Enterococcus faecium HDRsEf1 Represses CYP3A29 Expression in the Intestine through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α Signaling

Background CYP3A29 is crucial for drug metabolism in pigs and an important research model of human CYP3A4. Recent studies have shown that some probiotics can regulate the expression of CYP3A in mammals, but the mechanisms underlying their action are unclear. This study explored whether Enterococcus faecium HDRsEf1 could modulate the expression of CYP3A29 in pig intestine and potential mechanisms. Results we found that HDRsEf1 significantly reduced CYP3A29 expression in pig intestinal tissues and epithelial cells, dependent on cell-cell contact. In IPEC-J2 cells, HDRsEf1 treatment decreased the transcription activity of the CYP3A29 promoter, RXR-α expression and abrogated or mitigated the RXR-α or PXR over-expression-increased CYP3A29 expression. Both RXR-α/PXR over-expression synergistically increased CYP3A29 expression while RXR-α or PXR silencing reduced CYP3A29 expression. Co-immunoprecipitation revealed that RXR-α directly interacted with PXR. HDRsEf1, like a NF-kB inhibitor, significantly decreased the NF-kBp65 activation, RXR-α and CYP3A29 expression, which were abrogated by RXR-α silencing. The down-regulated CYP3A29 expression by HDRsEf1 was associated with increased A20 expression, which were abrogated by TLR1/2 silencing. Conclusions HDRsEf1 inhibits the expression of CYP3A29 through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α signaling in pig intestinal tissues. Our findings may provide new insights into the mechanisms by which probiotics regulate the expression of CYP450s, and suggest potential risks in the clinical application of probiotics


Introduction
Drug metabolism in the intestine or intestinal first-pass metabolism can cause low bioavailability of some oral drugs, which depends on the contents of relevant enzymes in the gastrointestinal lumen and epithelium as well as bacterial enzymes ). Probiotics can modulate gut microbiota and regulate immune responses, benefiting patients with intestinal diseases, diabetes, tumors and obesity (Marchesi et al., 2016). Probiotics have been widely used in humans and animals,and probiotics may alter the expression of intestinal drugmetabolizing enzymes (DMEs) to change the bioavailability of some oral drugs. However, the effect of 3 probiotics on the expression of intestinal DMEs and their mechanisms have not been clarified.
Accordingly, it is important to understand the effect and mechanisms of probiotics on the expression of intestinal DMEs.
Mammalian cytochrome P450s (CYP450s) are crucial for the oxidative metabolism of xenobiotics, including therapeutic drugs, environmental carcinogens and toxins (Guengerich, 1997). CYP3A4, one of the DMEs in humans, can metabolize > 50% of clinical drugs (Rodriguez-Antona et al., 2010). Pigs have a high similarity in physiology and anatomy to humans and become an important animal model for evaluating new drugs (Shang et al., 2013;Skaanild, 2006). Porcine CYP3A29 accounts for 30% of total CYP proteins and the major CYP3A activity in pig liver microsomes (Wu et al., 2016). Pig CYP3A29 and human CYP3A4 have similar tissue distribution, pharmacokinetic characteristics and regulatory mechanisms, and pig CYP3A29 is a suitable model for research of human CYP3A4 (Anzenbacher et al., 1998;He et al., 2018).
The nuclear receptors (NRs), such as retinoid X receptor alpha (RXR-α), pregnane X receptor (PXR), vitamin D receptor (VDR) and the constitutive androstane receptor (CAR), are important for regulating the expression of CYP450s (Zordoky and El-Kadi, 2009). The RXR-α can directly interact with PXR, VDR or CAR to form heterodimers and bind to the CYP450s gene promoter to regulate their expression (Lefebvre et al., 2010). For example, the VDR/RXR-α heterodimer can bind to the CLEM4-ER6 motif to regulate the expression of CYP3A4 (Pavek et al., 2010), and the CAR/RXRα and PXR/RXRα heterodimers can interact with pER6 and dXREM to regulate the expression of CYP3A4, respectively (Chen et al., 2010;Toriyabe et al., 2009). Our previous study has shown that the PXR/RXR-α can bind to the CYP3A29 promoter to induce its expression in porcine liver HepLi cells (Zhou et al., 2016).
Apparently, RXR-α expression is critical for the expression of CYP3A in the digestive system.
The nuclear factor-kappa B (NF-κB) signaling is crucial for inflammation, immunity, cell proliferation and apoptosis (Pereira and Oakley, 2008). It can regulate the expression of CYP450s by directly binding to the CYP450 promoters,or indirectly regulate the expression of NRs (Lee et al., 2000;Zhou et al., 2016;Zordoky and El-Kadi, 2009). In addition, the NF-κB can also enhance the activity of CYP450 at the post-transcriptional level by stabilizing CYP450 proteins (Zangar et al., 2008).
Interestingly, previous studies have revealed that some probiotics can inhibit the NF-κB activity (Kawano et al., 2019;Sun et al., 2017). However, whether and how probiotics could modulate the expression of CYP450s, such as CYP3A29 in pig intestinal tissues, have not been clarified.
Toll-like receptors (TLRs) act as a type of pattern recognition receptors (PRR) in the innate immune system and TLRs can recognize microbial components to initiate immune responses. TLR2 can directly interact with TLR1 or TLR6 to form a heterodimer and recognize conserved molecular patterns (such as peptidoglycan, lipoteichoic acid, etc.) on the cell wall of Gram-positive bacteria (Jin et al., 2007;Takeuchi et al., 1999). Engagement of TLR2 by the cell wall components of some probiotics can upregulate the expression of some NF-κB inhibitors, such as A20 (tumor necrosis factor-α-induced protein 3), IRAK-M and Tollip, to inhibit inflammation (Kawano et al., 2019;Sun et al., 2017). Actually, A20 is a deubiquitinating enzyme and can inhibit the NF-kB activation (Boone et al., 2004). Our previous study indicates that Enterococcus faecium HDRsEf1 enhances the expression of occluding, the intestinal tight junction protein, by activating the TLR2 (Yan et al., 2018). Accordingly, we hypothesize that HDRsEf1 can modulate the expression of CYP3A29 in pig intestinal tissues by modulating the NB-kB signaling.
In this study, we explored the effect of HDRsEf1 on CYP3A29 expression and potential mechanisms in pig intestinal tissues and epithelial IPEC-J2 cells. We found that HDRsEf1 decreased the expression of CYP3A29 in pig intestinal tissues by activating the TLR2 to up-regulate A20 expression, inhibiting the NF-kB activation to attenuate RXR-α expression.

