Insights on eukaryotic translation initiation factor 5A (eIF5A) in the brain and aging

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Insights on eukaryotic translation initiation factor 5A (eIF5A) in the brain and aging Augusto D. Luchessi a,⁎, Tavane D. Cambiaghi a , Adilson S. Alves a , Lucas T. Parreiras-E-Silva b , Luiz R.G. Britto a , Claudio M. Costa-Neto b , Rui Curi a a

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av Prof Lineu Prestes, 1524, 05508-900, São Paulo, SP, Brazil b Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil

A R T I C LE I N FO

AB S T R A C T

Article history:

Long-term memory, a persistent form of synaptic plasticity, requires translation of a

Accepted 13 June 2008

subset of mRNA present in neuronal dendrites during a short and critical period through a

Available online 24 June 2008

mechanism not yet fully elucidated. Western blotting analysis revealed a high content of eukaryotic translation initiation factor 5A (eIF5A) in the brain of neonatal rats, a period of

Keywords:

intense neurogenesis rate, differentiation and synaptic establishment, when compared to

eIF5A

adult rats. Immunohistochemistry analysis revealed that eIF5A is present in the whole

eIF-5A

brain of adult rats showing a variable content among the cells from different areas (e.g.

Dendritic translation

cortex, hippocampus and cerebellum). A high content of eIF5A in the soma and dendrites

Varicosity

of Purkinje cells, key neurons in the control of motor long-term memory in the

Brain aging

cerebellum, was observed. Detection of high eIF5A content was revealed in dendritic

Long-term memory

varicosities of Purkinje cells. Evidence is presented herein that a reduction of eIF5A content is associated to brain aging. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

The basic amino acid hypusine was discovered in 1971 in a trichloroacetic acid-soluble extract of bovine brain (Shiba et al., 1971). Hypusine was then found in other mammalian tissues as a free amino acid (in brain, liver, kidney, muscle, blood and intestine of rats) (Nakajima et al., 1971) and as a protein component (in brain, liver, kidney, heart, muscle, spinal cord, lung and spleen of rabbits) (Imaoka and Nakajima, 1973). Later, the eukaryotic translation initiation factor 5A (eIF5A) was identified as the unique hypusine-containing protein (Cooper et al., 1983; Park et al., 1981).

The hypusine residue is produced by the post-translational modification of a specific lysine residue in eIF5A chain in a reaction where spermidine is the substrate (Fig. 1). The 4-aminobutyl moiety of spermidine is transferred to the ɛ-amino group of the lysine50 (rat/human protein numbering) in the eIF5A precursor by deoxyhypusine synthase using NAD+ as cofactor. This is a reversible reaction that produces the intermediate deoxyhypusine residue. Subsequently, this intermediate residue is irreversibly hydroxylated at carbon 2 of the incoming 4-aminobutyl moiety by deoxyhypusine hydroxylase, producing the hypusine residue to complete production of the active eIF5A (Park, 2006). Free hypusine is derived from

⁎ Corresponding author. Fax: +55 11 30917285. E-mail address: [email protected] (A.D. Luchessi). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.057

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Fig. 1 – The eIF5A hypusination pathway. The deoxyhypusine synthase catalyzes the transfer of 4-aminobutyl moiety from spermidine to the ɛ-amino group of the lysine50 (rat/human protein numbering) residue of eIF5A precursor, using NAD+ as cofactor and producing the intermediate deoxyhypusine [Nɛ-(4-aminobutyl)-lysine] residue. Subsequently, the intermediate residue is hydroxylated at the carbon 2 of the incoming 4-aminobutyl moiety by deoxyhypusine hydroxylase, producing the hypusine [Nɛ-(4-amino-2-hydroxybutyl)-lysine] residue to complete the production of the active eIF5A.

