d-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity
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In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using l-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring l-isoform peptide due to the longer persistence of the d-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of l- and d-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both l- or d-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the d-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.
Duchenne muscular dystrophy (DMD) is a genetic form of muscle degeneration caused by the absence of the 427 kDa cytoskeletal protein dystrophin (1,2). Clinically, patients with DMD are confined to a wheelchair for mobility by their early teen years and succumb to the disease by their second or third decade of life, usually due to cardiorespiratory failure (3). The only currently recommended pharmaceutical treatment to slow muscle degeneration in DMD is glucocorticoids that inhibit inflammation and promote muscle protein synthesis (4, 5, 6), which leaves a clear need for further therapy development for DMD. Dystrophin localizes to the cytoplasmic face of the sarcolemma (i) to provide a structural link between intracellular F-actin and extracellular laminin through the dystrophinassociated protein complex (DAPC) (7,8), and (ii) to bind cellular signaling molecules such as nitric oxide synthase (9,10). In the absence of dystrophin, the DAPC is lost, leading to membrane instability, inflammation, degeneration of muscle fibers and eventual necrosis and replacement of muscle fibers with connective and adipose tissue (11).
The absence of the dystrophin protein in the muscle of patients with DMD and in the mdx mouse leads to the activation of pathogenic signaling pathways in striated muscle tissue. Central among mechanisms of chronic inflammation is the activation of the transcription factor nuclear factor-κB (NF-κB). Elevated levels of NF-κB are observed in dystrophic tissues (12, 13, 14, 15, 16, 17), resulting in upregulation of proinflammatory cytokines (18, 19, 20, 21).
NF-κB is composed of subunit dimers sequestered in the cytoplasm by the inhibitor protein, IκB. In the classical pathway of NF-κB activation, the inhibitor of κB kinase (IKK) complex phosphorylates the IκB inhibitor protein, leading to its ubiquitination and degradation. The nuclear localization signal of the NF-κB dimer is unmasked and, once free from the IκB inhibitor protein and the NF-κB dimer, it rapidly translocates to the nucleus of the cell and activates proinflammatory cytokine expression (21,22).
The demonstration of proof-of-principle that inhibition of the classical pathway of NF-κB could provide therapeutic benefit in patients with DMD was achieved by showing improved dystrophic muscle histopathology in dystrophin-deficient mdx mice haploinsufficient for the NF-κB p65 subunit compared with mdx control animals (12). Toward clinical translation, peptide-mediated treatments were developed to inhibit NF-κB activation by interfering with the formation of the IKK complex. The IKK complex consists of α and β catalytic subunits that are bound by a regulatory γ subunit, also called the NF-κB essential modulator (NEMO) (23). The NEMO-binding domain (NBD) peptide consists of the protein-binding domain of the IKKβ subunit for the IKKγ subunit of the IKK complex. Once bound, the NBD peptide prevents association of the IKK complex, inhibiting the catalytic activity required for the phosphorylation of the IκB inhibitor protein, thus inhibiting NF-κB activation (24).
In previous studies in the mdx mouse, systemic administration of the NBD peptide fused to a protein transduction domain (PTD) peptide comprised of 8K residues showed improvement in limb and diaphragm skeletal muscle pathology and function (12,17,25). To date, only l-isoform peptides have been tested in the muscular dystrophy model. We hypothesized that d-isoform peptides would have greater therapeutic potential due to their longer bioavailability in vivo (26, 27, 28, 29). We therefore directly compared an all-l-isoform 8K-NBD peptide with an all-d-isoform 8K-NBD peptide. Despite pathological improvements in dystrophic muscle with d-8K-wild-type-NBD peptide, kidney toxicity was observed.