Materials And Methods
Special chemicals, and reagents The relative levels of target gene to the control β-actin mRNA transcripts were determined by qRT-6 PCR using specific primers (Table S1) and iQ™ SYBR Green PCR Supermix in the Bio-Rad CXF real-time PCR detection system (Bio-Rad, CA, USA) after we extracted total RNA from individual groups of cells with the TRIzol reagent (Invitrogen) and reversely transcribed (RT) into cDNA using the Superscript reverse transcriptase (Takara, Otsu, Japan). The data were analyzed by 2-ΔΔCt method.

Western blotting
We extracted the jejunal tissue proteins as our previously described (Yan et al., 2018) and the proteins from individual groups of cells after lyzing IPEC-J2 cells with RIPA Buffer (Pierce, Rockford, IL, USA). Similarly, we also extracted nuclear and cytoplasmic proteins from individual groups of cells using specific extraction kit (EpiZyme, Shanghai, China). The protein samples (20 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred onto polyvinylidene fluoride membranes. After being blocked with 5% non-fat dry milk in TBST, the membrane was incubated with primary antibodies at 4℃ overnight and reacted with secondary antibodies, followed by visualizing using ECL chemiluminescence system (Bio-Rad).
Transfection and luciferase assay IPEC-J2 cells were maintained in Opti-MEM medium until 80% of confluence and transfected with pGL3-CYP3A29 that contained the CYP3A29 promoter or the NF-kBp65 promoter using Lipofectamine 2000 for 18 h. Furthermore, IPEC-J2 cells were co-transfected with pGL3-3A29-2007 and plasmid for Renilla luciferase expression for 18 h. The cells were treated with, or without, 108 CFU/mL HDRsEf1 for 6 h and the CYP3A29 promoter activity was measured using the Dual-Luciferase Reporter Assay System. In addition, IPEC-J2 cells were transfected with a control or plasmid for RXR-α, PXR, VDR or CDR over-expression for 18 h and treated with, or without, 108 CFU/mL HDRsEf1 for 6 h. The impact of RXR-α, PXR, VDR or CDR over-expression on CYP3A29 expression was determined.

