosis; and it has a blood-brain-barrier penetrable targeted drug that has entered early phase human clinical trials already. Then we utilized RNA-interference approach to verify the cancer-dependency of SSRP1 in multiple MYCamp-G3-MB lines and further confirmed the therapeutic efficacy of FACT-targeted curaxin drug CBL0137 on treating preclinical models of MYCamp-G3-MB in vitro and in vivo, including an orthotopic intracranial xenograft model. Mechanistically, transcriptome analyses showed CBL0137 preferentially suppressed cell-cycle and DNA-repair related biological processes. Moreover, it selectively disrupted transcription of MYC and NEUROD1, two critical oncogenic transcription factors of MYCamp-G3-MB, via depleting FACT complex from their promoter regions. In summary, our study demonstrates FACT-targeted CBL0137 works effectively on treating MYCamp-G3-MB, presenting another promising epigenetic-targeted therapeutic strategy against the most devastating form of MB.Medulloblastoma (MB) is the most common malignant pediatric brain tumor that can be categorized into four major molecular subgroups. Group 3 MB with  Each MB subgroup manifests distinct demographic, pathological, and clinical characteristics, such as age, gender, cell of origin, oncogenic driver, tendency of metastasis, response to conventional therapy and prognosis. Among the four subgroups, group 3 medulloblastoma (G3-MB) exhibits the worst prognosis and therefore needs to identify novel effective therapy most urgently. Notably, G3-MB has been shown to be significantly associated with MYC amplification, which indicates an extremely poor prognosis4. The dismal outcome is associated with the high rate of metastasis and recurrence and the late diagnosis at the advanced stage of the malignancy5. Worse still, while chemotherapy and radiation regimens prove less efficacious in patients with MYC-amplified G3-MB (MYCamp-G3-MB), further intensification of chemotherapy or radiation could exacerbate the adverse systemic toxicity6.As the most common malignant pediatric brain tumor, medulloblastoma (MB) is highly heterogeneous and consists of four major molecular subgroups including Sonic Hedgehog (SHH), Wingless (WNT), group 3 (G3), and group 4 (G4)9. Targeting BRD4 with BET inhibitor results in transcriptional inhibition of MYC, which is the oncogenic driver and crucial cancer-dependent gene of MYCamp-G3-MB10. HDAC inhibition attenuates growth of MYCamp-G3-MB in part by inducing expression of the FOXO1 tumor suppressor gene9. SETD8 inhibition targets H4K20me chromatin occupancy at key genes involved in tumor invasiveness and pluripotency of MYCamp-G3-MB8. Together, these studies demonstrate that epigenetic targeted therapy represents a promising avenue for developing novel effective therapy against MYCamp-G3-MB.Researchers have been making a great amount of effort to developing targeted therapies for treating MYCamp-G3-MB in recent years. Of note, multiple epigenetic modulators have been demonstrated to effective therapeutic targets, such as BRD4, HDACs, and SETD811. It also regulates DNA replication and DNA repair. FACT complex is upregulated in many cancer types and plays a crucial role in tumorigenesis. Curaxin CBL0137, a FACT-targeted small-molecule antagonist, has been shown to exhibit broad antitumor activity. It exerts its inhibitory effect through altering DNA architecture and trapping FACT complex in chromatin without inducing DNA chemical modification12. The trapping could then deplete FACT from transcriptional active regions of various oncogenes in different cancer types and subsequently block their transcription13. More importantly, CBL0137 has entered into Phase I clinical trials on adult patients with hematological malignancies and solid tumors . And it is able to effectively infiltrate through the blood-brain barrier (BBB) and thereby suitable for treating brain tumors14.SSRP1 and SPT16 constitute a heterodimer histone-chaperone complex called facilitates chromatin