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PROTOCOL

Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells Darren R Williams, Gun-Hee Kim, Myung-Ryul Lee & Injae Shin Department of Chemistry, Yonsei University, Seoul 120-749, Korea. Correspondence should be addressed to I.S. ([email protected]).

© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 17 April 2008; doi:10.1038/nprot.2008.47

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye, FM 1-43, which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus, small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening, which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d, which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43.

INTRODUCTION Neurons are not regenerated efficiently and their gradual or rapid loss causes neurologic diseases, such as stroke, amyotrophic lateral sclerosis, spinal cord injury, Parkinson’s and Alzheimer’s diseases. These diseases may be attenuated or even cured by cell therapies using stem cells1,2. However, for the medical applications of stem cells, several technical issues should be addressed, such as the requirement for a source that supplies a sufficient amount of stem cells, a precise control over differentiation, a suppression of host rejection against allogenic cells and the prevention of tumor induction3,4. These technical problems and the ethical issues associated with the use of discarded embryos impose restrictions on the implementation of cell therapies that employ embryonic stem cells. Adult stem cells have the potential to differentiate into several cell types and thus could be used to treat various diseases, such as hematologic disorders, neurologic diseases and diabetes5,6. In comparison to embryonic stem cells, however, these stem cells are less efficient with regard to growing and differentiating into various types of cells. An alternative approach that is more attractive and convenient for the production of neurons is to use small molecules with neurogenesis-inducing activity in cells that can be obtained from readily available adult tissues. Generation of neurogenic cells from nonstem cells using a small molecule inducer can avoid the problems associated with stem cell-based therapies. Recently, a small molecule (named neurodazine (Nz)) that induces the development of neuron-like properties in muscle cells has been discovered from a pool of small molecules by a fluorescent highthroughput screening method using a fluorescent dye (Fig. 1a)7. Herein, we describe the detailed protocol we have used to identify neurogenesis-inducing small molecules in muscle cell lines. This approach relies on a strategy for fluorescent highthroughput screening. Our screening system uses the fluorescent dye FM 1-43, which has been used to study the neuronal property of depolarizationinduced synaptic vesicle recycling (Fig. 1b)8–11. The dye can be internalized from the culture medium during synaptic vesicle

recycling, in response to a high concentration (B80 mM) of potassium ions in the medium8–11. Although FM1-43 can be internalized by endocytosis, in the time course of our protocol (5 min exposure to FM1-43) and the presence of a high external concentration of potassium ions to induce synaptic terminal exocytosis and rapid recycling back into the nerve terminal (with a concomitant entry of FM1-43 into the cell via this process), cells that possess the neurogenic function display increased FM1-43 fluorescence. Therefore, the screening method using FM 1-43 allows neurogenesis-inducing small molecules to be facilely and rapidly identified from a large number of compounds by measuring the fluorescent intensity of treated cells. Our protocol uses the murine skeletal muscle C2C12 cells, which are well-characterized, readily available and have been shown previously to possess the potential to differentiate into nonmuscle cell types10,12–14. The neurogenesis of PC12 cells induced by nerve growth factor is employed in parallel with C2C12 cells as a positive control for this screening (see Fig. 2 for the general outline of the PROCEDURE)15,16. It should be noted that other types of muscle

a

MeO H N

Cl O

N MeO

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Bu +

Et Et

N+ Et

N 2Br –

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Figure 1 | Structures of neurodazine, a compound identified using the fluorescent high-throughput screening method and the fluorescent dye FM 1-43. (a) Neurodazine, (b) FM 1-43. NATURE PROTOCOLS | VOL.3 NO.5 | 2008 | 835


PROTOCOL cells can be used in the screening protocol; however, some muscle cell types, such as those derived as the primary culture from muscle tissue, require specially coated culture plates that may interfere with the fluorescent signal from FM 1-43. For example, muscle satellite cells derived from humans are usually cultured on Matrigel (BioCoat)17, and they may, thus, have to be transferred to an uncoated microplate and allowed to attach fully to the plate’s surface before testing with FM 1-43. It should also be noted that the neuronal properties of the cells developed from skeletal muscle through treatment with small molecules, which are identified from this screening method, should be further investigated using other methods. For example, the neuronal properties of the compound-treated cells can be examined by immunostaining for neuron-specific markers in the cells, analyzing the expression level of neuronspecific markers in the cells, studying their neurophysiological properties, assessing the genome-wide changes in the expression pattern of the cells, detecting glutamateinduced calcium influx or measuring the action potential of the cells7,10,18,19.