RNA Interference
IPEC-J2 cells were cultured in 12-well plates overnight and transfected with 40 nM control scramble or gene-specific siRNA using Lipofectamine 2000 for 36 h. The cells were treated with, or without, 108 CFU/mL HDRsEf1 for 6 h. The efficacy of specific gene silencing and its impact on the expression of other genes were quantified by qRT-PCR and Western blot.

Co-immunoprecipitation (Co-IP)
The physical association between PXR and RXR-α was characterized by Co-IP using SureBeads™ Starter Kit Protein G (Bio-Rad) (Xie et al., 2019). Briefly, RXR-α, PXR and specific antibody IgG or control isotype IgG (10 µg each) were reacted and the formed immunocomplex was precipitated with the microbeads, followed by eluting the proteins with laemmli buffer for subsequent Western blot analysis.

Statistical analysis
Data are present as mean ± S.D. Comparison was performed by Student's t-test, one-way ANOVA, or two-way ANOVA. Significant difference was defined when a P-value of < 0.05.

HDRsEf1 reduces CYP3A29 expression in vivo and in vitro
Previous reports have shown that probiotics can regulate the expression of CYP450s in the intestinal tract of animals (Jourova et al., 2017;Kato et al., 2007). Because CYP3A29 is an important enzyme in the intestine of pigs, we examined whether HDRsEf1 could modulate the CYP3A29 expression in pig intestinal tissues. After fed with HDRsEf1-contained diet for 30 days, we found that the relative levels of CYP3A29 expression in the jejunum tissues of the probiotic group of pigs were significantly lower than that in the control group (Fig, 1A). Furthermore, treatment with 10 8 CFU/mL of HDRsEf1 for 6 h did not alter the viability of IPEC-J2, but treatment with HDRsEf1 for a longer time period obviously decreased the viability of IPEC-J2 cells (Fig. 1B). In addition, treatment with 10 6 , 10 7 and 10 8 CFU/mL of HDRsEf1for 2-6 h down-regulated CYP3A29 mRNA transcripts in IPEC-J2 cells in a dose-and timedependent manner (Fig. 1C). Moreover, treatment with 10 8 CFU/mL of HDRsEf1 for 2-6 h in a transwell system revealed that HDRsEf1 inhibited CYP3A29 expression in IPEC-J2 cells only when IPEC-J2 cells were co-cultured with HDRsEf1 in the same wells, but not in the separated transwells. Hence, HDRsEf1 down-regulated the expression of intestinal CYP3A29 in vivo and in vitro, dependent on cellcell contact.

HDRsEf1 down-regulates the expression of CYP3A29 at the transcriptional level
To investigate the effect of HDRsEf1 on the transcription of CYP3A29, we performed a transcriptional inhibition assay. We found that treatment with DRB, an inhibitor of RNA synthesis, completely abolished the HDRsEf1-down-regulated CYP3A29 transcription in IPEC-J2 cells ( Fig. 2A). We further cloned the potential sequences of the CYP3A29 promoter of Sus scrofa into the pGL3-BASIC (Fig. 2B).
After transfection, we performed luciferase reporter assays and found that all sequences exhibited varying levels of promoter activities and the highest levels of promoter activity were from the pGL3-3A29-371-transfected cells (Fig. 2B). More importantly, HDRsEf1 treatment significantly mitigated the pGL3-3A29-371 and pGL3-3A29-2007 (the longest sequence)-control luciferase expression following co-culture with IPEC-J2 cells, but not in separated transwell culture ( Fig. 2C and D). Together, such data indicated that HDRsEf1 inhibited the transcription activity of the CYP3A29 promoter, dependent on cell-cell contact.