the degradation of eIF5A, since no pathway for free hypusine synthesis has been found. Dipeptides containing hypusine, α(γ-aminobutyryl)-hypusine (or GABA-hypusine) (Sano et al., 1986; Sano et al., 1987) and α-(β-alanyl)hypusine (Ueno et al., 1991), were isolated from mammalian brain, but the biosynthesis pathway and function of these hypusine-containing peptides remain unclear. eIF5A was originally isolated from ribosomes of rabbit reticulocytes as one of the proteic components that are required for synthesis of methionyl-puromycin, an assay used to investigate factors involved in initiation of translation (Kemper et al., 1976). Its initial association with the translational process was suggested by experiments conducted in yeast where eIF5A physically interacts with the translating 80S ribosomal complex (Jao and Chen, 2006). A rapid depletion of eIF5A reduces by 30% the rate of protein synthesis (Kang and Hershey, 1994). eIF5A has been shown to be involved with nuclear export pathways (Elfgang et al., 1999; Hofmann et al., 2001; Rosorius et al., 1999; Ruhl et al., 1993; Schatz et al., 1998) and mRNA decay (Schrader et al., 2006; Zuk and Jacobson, 1998). The studies of eIF5A in yeast aimed to elucidate its function in mammals. In fact, the human eIF5A protein can replace the homologous yeast protein in vivo, revealing that both proteins are functionally similar (Schwelberger et al.,

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1993). However, on spite of the studies carried out up to now the precise molecular function of eIF5A in the translational process remains to be clarified. The concentration of hypusine in proteins of rat brain decreases from the first two weeks of postnatal life to adulthood (Sano et al., 1984). The decrease of hypusine follows a similar trend of changes in the quantity and specific activity of the deoxyhypusine hydroxylase (Abbruzzese, 1988). These observations suggest that eIF5A might play a role in brain development. Abbruzzese postulated that eIF5A may act on cell proliferation, based on previous findings that hypusination is intensified during activation of lymphocyte proliferation (Cooper et al., 1982), and that a high neurogenesis activity is observed in the first days of postnatal life (Altman, 1969). A recent in vitro study revealed that eIF5A and its hypusination are required for neurite growth and survival of PC12 cells (derived from rat pheochromocytoma) and primary hippocampal cells (from embryonic rats) in response to nerve growth factor (NGF) (Huang et al., 2007). In the present study, the content of eIF5A was evaluated in brains from neonatal (7 day-old) and adult (2–3 monthold) rats by western blotting. Immunohistochemistry for eIF5A was performed in certain brain regions of adult rats (e.g. cortex, cerebellum, and hippocampus). The content of eIF5A was also determined in cerebellum, cortex and hippocampus of adult (2–3 month-old) and aged (1.5 yearold) rats. Our results reveal insights on eIF5A in the brain and aging.

2.

Results

2.1. The content of eIF5A in the whole brain of rats decreases from neonatal to adult age Brains from neonate (7 day-old) and adult (2–3 month-old) rats were collected and the total brain extract was prepared. The same quantity of total proteins from brain extracts was used for the western blotting assay (Fig. 2). Brain from neonate rats presented a higher quantity of eIF5A than those from adult animals. The intensity of the bands obtained for the rat brains was normalized with the total protein distributed in the membrane. A decrease of about 80% in the content of the eIF5A in brain extracts from adult as compared to neonatal rats was found.

2.2. eIF5A is widely found in the whole brain of rats and is more abundant in certain cell groups Whole adult rat brains were analyzed by immunohistochemistry (immunoperoxidase method) using anti-eIF5A antibody. The protein eIF5A was widely distributed and presents variable contents in certain cell groups. Some cells of cerebellum, cortex and hippocampus showed differential and expressive contents of eIF5A (Fig. 3). In the hippocampus, a variable content of eIF5A was detected in cells from CA1 and CA3 areas and a high content in cells from dentate gyrus area. In the cerebellum, a high content of eIF5A was found in Purkinje cells showing a clear somatodendritic localization; in addition, eIF5A was highly detected in some punctual regions of the

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Fig. 2 – Content of eIF5A the brain of neonate and adult rats. Brain extracts from neonate (7 day-old) and adult (2–3 month-old) rats were prepared and the content of eIF5A was determined by western blotting analysis using 75 μg of total protein for each sample. The results of 4 animals, from each group, were quantified and normalized by the total protein content distributed in the blot membrane colored by Ponceau-S. The data are presented as mean ± SEM. *p < 0.0001.