For a positive control Culture C2C12 cells

Seed C2C12 cells in a 96-well plate

Seed PC12 cells in a 96-well plate

Incubate the cells in culture media for 1 d

Incubate the cells in culture media for 1 d

Change the culture media with the screening media and add compounds to the cell culture

Replenish with the fresh culture media and add NGF to the cell culture

Incubate for 2 d

Change the culture media with the screening media and add compounds to the cell culture

Incubate for 3 d

Change the media with Krebs-Ringer buffer containing FM 1-43

Measure fluorescence intensity of the treated cells using a microplate reader

Incubate for 1 d


Incubate for 1 d


Incubate for 3 d

Figure 2 | Flow diagram of this protocol.

MATERIALS REAGENTS

. C2C12 Mus musculus (mouse) myoblasts (American Type Culture Collection (ATCC), cat. no. CRL-1772)

. PC12 Rattus norvegicus (rat) adrenal gland pheochromocytoma cells (ATCC, cat. no. CRL-1721)

. DMEM (Invitrogen, cat. no. 11995) . Ham’s F12K media (Invitrogen, cat. no. 31765035) . Horse serum (HS; Sigma-Aldrich, cat. no. H1138) . Trypsin–EDTA solution (Sigma-Aldrich, cat. no. T4174) . 100 Penicillin/streptomycin (Invitrogen, cat. no. 15140) . N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM 1-43; Invitrogen, cat. no. F35355)

. Nerve growth factor (NGF; Sigma-Aldrich, cat. no. N2513) . Potassium chloride (KCl; Sigma-Aldrich, cat. no. P9333) . DMSO (Sigma-Aldrich, cat. no. 276855) . Sodium chloride (NaCl; Sigma-Aldrich, cat. no. S7653) . Magnesium chloride (MgCl2; Sigma-Aldrich, cat. no. 449172) . Sodium phosphate, monobasic (NaH2PO4; Sigma-Aldrich, cat. no. S8282)

. Sodium sulfate (Na2SO4; Sigma-Aldrich, cat. no. 2044447) . Calcium chloride (CaCl2; Sigma-Aldrich, cat. no. C1016) . Sodium bicarbonate (NaHCO3; Sigma-Aldrich, cat. no. S6297) . Glucose (Sigma-Aldrich, cat. no. G7528) . Culture medium for C2C12 cells (see REAGENT SETUP) . Culture medium for PC12 cells (see REAGENT SETUP) . Screening medium for C2C12 cells (see REAGENT SETUP) . Krebs-Ringer buffer (see REAGENT SETUP) . Krebs-Ringer buffer containing 100 mM potassium ions (see REAGENT SETUP) EQUIPMENT . Incubator for mammalian cell culture (Sanyo, cat. no. MCO-5AC)

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Culture PC12 cells

. Clean bench (Samki-Lab, cat. no. CB2012-840) . Fluorescence microscope (Nikon Eclipse TE2000; Nikon) . Centrifuge (5804R; Eppendorf) . Culture flask (25 cm2; Nunc, cat. no. 136196) . Conical centrifuge tube (15 ml; SPL life Sciences, cat. no. 50015)

. 96-Well plate (Nunc, cat. no. 167008) . Fluorescent microplate reader (SpectraMax GeminiEM; Molecular Devices)

. 8-Channel micropipette (40–200 ml; Nichirto, E98-01382) REAGENT SETUP Culture medium for C2C12 cells DMEM supplemented with 10% (vol/vol) FBS, 50 U ml 1 of penicillin and 50 mg ml 1 of streptomycin. Store at 4 1C and use within 1 month. Screening medium for C2C12 cells DMEM supplemented with 2% (vol/vol) FBS, 50 U ml 1 of penicillin and 50 mg ml 1 of streptomycin. Store at 4 1C and use within 1 month. Culture medium for PC12 cells Ham’s F12K media supplemented with 7% (vol/vol) HS, 7% (vol/vol) FBS, 50 U ml 1 of penicillin and 50 mg ml 1 of streptomycin. Store at 4 1C and use within 1 month. Krebs-Ringer buffer 115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 25 mM NaHCO3 and 10 mM glucose (glucose should be added freshly before use). Krebs-Ringer buffer containing 100 mM potassium ions 20.9 mM NaCl, 100 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 25 mM NaHCO3 and 10 mM glucose (glucose should be added freshly before use). Note that the amount by which the concentration of K+ increases in this buffer compared to the ‘regular’ Krebs-Ringer buffer described earlier (see change in concentration of KCl), is the same as the amount by which the concentration of Na+ decreases (see corresponding change in NaCl concentration).