transcription (FACT), which can promote transcriptional elongation by facilitating disassembly and reassembly of nucleosomesIn the present study, we carried out systemic bioinformatic analyses of public-available MB tumor datasets as well as whole-genome functional genomic screening data of patient-derived primary tumor lines, and identified SSRP1 as the top drug target candidate among epigenetic modulators in MYCamp-G3-MB. We then confirmed its cancer-dependency and further revealed SSRP1 inhibition with CBL0137 as another effective epigenetic-targeted therapeutic strategy against MYCamp-G3-MB.http://r2.amc.nl, referenced accessions: Pomeroy dataset15, u133p2dataset ) and GEO (GSE8521719 and GSE2287520) for the data analysis in the current study.Medulloblastoma tumor gene expression datasets were obtained from R2 Genomics Analysis and Visualization Platform . The medulloblastoma patients were divided into low- and high-expression groups based on the median expression level. To evaluate the difference in survival time between the high-expression and low-expression medulloblastoma groups, we used log-rank tests and plotted Kaplan–Meier survival curves.https://depmap.org/portal/). A CERES score less than −0.5 indicated that cell growth or viability was inhibited after the depletion of target gene.The effects of SSRP1 and SUPT16H depletion on medulloblastoma cell growth and viability were evaluated in including 4 medulloblastoma cell lines, based on the CRISPR-Cas9 screening DepMap (Cancer Dependency Map) dataset download from Project Achilles  was used to perform the reverse transcription reactions. An ABI 7900 Real-Time PCR instrument was used incubate the qPCR mixture at 95 °C for 3 min as the initial denaturation step, followed by 40 PCR cycles . The 2−ΔΔCq method was used to calculate the expression of the corresponding relative mRNA, and the relative mRNA expression level was normalized to the expression of GAPDH. The results of qPCR represent three independent experiments. The qPCR primer sequences used in the current study are listed as follows: SSRP1, 5′-TTGAGAGGGAGGAGTACGGG-3′ (forward); 5′-CTAGCTTGGGTTCATGCCCT-3′ (reverse); GAPDH, 5′-TGACTTCAACAGCGACACCCA-3′ (forward); 5′- CACCCTGTTGCTG TAGCCAAA-3′ (reverse); NEUROD1, 5′-GGTGCCTTGCTATTCTAAGACGC-3′ (forward); 5′-GCAAAGCGTCTGAACGAAGGAG-3′ (reverse); MYC, 5′-AGAGTCTGGATC ACCTTCT GCT-3′ (forward); 5′-ACACTGTCCAACTTGACCCTCT-3′ (reverse).TRIzol reagent  was used to isolate the total RNA from the cells, following the manufacturer’s instructions. SYBR Green Master  and High Capacity cDNA Reverse Transcription Kit  were used to perform reverse transcription and qPCR assays. The Bio-Rad S10002, 0.34 M sucrose, 10% glycerol, 1 mM DTT, and protease inhibitors) with 0.2% Triton X-100 on ice for 10 min. Nuclei were collected in pellet by low-speed centrifugation . Nuclei were washed once in buffer A, and then lysed in buffer B . After 30 min on ice, insoluble proteins were removed from the nuclear extract by high-speed centrifugation .Cytoplasmic and nuclear extracts were prepared as following: medulloblastoma cells were swollen in buffer A  and MYC (5605S) were purchased from Cell Signaling Technology . Antitubulin primary antibody (ab6046) was purchased from Abcam .For the generation of shRNA plasmids, double-strand oligonucleotides were annealed and cloned into the TRC2-pLKO-puro vector. The oligonucleotides of shRNA were synthesized by Sangon Biological Engineering Technology . The target oligonucleotides are as the following:shSCR-Forward: CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTGshSCR-Reverse: AATTCAAAAACCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGshSSRP1-1-Forward: CCGGCGCTTCGATGAGATCTCCTTTCTCGAGAAAGGAGATCTCATCGAAGCGTTTTTGshSSRP1-1-Reverse: AATTCAAAAACGCTTCGATGAGATCTCCTTTCTCGAGAAAGGAGATCTCATCGAAGCGshSSRP1-2-Forward: CCGGGCCATGGACTTAAACTGCTTACTCGAGTAAGCAGTTTAAGTCCATGGCTTTTTGshSSRP1-2-Reverse: AATTCAAAAAGCCATGGACTTAAACTGCTTACTCGAGTAAGCAGTTTAAGTCCATGGC.Oligonucleotides