HDRsEf1 down-regulates CYP3A29 expression via RXR-α
NRs are crucial for the expression of CYPP450s (Honkakoski and Negishi, 2000). We found that HDRsEf1 treatment significantly decreased the relative levels of RXR-α, but not PXR, CAR and VDR mRNA transcripts and protein expression (Fig. 3A) and particularly reduced nuclear RXR-α protein levels in IPEC-J2 cells (Fig. 3B). Given that RXR-α often interacts with PXR, VDR and CAR to form heterodimers and regulate the expression of CYP450s (Lefebvre et al., 2010) we further tested whether altered their expression could modulate the HDRsEf1-inhibited CYP3A29 expression in IPEC-J2 cells. We found that RXR-α or PXR over-expression increased CYP3A29 expression while HDRsEf1 treatment abrogated or significantly reduced RXR-α, PXR and CYP3A29 expression in IPEC-J2 cells ( Fig. 3C and 3D). In contrast, RXR-α or PXR silencing by specific siRNA also significantly decreased the relative levels of CYP3A29 expression in IPEC-J2 cells (Fig. 3E and 3F). However, altered VDR or CAR expression did not affect the expression of CYP3A29 in IPEC-J2 cells (Fig. S1). Interestingly, both RXRα and PXR over-expression further significantly increased the activity of pGL-3A29-2007 (Fig. 3G) and CYP3A29 protein expression (Fig. 3H), compared to RXR-α or PXR over-expression in IPEC-J2 cells. Co-IP assay revealed that RXR-α directly interacted with PXR in IPEC-J2 cells (Fig. 3I). Collectively, HDRsEf1 down-regulated CYP3A29 expression, dependent on inhibiting RXR-α expression and its interaction with PXR in IPEC-J2 cells.

HDRsEf1 down-regulates RXR-α expression by attenuating the NF-κB signaling
The NF-κB activation can significantly up-regulate the expression of RXR-α (Ren et al., 2016) while it can be inhibited by some probiotics (Finamore et al., 2014;Kawano et al., 2019). Therefore, we speculated that the down-regulated RXR-α expression by HDRsEf1 may stem from its down-regulation on the NF-κB activation. We found that HDRsEf1 treatment significantly decreased the NF-κ Bp65driven luciferase expression in IPEC-J2 cells (Fig. 4A). Furthermore, HDRsEf1 treatment significantly reduced NF-kBp65, RXR-α and CYP3A29 expression and NF-kBp65 phosphorylation in IPEC-J2 cells ( Fig. 4B-D). A similar pattern of inhibition on RXR-α and CYP3A29 expression and NF-kB activation was achieved by treatment with BAY-7082, an inhibitor of NF-kB, in IPEC-J2 cells ( Fig. 4B and 4D). In addition, although the over-expressed NF-kBp65 masked endogenous NF-kBp65 detection in an automated imaging condition the NF-kBp65 over-expression significantly increased RXR-α and CYP3A29 expression, which were significantly mitigated by HDRsEf1 treatment in IPEC-J2 cells ( Fig. 4E and 4F). Interestingly, HDRsEf1 failed to modulate significantly the relative levels of CYP3A29 expression in the RXR-α-silenced IPEC-J2 cells (Fig. 4G and 4H). Thus, HDRsEf1 attenuated the NF-kBp65 activation to inhibit the CYP3A29 and RXR-α expression in IPEC-J2 cells.

HDRsEf1 induces A20 to inhibit CYP3A29 expression, dependent on TLR1/2
Engagement of TLRs by Gram-positive bacteria can regulate the NF-κB activity in animal intestinal tissues (Bron et al., 2017). Finally, we examined whether and how any of the TLRs participated in the HDRsEf1-decreased CYP3A29 expression in IPEC-J2 cells. We detected TLR1, 2, 3, 4, 5 and 6 gene mRNA transcripts, which were effectively silenced by their specific siRNA in IPEC-J2 cells (Fig. 5A).
HDRsEf1 treatment significantly decreased CYP3A29 mRNA transcripts in the TLR3, 4, 5 and 6silenced and wild-type IPEC-J2 cells, but not in the TLR1 or 2-silenced cells (Fig. 5B). Such data suggest that the down-regulated CYP3A29 expression by HDRsEf1 may depend on the sufficient expression of TLR1/2 in IPEC-J2 cells.
Given that engagement of TLRs by some Grams-positive bacteria can enhance the expression of some NF-κB inhibitors, attenuating the NF-kB activation (Finamore et al., 2014) we further determined the effect of HDRsEf1 on the expression of A20, IRAK-M and Tollip in IPEC-J2 cells. We found that HDRsEf1 treatment significantly increased A20, but not IRAK-M and Tollip, mRNA transcripts and protein expression in IPEC-J2 cells (Fig. 5C). The enhanced A20 mRNA transcripts by HDRsEf1 were abrogated by TLR1 or TLR2 silencing in IPEC-J2 cells, indicating that HDRsEf1 increased A20 expression in a TLR1/2-dependent manner (Fig. 5D). Actually, A20 silencing by specific siRNA increased CYP3A29 and RXR-α expression and abrogated the HDRsEf1-reduced CYP3A29 and RXR-α expression in IPEC-J2 cells ( Fig. 4E and 4F). In contrast, A20 over-expression decreased CYP3A29 and RXR-α expression ( Fig. 4E and 4F). Therefore, HDRsEf1 up-regulated A20 to down-regulate CYP3A29 expression in IPEC-J2 cells in a TLR1/2-dependent manner.