PROTOCOL PROCEDURE Culturing C2C12 cells and PC12 cells 1| Thaw the frozen C2C12 cells and PC12 cells in two 1-ml cryovials for 5 min at 37 1C. PC12 cells are used as a positive control for this screening. 2| Transfer C2C12 cells to a 15-ml conical centrifuge tube containing 10 ml culture medium for C2C12 cells (DMEM supplemented with 10% FBS, 50 U ml 1 of penicillin and 50 mg ml 1 of streptomycin) and PC12 cells to a 15-ml conical centrifuge tube containing 10 ml culture medium for PC12 cells (Ham’s F12K medium supplemented with 7% HS, 7% FBS, 50 U ml 1 of penicillin and 50 mg ml 1 of streptomycin).


3| Centrifuge C2C12 cells and PC12 cells at 180g for 5 min at 25 1C and discard the supernatants by decanting. Steps 1–3 are implemented to remove DMSO used to help preserve cell integrity during the freezing process. 4| Suspend C2C12 and PC12 cell pellets in 5 ml of the culture media for C2C12 cells and PC12 cells (see Step 2), respectively, and transfer each cell suspension into a culture flask. 5| Incubate C2C12 cells and PC12 cells in the culture flask inside an incubator filled with 5% CO2 at 37 1C until they are 70% confluent (usually 24–48 h). 6| Remove the culture media from the cell culture by decanting and wash the cells with 8 ml sterilized PBS (pH 7.4). 7| Treat C2C12 cells and PC12 cells with 1 ml Trypsin–EDTA solution to detach cells from the surface of the culture flask and incubate the cells at 37 1C until the cell layer is dispersed (usually 5–15 min). 8| Add 6–8 ml culture medium for C2C12 cells or PC12 cells (see Step 2) to each cell culture to stop the trypsin reaction and transfer each cell culture into a 15-ml conical centrifuge tube. 9| Centrifuge C2C12 cells and PC12 cells at 180g for 5 min at 25 1C and discard the supernatants by decanting. 10| Suspend C2C12 and PC12 cell pellets in 5-ml culture media for C2C12 cells and PC12 cells (see Step 2), respectively, and count the number of cells using a hemocytometer. Screening of neurogenesis-inducing small molecules using FM 1-43 11| Seed 200 ml of C2C12 cells at a density of 104 cells ml 1 in a 96-well plate. Cells should be seeded in triplicate for each compound to be screened. For a positive control, seed 200 ml of PC12 cells at a density of 105 cells ml 1 in parallel with C2C12 cells in a 96-well plate. m CRITICAL STEP If C2C12 cells are seeded at a density higher than that prescribed (4104 cells per well in a 96-well plate), widespread contact among the cells will occur, and this will prompt cells to fuse together into immature muscle fibers called myotubes. Cells that form myotubes are, in turn, likely to be inert to any neurogenic stimulus as they have already embarked upon the myogenic differentiation pathway. 12| Incubate C2C12 cells and PC12 cells inside an incubator filled with 5% CO2 for 24 h at 37 1C. Cells will attach to the plate surface during this time. 13| Remove the medium from the culture of C2C12 cells, and using an 8-channel micropipette, add to the cells 200 ml of the screening medium for C2C12 cells (DMEM supplemented with 2% FBS, 50 U ml 1 of penicillin and 50 mg ml 1 of streptomycin). With regard to the culture of PC12 cells, remove the medium as earlier, and replenish the culture with 200 ml of fresh culture medium for PC12 cells (see Step 2). m CRITICAL STEP Screening medium for C2C12 cells should contain a low percentage of FBS (2%) to reduce the rate of cell proliferation, thus reducing the likelihood of formation of myotubes by suppressing widespread contact among the cells under low cell density conditions (104 cells ml 1 in a 96-well plate). 14| Add 0.6 ml of 1 mM DMSO solutions of the compounds to be tested to 200 ml of C2C12 cell culture in a 96-well plate in triplicate using a micropipette (final concentration of the compounds: 3 mM). As for the positive control, add 0.5 ml of 40 mg ml 1 NGF dissolved in culture medium for PC12 cells to 200 ml of PC12 cell culture (final concentration of NGF: 100 ng ml 1). Two wells of the cells (one for C2C12 and one for PC12 cells) should be left untreated as a negative control. It is recommended that an automated liquid handling system is used to add a large number of compounds to the cell culture with high speed and precision. 15| Incubate the treated C2C12 cells and PC12 cells for 24 h at 37 1C. Identify toxic compounds by observing the detachment of C2C12 cells using microscopy. Toxic compounds are later retested at lower concentrations. In the case of PC12 cells, change the culture medium with the fresh culture medium for PC12 cells (see Step 2) containing 100 ng ml 1 NGF. ? TROUBLESHOOTING NATURE PROTOCOLS | VOL.3 NO.5 | 2008 | 837