were annealed and then cloned into the TRC2-pLKO-puro plasmid to generate lentiviral shRNA plasmids. Moreover, we seeded the MB cells in the 6-well plates at the confluency of about 70%. The cells were transfected with lentiviral shRNA plasmids 24 h after seeding. The transfection was performed in Gibco Opti-MEM reduced serum medium using 1.67 μg target lentiviral vector, 1 μg psPAX2, and 0.67 μg pMD2.G and PEI max (Polysciences) transfection reagents. After 6 h of incubation, the medium was removed. The first and second viral supernatants were collected 24 h and 48 h after the transfection, respectively. A 0.45-μm membrane was used to filter the harvested viral supernatant, which was then concentrated by PEG6000, resuspended, aliquoted and stored at −80 °C. Fortyeight hours of post-infection, the target cell lines infected with lentiviral shRNA were cultured in 1.0 μg/ml puromycin-containing growth medium.D425, D458, and HDMB03 cell lines were kind gifts from Yoon-Jae Cho . The D425 and D458 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin–streptomycin and 10% FBS. The histological features of the primary tumors of HDMB03 showed large cell medulloblastoma, with its gene expression markers consistent with the expression pattern of MYC-amplified MB. We maintained the HDMB03 cells in culture media with Neurobasal medium supplemented with B27 (Gibco), FGF , EGF , Heparin , and LIF .CBL0137 (HY-18935A) was purchased from MedChem Express . For CBL0137 treatment in vivo, stock solution was diluted with 10% cyclodextrin at the dose of 70 mg/kg every 4 days by intravenous injection.d-luciferin  and were imaged on the Xenogen IVIS2000 system (Perkin-Elmer) 72 h after the injection to confirm engraftment. The nude mice were randomly assigned into the vehicle group and CBL0137 group. Tumor growth in both two groups was observed by IVIS imaging after 0, 7, 14, 21, 28, and 35 days of treatment. Finally, log-rank test was performed to evaluate the statistical significance in the Kaplan–Meier analysis.In vivo efficacy experiments were performed according to the following protocols. The protocol was approved by Institutional Animal Care and Use Committee at Shanghai Jiao Tong University. We used a GFP-luciferase lentiviral expression construct to infect the HDMB03 cells to obtain the GFP-luciferase-positive cells by Fluorescence Activated Cell Sorting (FACS). The GFP-luciferase-positive cells were injected into the cerebella  with stereotaxic guidance. Subsequently, the mice were treated with 2 × 0.5.Cell suspension was mixed with an equal volume of Matrigel  and was subcutaneously injected into each side of the dorsal flank. Calliper was used to measure the tumor volumes twice per week, and the tumor volumes were calculated as length × widthFor the immunohistochemical analysis of xenografted medulloblastomas, the mice were euthanized and the tumors were dissected and were either frozen in RNAlater (Qiagen) or preserved in 4% paraformaldehyde. The tumor tissues were embedded in paraffin.http://software.broadinstitute.org/gsea/msigdb/annotate.jsp. Gene set enrichment analysis was performed according to the instructions on the website http://www.broadinstitute.org/gsea/index.jsp.D458 and HDMB03 cells were treated in duplicate with DMSO or CBL0137 (1 µM) for 24 h. Total RNA was extracted using TRIzol reagent. SmartQuerier Biotech  performed the RNA sequencing. STAR (v2.5.3a) was used to map the RNAseq reads to the hg38 reference genome. Moreover, cufflinks was used to calculate the values of FPKM (fragments per kilobase million) for each gene. Differential expression analysis was performed using DESeq2 (v1.20.0). Gene ontology, KEGG pathway and Hallmark analyses were carried out using the Investigate Gene Sets tool on the website The Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit was used to measure cell proliferation. FITC Annexin V Apoptosis Detection Kit  was used to analyse apoptosis. Cell cycle analysis was carried out using the cell cycle