Discussion
Although many people believe that probiotics are highly safe for animals and humans a few people understand the effects of probiotics on the expression of DMEs in the intestine due to intestinal firstpass metabolism. Actually, probiotics can modulate the expression of intestinal DMEs (Jourova et al., 2017;Kato et al., 2007), but the mechanisms underlying the action of probiotics remain unclear. Our results indicated that HDRsEf1 down-regulated the expression of CYP3A29 in pig intestinal tissues through the TLR1/2-induced A20 expression to attenuate the NF-κB/RXRα pathway.
TLRs can recognize bacterial components (Iwasaki and Medzhitov, 2004). TLR2 can interact with TLR1 or TLR6 to form a heterodimer (Jin et al., 2007), and recognize Gram-positive bacterial cell wall components, such as peptidoglycan and lipoteichoic acid (Asong et al., 2009;Ghose et al., 2009). In this study, we found that HDRsEf1 enhanced A20 expression in pig intestinal epithelia cells, dependent on TLR1/2. Given that A20 is an inhibitor of the NF-kB signalling by degrading TRAF-6(Shembade et al., 2010) the enhanced A20 expression by HDRsEf1 should contribute to its inhibition on the NF-kBp65 activation and CYP3A29 expression in IPEC-J2 cells. In fact, we found that HDRsEf1, like the NF-kB inhibitor of BAY11-7082, significantly attenuated the NF-kB activation and RXR-α expression in IPEC-J2 cells. Such data extended previous observations that probiotics, such as Lactobacillus amylovorus and paracasei, can bind to TLR2 to regulate the expression of NF-κB inhibitors, including A20, SOCS1 and SOCS3 in macrophages (Kawano et al., 2019) , (Sun et al., 2017) and support the notion that Gram-positive bacteria can negatively regulate inflammation through the TLR1/2 to induce the expression of the NF-kB inhibitors (Finamore et al., 2014;Ren et al., 2016).
Furthermore, we found that HDRsEf1 significantly reduced the expression of RXR-α and CYP3A29 in IPEC-J2 cells, which were abrogated by NF-kBp65 overexpression, suggesting that the enhanced NF-kBp65 activity may up-regulate RXR-α expression and subsequent CYP3A29 expression. These, together with the fact that HDRsEf1 failed to decrease CYP3A29 expression in the RXR-α-silenced IPEC-J2 cells, indicated that the NF-κBp65 signaling enhanced the CYP3A29 expression indirectly by enhancing RXR-α expression in intestinal tissues. Previous reports indicate that Staphylococcus aureus-derived LTA can reduce RXR-α expression in mouse liver by inhibiting the JNK and NF-κB pathways (Ghose et al., 2009), while IL-1β enhances RXR-α expression by activating the NF-kB signaling in gastric carcinoma tissues (Ren et al., 2016). Apparently, the NF-kB signaling positively regulates the expression of RXRα, which can attenuate the inhibition of NF-kB activation (Ning et al., 2013), maintaining the balance of intracellular inflammatory and anti-inflammatory responses.
Interestingly, we found that HDRsEf1 did not modulate PXR expression in IPEC-J2 cells. These data were in disagreement with previous reports that the phosphorylated NF-κBp65 can down-regulate PXR expression and inhibit the formation of PXR/RXR-α heterodimers and CYP3A4 transcription in HepG2 cells (Gu et al., 2006). The discrepancy suggests that the activated NF-κBp65 may have different regulatory roles in the expression of NRs in different types of cells. Given that the activated NF-κB usually regulates the transcription of the NRs (Zhou et al., 2016) , (Vogel et al., 2014) we are interested in further investigating how the activated NF-κBp65 regulates RXR-α expression in pig intestinal tissues.
In this study, we found that HDRsEf1 significantly reduced RXRα expression, particularly for nuclear RXRα protein levels, but did not affect the expression of other NRs tested in IPEC-J2 cells.
Furthermore, RXRα or PXR overexpression and silencing significantly modulated CYP3A29 expression in IPEC-J2 cells and both RXR-α and PXR over-expression synergistically enhanced the CYP3A29 promoter activity. In addition, RXR-α directly interacted with PXR in IPEC-J2 cells. Together, such data suggest that RXR-α and PXR may form heterodimer for the CYP3A29 transcription in IPEC-J2 cells, consistent with our findings in HepLi cells (Zhou et al., 2016). However, we found that altered VDR or CAR expression did not affect the CYP3A29 expression in IPEC-J2 cells, implicating that the VDR/RXR-α and CAR/RXR-α might not be involved in up-regulating CYP3A29 expression although they are crucial for CYP3A4 transcription in human intestinal cells and HepG2 (Chen et al., 2010;Pavek et al., 2010). Therefore, the NRs may have interspecies difference in regulating the transcription of CYP3As even if there are organ variants in the same species.
We found that HDRsEf1 down-regulated the expression of CYP3A29 in IPEC-J2 in a time-and dosedependent manner. Although HDRsEf1 supernatants have been reported to mitigate the ETEC K88acup-regulated IL-8 expression (Tian et al., 2016), we found that HDRsEf1 inhibited the CYP3A20 expression in IPEC-J2 cells, dependent on cell-cell contact. Our findings were consistent with a recent report that Lactobacillus helveticus SBT2171 cell wall components inhibit inflammatory responses in peritoneal macrophage (Kawano et al., 2019). The difference between our and those with ETEC K88ac may stem from the presence of more pathogen-associated molecular patterns on the cell wall of Gram-positive bacteria. We will continually explore which cell wall components of HDRsEf1 can bind to TLR1/2 on intestinal epithelial cells to inhibit CYP3A29 expression.