PROTOCOL a

b

c

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16| Incubate the treated C2C12 cells and PC12 cells for an additional 24 h at 37 1C. Using an 8-channel micropipette, replenish the culture of C2C12 cells with 200 ml of the fresh screening medium for C2C12 cells and add 0.6 ml of 1 mM DMSO solutions of the compounds to be tested to the cell culture. In the case of PC12 cells, change the culture medium with 200 ml of the fresh culture medium for PC12 cells (see Step 2) containing 100 ng ml 1 NGF. ? TROUBLESHOOTING 17| Incubate the treated C2C12 cells and PC12 cells for additional 3 d at 37 1C. It should be noted that C2C12 cells and PC12 cells are incubated for 5 d after the initial treatment with compounds and NGF, respectively. 18| Remove the culture medium from the C2C12 and PC12 cell culture by gently inverting the plate and blotting onto tissue paper. 19| Wash the cells for 30 s two times with 100 ml Krebs-Ringer buffer prewarmed to 37 1C. ? TROUBLESHOOTING 20| Add 100 ml of prewarmed Krebs-Ringer buffer containing 100 mM potassium ions and 2 mM FM 1-43 to both C2C12 cells and PC12 cells. 21| Incubate the cells for 5 min at 37 1C. 22| Wash the cells for 30 s three times with 100 ml of prewarmed Krebs-Ringer buffer to remove excess FM 1-43. 23| Add 100 ml of prewarmed Krebs-Ringer buffer to the cells. 24| Measure the fluorescent intensity of the cells using a fluorescent microplate reader (excitation wavelength: 470 nm, emission wavelength: 540 nm). Cell images are obtained using a light microscope or a fluorescence microscope (fluorescence is excited by light from a mercury lamp filtered at wavelength 510–560 nm and emission is collected at wavelength 590 nm by a charge-coupled device camera). ? TROUBLESHOOTING

TIMING Steps 1–5: 48 h Steps 6–10: 1 h Steps 11 and 12: 24 h Steps 13 and 14: 1 h Steps 15–17: 5 d Steps 18–24: 1 h

a

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Fluorescence intensity

Fluorescence intensity


Figure 3 | Phase contrast (left) and fluorescence microscopic images (right) of cells after treatment with FM 1-43. (a,b) These images are of C2C12 cells after 5-d incubation (a) without any compound (negative control) and (b) with 2 mM neurodazine, followed in both cases through treatment with 2 mM FM 1-43 in the presence of 100 mM potassium chloride (KCl). (c,d) These images are of PC12 cells after 5-d incubation (c) without nerve growth factor (NGF) and (d) with 100 ng/ml NGF, followed in both cases through treatment with 2 mM FM 1-43 in the presence of 100 mM KCl. (Scale bar = 50 mm).

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20

40 60 80 [KCI] (mM)

100

Figure 4 | Quantitative analysis of fluorescence intensities of the neurogenesis-inducing compound-treated C2C12 cells and PC12 cells. (a) Fluorescence intensities of C2C12 cells after 5-d incubation either with (filled bar) or without (empty) 2 mM neurodazine and PC12 cells after 5-d incubation either with ( ) or without ( ) 100 ng ml 1 nerve growth factor (NGF), followed by treatment of the cells with 2 mM FM 1-43 in the presence of 100 mM potassium chloride (KCl). (b) Potassium ion concentrationdependent fluorescence intensities of C2C12 cells (circles) treated either with (solid lines) or without (dotted lines) neurodazine for 5 d and PC12 cells (triangles) treated either with (solid lines) or without (dotted lines) NGF for 5 d, followed by treatment of the cells with 2 mM FM 1-43 (ref. 7).

PROTOCOL ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1.