Materials and Methods
PTD-NBD Peptides and Mice
Peptides containing the 8K PTD peptide fused to either a wild type or mutated NBD peptide were synthesized at the Peptide Synthesis Facility (University of Pittsburgh, Pittsburgh, PA, USA). Both l- and d-isoforms of the 8K-wild-type-NBD peptide that were used for this study had the amino acid sequence: KKKKKKKK-GG-TALDASALQTE, with the PTD bridged to the NBD peptide with a diglycine spacer. For both l- and d-isoforms of the 8K-mutant-NBD peptides, two tryptophan amino acids (underlined) were substituted for two of the alanine residues in the NBD portion of the 8K-NBD peptide: KKKKKKKK-GG-TALDWSWLQTE. Lyophilized peptide stocks were resuspended in sterile, distilled water to a concentration of 40 mmol/L.
Mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed in a pathogen-free room within the Biomedical Science Tower-South Animal Facility at the University of Pittsburgh with food and water provided ad libitum. l- or d-8K-wild-type-NBD peptides were administered in vivo to male or female dystrophic, C57BL/10ScSn-Dmdmdx/J (mdx) or normal, C57BL/6J (C57) mice, beginning at 28 d of age and continuing for 4 wks with treatments of three intraperitoneal (i.p.) injections per week (Monday, Wednesday and Friday) at either a peptide to body weight dosage of 1 mg/kg (low dose) or 10 mg/kg (high dose). The l- or d-8K-mutant-NBD peptides were administered to mdx or C57 mice at a dose of 10 mg/kg. Age-matched untreated or saline-treated mdx and C57 mice were used as controls. Urine samples were collected at multiple intervals during the studies. Following 4 wks of saline or 8K-NBD peptide treatments, mice were euthanized to collect muscles, kidneys and blood. The quadriceps muscle was used for these studies. All animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC).
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assays (EMSA) were performed as described previously (17). For quadriceps muscles, a 25 to 30 mg frozen piece of tissue was pulverized in liquid nitrogen by mortar and pestle. For kidneys, a 20-mg section of kidney cortex was processed in the same manner as muscle. Nuclear protein was extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Fisher Scientific) and multiple dilutions in duplicate were assayed for protein content using the BCA Protein Assay Kit (Fisher Scientific). Nuclear extracts of quadriceps muscle (30 µg) or kidney cortex (10 µg) were incubated with 5× Gel Shift Buffer (Promega), followed by incubation with an α-32P-deoxycytodine triphosphate (32P-dCTP)-radiolabeled, double-stranded DNA probe containing the NF-κB binding sequence) (32P-dCTP, MP Biomedicals). For the NF-κB probe, two oligonucleotides were annealed and the overhang was filled in with 32P-dCTP using Klenow fragment (Life Technologies), as described previously (30). Samples were electrophoresed on a nondenaturing, 6% polyacrylamide gel at 300 V for approximately 90 min. Densitometry of shifted-NF-κB bands were analyzed using ImageJ (NIH) software. Probe sequences for both oligonucleotides, with the NF-κB binding sequence underlined, are: NF-κB Oligo#1: 5′-CAGGGCTGGG GATTCCCCATCTCCACAGTTTCACT TC-3′; NF-κB Oligo#2: 5′-GAAGTGAAAC TGTGG-3′ (IDT, Inc.).
Kidneys were harvested from untreated age-matched, saline-treated and l- and d-8K-wild-type-NBD peptide- and l- and d-8K-mutant-NBD peptide-treated mdx and C57 mice following 4 wks of treatment. Kidneys were washed in 1× PBS and digitally imaged. Following imaging, kidneys were processed for multiple applications, including: (1) bright-field histology, (2) transmission electron microscopy (TEM) and (3) nuclear extraction and detection of NF-κB activation by EMSA.