Conclusion
Our data indicated that HDRsEf1 inhibited the CYP3A29 expression in pig intestinal tissues and IPEC-J2 cells in a dose-, time-and cell-cell contact-dependent manner. HDRsEf1 through TLR1/2-induced A20 expression attenuated the NF-kBp65 activation to reduce RXR-α expression and limit the formation of RXR-α/PXR heterodimer, inhibiting CYP3A29 transcription in intestinal epithelial cells (Fig. 7). Our findings may provide new insights into the mechanisms by which probiotics regulate the expression of CYP450s and highlight the possible risks of probiotics for oral drug overdose in the clinic.   IPEC-J2 cells were transfected with control or TLR1/2 specific siRNAs for 30 h, and treated with, or without, 108 CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29 mRNA transcripts were quantified by qRT-PCR. (E, F) IPEC-J2 cells were transfected with 20 μM control or A20-specific siRNA for 30 h, and treated with, or without, 108 CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29 expression were quantified by qRT-PCR and Western blotting. (E, F) IPEC-J2 cells were transfected with the plasmid for A20 over-expression or control vector for 24 h. The relative levels of CYP3A29, RXR-α and A20 expression were quantified by qRT-PCR and Western blotting. Data are expressed as the mean ± SD of each group from three separate experiments. *p<0.05; **p<0.01; ***p<0.001. Figure 6 Schematic illustration of the possible mechanisms underlying the suppression of HDRsEf1 on CYP3A29 expression in the intestinal tissues. HDRsEf1 binds to TLR1/2 to up-regulate A20 expression, which inhibits the NF-κB activation by degrading TRIF6 and down-regulates RXRα expression to reduce the RXR-α/PXR heterodimer formation and CYP3A29 expression.

Supplementary Files
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