TABLE 1 | Troubleshooting table. Step 15

Problem Death of cells during incubation with compounds

Possible reason Compounds are cytotoxic

16 and 19

Detachment of cells from the wells while The cell monolayers are disrupted changing media and washing cells due to a low cell density

Solution Incubate cytotoxic compounds at a lower concentration after initial screening Be careful not to disrupt the cell monolayers while you replace the media and wash the cells Eject the media or buffer gently from the pipette aiming at the side of each well in the 96-well plate rather than directly at the cell layers

24

C2C12 cells treated with compounds mainly undergo myogenesis after 5-d incubation

Myogenesis of C2C12 cells is not suppressed

Lower the cell seeding density (we advise to start at B2 103 cells per well in a 96-well plate) Use screening media containing a lower concentration of FBS (down to 1%) Replace FBS with horse serum (2%) to reduce proliferation during the screening process

ANTICIPATED RESULTS Using this screening method, a small molecule named Nz shown in Figure 1a that induces neurogenesis in skeletal muscle was identified from B300 synthetic compounds (the synthesis of Nz was reported elsewhere7). In Figure 3 are reported the images of C2C12 cells treated with FM 1-43 at 100 mM KCl after 5-d incubation either without or with Nz. The cells treated with Nz exhibit a fluorescent signal unlike untreated cells. The synaptic vesicle recycling property of Nz-treated C2C12 cells using FM 1-43 is compared to that of PC12 cells treated with NGF (a well-characterized model of neurogenesis) at a fixed concentration (100 mM) or over a range of concentrations (10–100 mM) of external potassium ions (Fig. 4). The degree of synaptic vesicle recycling of Nz-treated C2C12 cells is similar to that displayed by the cells derived from NGF-treated PC12 cells. It should be noted that a positive result of this FM 1-43 experiment alone is insufficient to classify cells as neurogenic. To further characterize the neuronal properties of the compound-treated cells, other experiments such as immunostaining for neuronspecific markers in the treated cells, expression of neuron-specific markers in the cells and analysis of the genome-wide changes in expression pattern of the cells should also be performed (the complete experiments for these were reported elsewhere7).

ACKNOWLEDGMENTS This work was supported by a grant of the NRL program (MOST/KOSEF). G.-H.K. thanks the BK 21 program (KRF). Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Kim, J.H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418, 50–56 (2002). 2. Reubinoff, B.E. et al. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19, 1134–1140 (2001). 3. Swijnenburg, R.J. et al. Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 112 (9 suppl.), I166–I172 (2005). 4. Gray, J.A. et al. Conditionally immortalized, multipotential and multifunctional neural stem cell lines as an approach to clinical transplantation. Cell Transplant. 9, 153–168 (2000). 5. Clarke, D. & Frise´n, J. Differentiation potential of adult stem cells. Curr. Opin. Genet. Dev. 11, 575–580 (2001). 6. Pessina, A. & Gribaldo, L. The key role of adult stem cells: therapeutic perspectives. Curr. Med. Res. Opin. 22, 2287–2300 (2006). 7. Williams, D.R. et al. Synthetic small molecules that induce neurogenesis in skeletal muscle. J. Am. Chem. Soc. 129, 9258–9259 (2007). 8. Betz, W.J. & Bewick, G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 200–203 (1992). 9. Murthy, V.N. & Stevens, C.F. Synaptic vesicles retain their identity through the endocytic cycle. Nature 392, 497–501 (1998).

10. Watanabe, Y. et al. Conversion of myoblasts to physiologically active neuronal phenotype. Genes Dev. 18, 889–900 (2004). 11. Gaffield, M.A. & Betz, W.J. Imaging synaptic vesicle exocytosis and endocytosis with FM dyes. Nat. Protoc. 1, 2916–2921 (2006). 12. Yaffe, D. & Saxel, O. Serial passing and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725–727 (1977). 13. Katagiri, T. et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127, 1755–1766 (1994). 14. Chen, S., Zhang, Q., Wu, X., Schultz, P.G. & Ding, S. Dedifferentiation of lineage-committed cells by a small molecule. J. Am. Chem. Soc. 126, 410–411 (2004). 15. Dichter, M.A., Tischler, A.S. & Greene, L.A. Nerve growth factor-induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature 268, 501–504 (1977). 16. Zhou, T. et al. Neurons derived from PC12 cells have the potential to develop synapses with primary neurons from rat cortex. Acta Neurobiol. Exp. (Wars) 66, 105–112 (2006). 17. Bonavaud, S. et al. Preparation of isolated human muscle fibers: a technical report. In Vitro Cell. Dev. Biol. Anim. 38, 66–72 (2002). 18. Klein, M. & Kandel, E.R. Presynaptic modulation of voltage-dependent Ca2+ current: mechanism for behavioral sensitization in Aplysia califonica. Proc. Natl. Acad. Sci. USA 75, 3512–3516 (1978). 19. Roy, N.S. et al. In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat. Med. 6, 271–277 (2000).

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