Purkinje dendrites. Possibly, these regions correspond to dendritic varicosities. Some cells showed a more elevated content of eIF5A in nuclear and/or perinuclear regions than in the cytosol. The primary antibody was omitted from control cerebellum and a very low level of non-specific staining was observed. The antibody used was previously tested for unspecific labeling by western blotting analyses of total rat embryos in the presence of purified recombinant eIF5A (data not shown).

2.3. Varicosities associated to Purkinje cell dendrites reveal a high content of eIF5A Cerebellums from adult rats were analyzed by immunohistochemistry (immunofluorescence method) using anti-eIF5A antibody and anti-NFP antibody that reacts with neurofilament proteins (NFP): NF-L (low), NF-M (middle) and NF-H (high) (Fig. 4). The NFP fluorescence labeling clarified the Purkinje cell morphology and confirmed the somatodendritic localization

Fig. 3 – Determination of eIF5A in various brain areas of adult rats. The whole brain of adult rats was analyzed by immunohistochemistry (immunoperoxidase method) and the eIF5A content was determined in the cerebellum (CER), prefrontal cortex (PFC), hippocampus (areas CA1, CA3 and dentate gyrus). The arrow “α” indicates Purkinje cells from cerebellum; the arrow “β” indicates the detection of eIF5A in their dendrites and the arrow “δ” indicates putative labeled varicosities. The primary antibody was omitted from control cerebellum.

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Fig. 4 – Localization of eIF5A in the varicosities of Purkinje cells. The cerebellum of adult rats was analyzed by immunofluorescence using the detection of neurofilament proteins (NFP) as control. The arrows “α” indicate Purkinje cells; the arrows “β” indicate the detection of eIF5A in their dendrites and the arrows “δ” indicate varicosities. The primary antibodies were omitted from controls.

of eIF5A, including its high detection on dendritic varicosities. The primary antibody was also omitted from control and a very low level of non-specific labeling was observed.

2.4. A decrease of the eIF5A content was observed in the rat cerebellum and cortex during aging Adult (2–3 month-old) and aged (1.5 year-old) rat brains were collected and the cortex, hippocampus and cerebellum were carefully dissected out and used for total protein extract preparation. The same quantity of total proteins from each extract was used for western blotting assay to evaluate the eIF5A content (Fig. 5). The content of eIF5A in the cortex and cerebellum was decreased in brains from aged as compared to adult rats. There was no detectable change in the hippocampus. The intensity of the bands obtained from four animals of each group was normalized with β-actin. Based on the evidence that β-actin expression is increased during neonatal age (Poddar et al., 1996), this protein was not used as control in the experiments with neonate rat brains. A decrease of about 50% in the eIF5A content was found in the cortex and cerebellum of aged rats.

3.

Discussion

The involvement of eIF5A (eIF-4D, previous name) with the protein synthesis process was showed by its in vitro hypusinedependent stimulatory effect on methionyl-puromycin synthesis, an assay used to study factors involved in the forma-