Urinalysis by SDS-PAGE
Urine samples were collected from age-matched untreated, saline-treated and l- and d-8K-wild-type-NBD peptide-and l- and d-8K-mutant-NBD peptidetreated mdx and C57 mice at multiple time points during the peptide injection regimen as specified in the Results section. Urine samples were stored at −80°C until analysis. A 7.5 µL urine sample from each animal at specific time points in the study was electrophoresed on a 4% stacking/10% resolving SDS-PAGE gel, followed by whole-gel protein staining using Coomassie Blue gel staining reagent (45% methanol, 10% glacial acetic acid, 0.25% Coomassie Blue R-250). Coomassie Blue-stained gels were imaged on an Odyssey CLx Infrared Imaging System, utilizing the 700-nm channel (LI-COR Biosciences). The albumin band was confirmed by immunoblotting with a 1:1000 diluted goat anti-mouse albumin antibody (Bethyl Laboratories Inc.), followed by a 1:30,000 diluted IRDye 800CW goat anti-mouse IgG (H + L) conjugated secondary antibody (LI-COR Biosciences). Urine protein bands were either quantified by densitometry using ImageJ software or individual protein bands were counted in each gel lane to compare levels of urinary protein excretion.
Serum Creatinine Analysis
Blood samples were harvested from mdx and C57 mice treated with saline, l- and d-8K-wild-type-NBD peptides or l- and d-8K-mutant-NBD peptides, via cardiac puncture immediately following euthanization. Following a clotting step, blood samples were centrifuged at 8,000g to harvest serum for biochemical analysis. Serum samples from salinetreated and l- and d-8K-wild-type-NBD peptide- and l- and d-8K-mutant-NBD peptide-treated mdx and C57 mice were analyzed using the Creatinine (serum) Colorimetric Assay Kit (Cayman Chemical), according to the manufacturer’s instructions.
For bright-field histological analysis of skeletal muscle tissue, 10-µm cryosections of quadriceps tissues were stained either with hematoxylin and eosin (H&E) to observe morphological characteristics or with an Alexa Fluor 488 anti-mouse IgG antibody to assess levels of necrosis or with embryonic myosin heavy chain (eMyHC) to measure levels of regenerating muscle tissue as described previously (17). For bright-field histological analysis of the kidney, one half of one kidney was stored in 10% buffered formalin, paraffin embedded and sectioned at 3 to 4 µm. Sections were stained with H&E and periodic acid-Schiff (PAS) and Masson trichrome. All kidney histology was performed by the UPMC Presbyterian Hospital Pathology Department Histology Laboratory (Pittsburgh, PA, USA). Images were taken at 200× magnification.
Transmission Electron Microscopy
For electron microscopy studies, 1-mm3 cubes of tissue were excised from the kidney cortex of saline-treated and l- and d-8K-wild-type-NBD peptide- and l- and d-8K-mutant-NBD peptide-treated mdx mice and stored in Karnovsky fixative (2.5% gluteraldehyde, 2% paraformaldehyde in cacodylate buffer) and postfixed in 1% osmium tetroxide. The tissue was dehydrated through a series of ethanol changes, soaked in propylene oxide, then embedded in Epon plastic resin and cured overnight at 60° C. Thick sections (1 µm) were cut with a diamond knife using a Reichert Ultracut S ultramicrotome, stained with toluidine blue and imaged at 400× magnification. One plastic block was selected for thin sectioning. The plastic block was trimmed with a razor blade and thin sectioned using a diamond knife, cutting sections at 85-nm thickness. The sections were stained with uranyl acetate and lead citrate, and examined under a Philips 208 transmission electron microscope, and imaged at 60 kV. Digital images were captured by an AMT digital camera. For electron microscopy, representative images, taken at multiple magnifications (2200×, 2800×, 5600×), were utilized to observe any potential ultrastructural abnormalities in kidney cortex as a result of 8K-NBD peptide-mediated therapy upon comparison with saline-treated controls. All electron microscopy was performed by the UPMC Presbyterian Hospital Pathology Department Electron Microscopy Laboratory (Pittsburgh, PA).
For statistical comparison, a one-way analysis of variance (ANOVA) was utilized. All data is presented as values ± standard error of the mean (s.e.m.). Statistical significance was specified for p < 0.05. The number of animals for each analysis is noted in each figure.