staining kit . BD Fortessa FACS machine was used to analyse fluorescence-activated cell sorting (FACS), and FlowJo software was used to analyse the data.3 cells in each well of 96-well plates were cultured in the media containing puromycin (1 µg/ml). Finally, cell viability was measured in triplicate by assessing the ATP content using CellTiter-Glo (Promega) and the results were normalized to the baseline.We seeded 5000 cells in 96-well plates in serial dilutions of CBL0137 in triplicate to assess the cellular responsiveness to CBL0137. Then, we measured cell viability by assessing ATP content 72 h later by using CellTiter-Glo , according to the manufacturer’s instructions. Nonlinear dose response curves were applied by using GraphPad Prism. MB cells were infected with lentiviral plasmids encoding shRNA to assess the dependence of cells on SSRP1. After 96 h, 5 × 10After 28 days of treatment in the intracranial orthotopic model, two mice from each group received ice-cold PBS and 4% formaldehyde via intracardiac perfusions. H&E staining for paraffin-embedded tissue sections was carried out by Runnerbio biotech. Comp. . IHC was performed with the following primary antibodies: Ki67 , cleaved caspase-3  and MYC .HDMB03 cells were cross-linked with 1% paraformaldehyde for 8 min at room temperature and quenched with 0.125 M glycine. HDMB03 cells were re-suspended in MNase reaction buffer plus MNase  at 37 °C for 10 min. Digestion was terminated by the addition of EGTA. Chromatin was sonicated at 4 °C for 250 s with 20 s ultrasonication at 30 s intervals (Active Motif), and the resulting chromatin was immunoprecipitated at 4 °C for 12 h with the antibody against SSRP1  or IgG . Antibody-chromatin complexes were pulled-down using Pierce ChIP-grade Protein A/G Magnetic Beads , and then washed and eluted by elution buffer. After cross-link reversal and proteinase K treatment, immunoprecipitated DNA was extracted with PCR Purification Kit (Vazyme Biotech). The DNA fragments were further analysed by real-time quantitative PCR using the primers as listed in Supplementary Table t-test was used to compare the differences between two groups, and log-rank test was used in the survival analysis. A P value less than 0.05 was regarded as statistically significant .GraphPad was used to analyse the experimental data which were presented in the form of mean ± standard deviation (SD). Two-tailed Student’s 22 and performed bioinformatic analyses as follows: (1) identifying tumor-essential epigenetic genes (corrected CERES score < −0.5) from public available genome-scale CRISPR–Cas9 screening data of four MYC amplified or upregulated G3-MB cell lines  from DepMap project ; (2) identifying significantly upregulated epigenetic genes of MYCamp-G3-MB versus normal cerebellum  from published medulloblastoma dataset (R2: Pomeroy dataset15); (3) identifying survival-associated epigenetic genes whose higher transcript levels are correlated with worse prognosis in published medulloblastoma dataset   were found to meet all above-mentioned criteria, whereas only SSRP1 has targeted drug tested in human clinical trial and its role in tumorigenesis or cancer therapy has not been reported in MYCamp-G3-MB before  Fig. . Eight eore Fig. . Thus, w24. Intriguingly, even though both FACT subunits coding genes are highly cancer-dependent in all the four MYC amplified or upregulated G3-MB cell lines, only SSRP1 but not SUPT16H (the coding gene of SPT16) is significantly upregulated in MYCamp-G3-MB compared to normal cerebellum or most of the rest MB subtypes  across multiple published MB datasets   Fig. , S2 and on) Fig. .SSRP1 in MYCamp-G3-MB as measured by functional genomic screening . The knockdown efficiency of two individual shRNA against SSRP1 were confirmed by both RT-qPCR and WB assays  were detected by RNAseq and 1158 and 2032 genes were significantly downregulated by CBL0137  in HDMB03 and D458, respectively Fig. . MoreoveE2F127, CDC728, CDCA529, CDKN330, and POLE31) were