tion of the first peptide bond (Benne et al., 1978; Kemper et al., 1976; Park, 1989; Smit-McBride et al., 1989). Other studies confirmed the participation of eIF5A in the translational process (Jao and Chen, 2006; Kang and Hershey, 1994) but the molecular mechanisms involved still remain to be elucidated. The eIF5A functions in the brain were previously addressed in two studies. In 1984, Sano et al. showed that brains from neonatal rats present a high content of hypusine in proteins (Sano et al., 1984). Abbruzzese showed that the quantity and specific activity of the deoxyhypusine hydroxylase are increased during the first day of postnatal life (Abbruzzese, 1988). Therefore, the possibility that eIF5A, the only protein that contains hypusine, plays an important function in brain development prompted us to carry out the present study. The eIF5A content in the whole brain of neonate (7 day-old) was compared to that of adult (2– 3 month-old) rats. The eIF5A content was markedly higher in the whole brain and in cortex, hippocampus and cerebellum (data not show) of neonates. Cooper et al. observed an increase of the hypusination process during activation of lymphocyte proliferation (Cooper et al., 1982). Based on this study, Abbruzzese postulated that eIF5A is associated with the intense neurogenesis observed in the first days of postnatal life (Abbruzzese, 1988; Altman, 1969). Several other studies have shown that eIF5A and its hypusination are important for the cell proliferation process (Balabanov et al., 2007; Bevec et al., 1994; Caraglia et al., 2003; Cracchiolo et al., 2004; Hanauske-Abel et al., 1994; Jasiulionis et al., 2007; Kang and Hershey, 1994; Lee et al., 2002; Nishimura et al., 2005; Shi et al., 1996a; Torrelio et al., 1984). During the

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Fig. 5 – Effect of aging on eIF5A brain content. Cerebellum, cortex and hippocampus extracts from adult (2–3 month-old) and aged (1.5 year-old) rats were prepared and the eIF5A content in 75 μg of total protein was compared by western blotting analysis using β-actin as an internal control. The results of 4 animals, from each group, were quantified and normalized in relation to the β-actin content. The data are presented as mean ± SEM. *p < 0.005.

first days of postnatal life, there is also intense neuronal differentiation and a recent in vitro study showed that eIF5A and its hypusination are also involved in this process (Huang et al., 2007). Searching for more information on eIF5A function, an immunohistochemistry analysis was carried out to investigate the distribution of this protein in the whole brain of adult rats. Evidence was obtained that eIF5A is found in hippocampal cells: CA1, CA3 and dentate gyrus areas. The hippocampus plays a central role in declarative long-term memory control and presents high rates of neurogenesis and differentiation (dentate gyrus), in comparison to other brain areas. However, eIF5A function cannot be associated to cell proliferation and differentiation only. This protein was also detected in certain adult brain areas that present poor neurogenesis and differentiation activity such as cortex and

cerebellum. The detection of eIF5A in Purkinje cells, a cerebellar key cell in the control of motor long-term memory, is noteworthy. These are non proliferating cells and present very high eIF5A content. This observation clearly supports the idea that the high eIF5A requirement is not restricted to highly proliferating cells. In fact, eIF5A production may alter in function of its requirement. Besides the soma localization, eIF5A was also detected in the dendrites of Purkinje cells. In addition, a high content was found in its dendritic varicosities, which represent portions involved in synaptic contacts. Some cells possessed, apparently, a more elevated content of eIF5A in the nuclear and/or perinuclear regions than in the cytosol (Fig. 2). This observation supports the proposition for an additional nuclear localization of eIF5A (Chen et al., 2003; Jao and Yu Chen, 2002; Jin et al., 2003; Parreiras-E-Silva et al., 2007;

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Rosorius et al., 1999; Ruhl et al., 1993; Shi et al., 1997; Shi et al., 1996b; Taylor et al., 2007). As mentioned above, there is a strong association between eIF5A with the protein synthesis process. The dendritic protein synthesis plays a central role in synaptic plasticity associated with the establishment of long-term memory (Bramham and Wells, 2007; Eyman et al., 2007; Frey et al., 1988; Glanzer and Eberwine, 2003; Kelleher et al., 2004; Sutton and Schuman, 2005; Sutton and Schuman, 2006). Experiments with immunofluorescence confirmed the elevated content of eIF5A in the varicosities associated to Purkinje cell dendrites revealed by co-localization with neurofilament proteins. So, eIF5A may be involved with translation of the mRNA subset found in dendrites and associated to the synapse. A decline in the rate of protein synthesis in forebrain, cerebellum and brain stem during aging, from adult (3 monthold) to aged (22.5 month-old) rats has been reported (Dwyer et al., 1980). Herein, a decrease of the eIF5A content in the cortex and cerebellum of aged rats (1.5 year-old) was found. This is the first indication for a decline of eIF5A production in a mammalian tissue associated with aging. Interestingly, there was no reduction of the eIF5A content in the hippocampus of aged rats. A decrease of this protein may occur in a more advanced age but certainly the changes in the eIF5A content induced by aging are not the same in all brain regions. In addition to the possible consequences for the cortex, the decrease of eIF5A content in cerebellum may be associated with reduced dendritic protein synthesis in the Purkinje cells. The reduction of the eIF5A content may contribute to the impairment of longterm motor memory that is observed during aging.