All supplementary materials are available online at https://doi.org/www.molmed.org .
l- and d-8K-Wild-type-NBD Peptide-Mediated Therapy Reduced NF-κB Activation in Muscle Cells in mdx Limb Muscle In Vivo
Treatment with d-8K-Wild-type-NBD Peptides Improved Skeletal Muscle Histopathology of mdx Mice Similar to Treatment with l-8K-Wild-type-NBD Peptides
Quadriceps muscle showed decreased necrosis and increased regeneration in mdx mice that were treated with 4 wks of high-dose d-8K-wild-type-NBD peptide therapy in comparison to mdx mice that were treated with either d-8K-mutant-NBD peptide or saline control (Figures 1B–J). In a quantitative study comparing 4 wks of treatment of mdx mice (n = 8 mice per group) with either d- or l-isoform 8K-NBD peptide at low dose or high dose, the following results were obtained in comparison with untreated mdx mice collected in parallel. The average reduction in area of necrosis observed with d-8K-NBD peptide treatment was 32.7% for low dose and 55.0% for high dose. In comparison, l-8K-NBD peptide treatment reduced the area of necrosis by 50.5% for low dose and 39.6% for high dose. Treatment with d-8K-NBD peptide therapy resulted in an increased level of regeneration of 153.3% (p = 0.041) for low dose and 24.4% (not significant) for high dose. In comparison, l-8K-NBD peptide therapy resulted in an increased level of regeneration of 120.0% (p = 0.046) for low dose and 77.8% (not significant) for high dose.
Treatment with d-8K-Wild-type-NBD Peptide Caused Gross Pathological Changes to Kidney
Treatment with d-8K-Wild-type-NBD Peptide Activated NF-κB in the Kidney Cortex
EMSA analysis of nuclear extracts of the kidney cortex from mdx or C57 mice treated with d-8K-wild-type-NBD peptide had increased levels of NF-κB activation (Figure 2 and Supplementary Figure 1). NF-κB activation remained at normal levels or was mildly elevated in the kidney cortex of l-8K-wild-type-NBD or l- or d-8K-mutant-NBD peptide-treated mdx (Figure 2) or C57 (Supplementary Figure 1) mice compared with saline-treated controls.
l- and d-8K-Wild-type-NBD Peptide-Mediated Therapy Induced Proteinuria
l- and d-8K-Wild-type-NBD Peptide-Mediated Therapy Induced Elevations in Serum Creatinine
d-8K-Wild-type-NBD and d-8K-Mutant-NBD Peptide Treatment Caused Histological Changes in Mouse Kidney Cortex
d-8K-Wild-type-NBD and d-8K-Mutant-NBD Peptide Treatment Caused Ultrastructural Changes in the Mouse Kidney Cortex
The present study explores the efficacy and toxicity of a novel systemic therapy with NBD peptides synthesized with all-d isoform amino acids in the mdx mouse model of DMD. We confirm that blocking classical NF-κB signaling in dys-trophic muscle tissue of the mdx mouse model by systemic administration of 8K-NBD peptides improves muscle histopathology as shown by a reduction in necrosis and an increase in regeneration of skeletal muscle fibers (12,17,25). We directly compared the l-isoform with the d-isoform of 8K-NBD peptide at two doses. d-isoform peptides have greater resistance to degradation in comparison to peptides comprised of naturally occurring l-isoform amino acids (26, 27, 28, 29). In this study, we showed that the pathological activation of NF-κB signaling in mdx mouse skeletal muscle is reduced by either low- or high-dose 8K-NBD peptide composed of either l- or d-isoform amino acids. Our observation of gross kidney pathology and proteinuria with d-8K-wild-type-NBD peptide treatment led us to perform additional studies with l- and d-isomer mutant NBD peptides. The d-isomer, wild-type-NBD peptide treatment appeared to be the most toxic to the kidney with a lower level of toxicity observed with treatment with the d-isomer, mutant NBD peptide. Kidney toxicity ranged from subtle to not observed with treatment with the l-isoform of either 8K-wild-type-NBD peptide or 8K-mutant-NBD peptide. The kidney toxicity in normal C57 mice treated with l- and d-isomers of 8K-wild-type-NBD and 8K-mutant-NBD peptides followed a similar pattern to that observed in mdx mice.