performed. Both GSEA and RT-qPCR results showed that G1 phase and G1-S phase transition regulatory genes were significantly inhibited by CBL0137 only in HDMB03 cells but not D458 cells, suggesting the different cell cycle arrest outcomes might result from their different sensitivity to CBL0137 between the two MYCamp-G3-MB lines  were found to be 3rd and 4th enriched ones . Our results showed that CBL0137 could induce a dramatic decrease of MYC transcript at as early as 8 h post treatment in all tested lines  in D425 cells stably expressing shOTX2 for 48 or 96 h with previously published gene expression profile GSE2287520, and subjected them to GSEA test of RNAseq data of DMSO or CBL0137 treated HDMB03 or D458 cells. The results showed that CBL0137 treatment also resulted in significant downregulation of the two OTX2-NEUROD1 target gene sets in both MYCamp-G3-MB lines, indicating the suppression of their downstream oncogenic transcriptional programs by FACT inhibition as well  significantly upregulated in MYCamp-G3-MB compared to normal cerebellum; (2) significantly correlated with worse prognosis in MB; (3) exhibits crucial cancer-dependency in MYCamp-G3-MB cells. SSRP1 and SPT16 consist of the histone-chaperone FACT complex that plays important roles in regulating transcription, cell cycle and DNA repair in cancer cells40. Upregulation of either one of the two FACT genes could lead to FACT activation and is associated with more aggressiveness and worse prognosis in various cancer types41. In MYCamp-G3-MB, SPT16 does not fulfill the first two criteria despite exhibiting crucial cancer-dependency, whereas in another MYC family member driven malignant pediatric solid tumor, MYCN-amplified neuroblastoma (MYCNamp-NB), SPT16 is the major oncogenic player instead of SSRP142.Here we reported SSRP1 as another promising drug target candidate for epigenetic therapy against MYCamp-G3-MB. 26. Thus, FACT-targeted curaxin drug CBL0137 was further tested on multiple MYCamp-G3-MB models in vitro and in vivo and consistently exhibited robust therapeutic effects, including an orthotopic intracranial xenograft model. RNAseq analyses of CBL0137-treated MYCamp-G3-MB cells showed that cell cycle and DNA repair were the top two targeted biological processes, proving its on-target inhibitory effects. Moreover, targeting DNA repair indicates the potential combinatory use of CBL0137 with conventional therapies in treating MYCamp-G3-MB as demonstrated in other cancers, such as MYCNamp-NB, glioblastoma, small cell lung cancer and pancreatic cancer44. The therapeutic effect of CBL0137 on MYCamp-G3-MB cells was further demonstrated by its inhibition on transcription of two well-described oncogenic transcription factors (TFs), MYC and NEUROD1, via depleting FACT complex from their promoter regions. However, it remains unknown why CBL0137 treatment selectively targets these two oncogenic TFs without affecting others including OTX2, CRX, and NRL. Further anti-SSRP1 ChIPseq analyses on DMSO-treated or CBL0137-treated MYCamp-G3-MB cells may help better understand this discrepancy.In our study, SSRP1 was selected for further investigation also because of the availability of its targeted drug which possesses BBB penetrating capacity and has entered early-phase human clinical trialsTaken together, our study demonstrates that FACT-targeted curaxin drug CBL0137 works effectively in treating MYCamp-G3-MB preclinical models both in vitro and in vivo, providing evidence for initiating human clinical trials against this devastating form of MB in near future. Moreover, our study further supports that epigenetic-targeted therapy is a promising direction for identifying novel effective treatment for MYCamp-G3-MB, therefore, the other top candidates identified in our initial bioinformatic analyses are currently under active investigation to identify potential therapeutic targets for future drug development.Supplemental figure legendFigure S1Figure S2Figure S3Figure S4Supplementary table S1