4.

Experimental procedures

4.1.

Animals

Male Wistar rats were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo. The rats were maintained at 23 ± 2 °C under a cycle of 12 h light and 12 h darkness, and were allowed free access to food and water. The experimental procedure of this study was approved by the ethical committee of animal experimentation of the Institute of Biomedical Sciences, University of São Paulo (protocol number 007; v.2; p.28; August 2nd, 2006).

4.2.

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the supernatant was used to determine the total protein content by the Bradford method (Bradford, 1976).

4.3.

Western blotting

One volume of loading buffer (150 mM Tris–HCl pH 6.8; 6% SDS; 0.1% bromophenol blue) was added to two volumes of each extract and heated for 5 min at 98 °C. A volume containing 75 μg of total protein was resolved in 15% SDS-PAGE using the electrophoresis buffer (24.8 mM Tris; 192 mM glycine; 0.1% SDS) at 75 V. The proteins were transferred to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences, Piscataway, NJ, USA), at 100 V for 1 h, using the transfer buffer (24.8 mM Tris; 192 mM glycine; 10% methanol). The transferred proteins were stained with Ponceau-S (0.1% Ponceau-S; 5% acetic acid) and the total protein in each lane was quantified by using the ImageJ software developed by the US National Institutes of Health and available at http://rsb.info.nih.gov/ij/. The values obtained in the quantification were used to normalize the content of eIF5A in the whole brain of neonate and adult rats obtained at the end of the western blotting assay. The membrane was washed with TBS-T (10 mM Tris–HCl; 150 mM NaCl; 0.1% Tween-20) to remove coloring and was blocked with TBS-T, containing 5% nonfat milk for 1 h at room temperature or 4 °C overnight. The membrane was then incubated with rabbit anti-eIF5A antibody (Parreiras-E-Silva et al., 2007) diluted 1:1000 in TBST, containing 5% nonfat milk, for 1.5 h at room temperature. After four washings with TBS-T for 5 min each at room temperature, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, Saint Louis, MO, USA) diluted 1:5000 in TBS-T, containing 5% nonfat milk, for 1 h at room temperature. After four washings with TBS-T for 5 min each at room temperature, the secondary antibody was detected by chemiluminescence (ECL Western Blotting Detection Reagents, Amersham Biosciences). For β-actin detection, used as an internal control, the same membrane used for eIF5A was washed, blocked again and incubated with monoclonal anti-β-actin antibodies (Sigma-Aldrich) diluted 1:6000 in TBS-T, containing 5% nonfat milk, for 1 h at room temperature. The remaining process was performed as described for eIF5A detection, except for the use of horseradish peroxidase-conjugated goat antimouse IgG as secondary antibody (Amersham Biosciences). The band intensity on X-ray film was analyzed by using the ImageJ software.

Protein extraction

Animals were killed by decapitation and the whole brains were collected. The whole brain or dissected regions corresponding to cerebellum, cortex and hippocampus were immediately frozen in liquid nitrogen. The frozen tissues were homogenized in extraction buffer (100 mM Tris–HCl pH 7.5; 10 mM EDTA pH 8.0; 10 mM sodium pyrophosphate; 0.1 mM NaF; 10 mM sodium orthovanadate; 2 mM PMSF; 10 μg/mL aprotinin) and treated with 1% Triton X-100. The lysates were incubated on ice for 30 min with strong shaking every 10 min for 30 s. The lysates were centrifuged at 12,000 ×g for 20 min at 4 °C and the supernatants were collected and transferred to a −80 °C freezer until being used for western blotting analysis. An aliquot from

4.4.