The relationship of necrosis and regeneration in dystrophic skeletal muscle is interdependent and complex. We found enhanced regeneration of muscle fibers in d-isoform 8K-NBD peptide-treated mdx muscle, similar to what was previously published with treatment with l-isoform 8K-NBD peptides (12,17). The decrease in muscle fiber necrosis observed in the setting of enhanced regeneration in either d- or l-isoform 8K-NBD peptide-treated mdx muscle suggests that 8K-NBD treatment directly enhances muscle fiber regeneration.
Peptide treatment regimens of three injections per week beginning at approximately 4 wks of age and continuing for 4 wks were assessed in this study to coincide with the most active phase of muscle degeneration and regeneration in hind limb skeletal muscle of the mdx mouse (31). Interestingly, it was recently shown that inhibition of TNF receptorassociated factor 6 (TRAF6), an upstream NF-κB pathway molecule, improved mdx histopathology in young mice, however, mice with long-term inhibition of TRAF6 developed necrosis and fibrosis in muscle (32). Since none of our studies exceed 7 wks of treatment with NBD peptides, we have not tested the effect of long-term NBD peptide treatment in mdx mice.
While previous PTD-NBD peptide studies have focused on the ability of the NBD peptide to block NF-κB activation and improve dystrophic muscle histopathology in skeletal muscle, this is the first study to address potential renal toxicity of NBD peptide-mediated therapy. Previous in vivo studies of the biodistribution of l-isoform PTD-peptides show localization to kidneys 8 h after intraperitoneal injection (33) and studies examining d-isoform PTD-peptides in vivo revealed high levels of transduction in the kidney beginning at 1 h after systemic administration, with levels still detected at 24-h postinjection (34).
In the present study, we observed gross structural alteration of kidneys in both mdx and C57 mice following treatment with high dose of d-8K-wild-type-NBD peptide and, to a lesser extent, d-8K-mutant-NBD peptide. Microscopic examination of PAS-stained kidney cortex regions from high-dose d-8K-wild-type-NBD peptide-treated mice revealed damage to the microvillus brush border of many proximal tubules. In the highdose d-8K-mutant-NBD peptide-treated mice, we only observed a few disruptions of the microvillus brush border. H&E-stained sections revealed a general disorganization of the kidney cortex in mice treated with d-8K-wild-type-NBD and d-8K-mutant-NBD peptides, with d-8K-wild-type-NBD peptide-treated mice exhibiting greater disorganization, in addition to dilated tubules and interstitial lymphocyte infiltration. Ultrastructural analysis of the kidney cortex from d-8K-wild-type-NBD peptide and d-8K-mutant-NBD peptide-treated mdx mice revealed lysosomal inclusions in proximal tubules, while examination of glomeruli, red blood cells, mitochondria, capillaries and podocytes appeared unaffected. Comparison of the levels of observed toxicity in mice treated with d-8K-wild-type-NBD peptide and the d-8K-mutant-NBD peptide, suggests that contributions to renal toxicity can be attributed to both the d-isoform nature of the peptides and the wild-type NF-κB inhibitory peptide sequence.
Nuclear extracts of isolated kidney cortex tissue were analyzed by EMSA to assess activation of the classical NF-κB signaling pathway. In contrast to a decrease in the activation of NF-κB signaling that we observed in skeletal muscle of l- and d-8K-wild-type-NBD peptide-treated mice, there was increased activation of NF-κB signaling in kidney cortex of mice treated with d-8K-wild-type-NBD peptide and d-8K-mutant-NBD peptide, but not in mice treated with l-8K-wild-type-NBD peptide, l-8K-mutant-NBD peptide or saline. Previous in vitro studies have shown induction of the NF-κB signaling pathway in proximal tubule epithelial cells in the presence of bovine serum albumin (BSA) (35,36) or human serum albumin (HSA) (37,38), in a dose-dependent manner by EMSA. We detected elevated levels of albumin in urine from d-8K-wild-type-NBD peptide-treated mdx mice in conjunction with increases in NF-κB activation in nuclear extracts of kidney cortex. These results suggest that the kidney toxicity observed in d-8K-wild-type-NBD peptide-treated mdx mice may lead to proteinuria, which then activates the classical NF-κB signaling pathway in kidney tissue. Since the wild-type NBD peptide would be expected to block this activation, one can speculate that increases in NF-κB signaling would be even more prominent if the peptide causing renal toxicity did not include the NBD sequence. However, after examination of mice treated with d-8K-mutant-NBD peptide, there was a much lower level of kidney toxicity detected, in comparison to mice treated with d-8K-wild-type-NBD peptide. Therefore, it appears that the wild-type NBD peptide sequence significantly contributes to the pathogenic potential of the therapy.
We postulate that the kidney toxicity and increased NF-κB activation in response to the d-8K-wild-type-NBD peptide treatments may have been compounded by the increased half-life of the d-isoform peptide. d-isoform peptides, a non-naturally occurring form, may have accumulated to high levels in the proximal tubules of the kidney cortex of both mdx and C57 mice during treatment. A study examining levels of the amino acid d-serine in rats determined that high levels of d-serine can be nephrotoxic (39). Our 8K-NBD peptide constructs in this study contain a single serine amino acid in the NBD peptidebinding domain. The potential build up to nephrotoxic levels of d-serine in the kidneys of mice receiving a d-isoform peptide may account for a component of the observed toxicity. Additionally, proteinuric renal diseases have been shown to upregulate the NF-κB signaling pathway (40). Protein overload of the proximal tubules leads to the release of cytokines and chemokines that ultimately lead to inflammation and fibrosis of the tubular interstitium (41,42). It is unclear whether the observed proteinuria is glomerular or tubular in origin, however, albuminuria observed in early urinalysis samples of d-8K-wild-type-NBD peptide-treated mice would indicate a glomerular proteinuria. In the late urinalysis samples from the same mice, we no longer observed albuminuria, but rather, we observed a greater number of lower molecular weight proteins present in urine, which is evidence of tubular proteinuria. We postulate that early in the treatment regimen, glomerular damage led to the observed proteinuria/albuminuria of the d-8K-wild-type-NBD peptide and d-8K-mutant-NBD peptidetreated mice. We further suggest that the ultrastructural detection of lysosomal inclusions in the proximal tubules of the d-8K-wild-type-NBD peptide and d-8K-mutant-NBD peptide-treated mice may relate to the observed proteinuria following the full treatment regimen.
The goal of this study was to explore the therapeutic utility of a novel d-isoform of the 8K-NBD peptide whose l-isoform demonstrated efficacy in amelioration of dystrophic myopathy in the mdx mouse model of DMD (12,17,25). While the d-isoform of the 8K-wild-type-NBD peptide provided equivalent ability to decrease NF-κB activation in skeletal muscle compared with the l-isoform, its effects in the kidney revealed toxicity at a level not seen with the l-isoform 8K-wild-type-NBD peptide. Our finding of toxicity in both the mdx muscular dystrophy mouse model and normal mice suggest that future NBD peptide-based studies should focus on strategies other than the use of d-isoform peptides.
The authors take full responsibility for the contents of this manuscript, which do not represent the views of the Department of Veterans Affairs or the U.S. Government.
This work was supported in part by by the Kidney Imaging Core of the Pittsburgh Center for Kidney Research (NIH grant P30-DK079307). In addition, some of the transmission electron microscopy equipment used in this study was funded by grant # 1S10RR019003-01. The work was funded by the Department of Veterans Affairs (VA) Medical Center Merit Review Grant and departmental funds (PRC). We would like to thank Katie Clark and the UPMC Presbyterian Hospital Pathology Department Electron Microscopy Laboratory for their contributions to this manuscript.
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