Immunohistochemistry

4.4.1.

Brain slice preparation

The animals were deeply anesthetized with i.m. ketamine chloridrate (5 mg per 100 g b.w.) (Ketamina-Agener, União Química Farmacêutica Nacional, Embu-Guaçu, SP, Brazil) and xylazine chloridrate (2 mg per 100 g b.w.) (Rompun, Bayer, São Paulo, SP, Brazil), and transcardially perfused with phosphate buffer saline pH 7.4 (PBS), followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PB). The brain was excised, incubated with 4% paraformaldehyde in PB for 4 h and transferred to 30% sucrose in PB for 48 h at 4 °C. Frozen brain slices (30 μm) were cut using a cryostat and were

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free-floating incubated with 5% bovine serum albumin (BSA) in PB for 1 h at room temperature.

4.4.2.

Immunoperoxidase method

Brain slices were free-floating incubated overnight with rabbit anti-eIF5A antibody diluted 1:500 and normal donkey serum diluted 1:200 (Jackson Laboratories, West Grove, PA, USA) in PB containing 0.3% Triton X-100 at room temperature. After three washings with PB for 10 min each at room temperature, the sections were incubated with a biotinylated donkey antirabbit antibody (Jackson Laboratories) diluted 1:200 in PB, containing 0.3% Triton X-100, for 2 h at room temperature. The sections used for immunoperoxidase method were washed three times with PB for 10 min each at room temperature and incubated with avidin-biotin-peroxidase (ABC Elite, Vector Labs, Burlingame, CA, USA) for 2 h at room temperature. The reaction was developed with 0.05% 3,3¢-diaminobenzidine tetrahydrochloride (DAB) and a solution of 0.01% hydrogen peroxide in PB. Intensification was performed by using 0.05% osmium tetroxide in water. The sections were mounted on gelatinized slides, dehydrated, cleared and coverslipped. The primary antibody was omitted from the control.

4.4.3.

Immunofluorescence method

Brain slices were free-floating incubated overnight with rabbit anti-eIF5A antibody diluted 1:100, monoclonal mouse antineurofilament (NFP) diluted 1:1000 (Zymed Laboratories Inc, South San Francisco, CA, USA) and normal donkey serum diluted 1:50 (Jackson Laboratories) in PB containing 0.3% Triton X-100 at room temperature. After three washings with PB for 10 min each at room temperature, the sections were incubated with FITC-conjugated donkey anti-rabbit IgG (Jackson Laboratories) diluted 1:50 and TRITC-conjugated donkey anti-mouse IgG (Jackson Laboratories) in PB, containing 0.3% Triton X-100, for 2 h at room temperature. The sections were washed three times with PB for 10 min each at room temperature and mounted on gelatinized slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). The primary antibody was omitted from the controls.

4.5.

provement of Higher Education Personnel (CAPES) and T.D.C. was recipient of a fellowship from FAPESP.

Statistical analysis

The data are presented as mean ± SEM and analyzed by Student t-Test. Differences between values were considered as statistically significant for p < 0.05. All results were analyzed using the GraphPad Prism 4.0 statistical software (GraphPad Software, Inc., San Diego, CA, USA).

Acknowledgments The authors are grateful to J.R. de Mendonça and G.O. de Souza for their technical assistance; to Ms. R.H. Lambertucci for providing the aged rats and to Dr. S.M. Hirabara, Dr. A.H. Kihara and Dr. E.R. Kinjo for the valuable discussions during the experiments. This study is supported by The State of São Paulo Research Foundation (FAPESP) and by The National Council for Scientific and Technological Development (CNPq). A.D.L. was recipient of a fellowship from the Coordination for the Im-

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