Cg-Clp2 involved in immune defence of the molluscCrassostrea gigas Fabien Badariotti, Christophe Lelong, Marie-Pierre Dubos and Pascal Favrel Universite´ de Caen Basse-Normandie, IBFA, U
Trang 1Cg-Clp2) involved in immune defence of the mollusc
Crassostrea gigas
Fabien Badariotti, Christophe Lelong, Marie-Pierre Dubos and Pascal Favrel
Universite´ de Caen Basse-Normandie, IBFA, UMR M100 IFREMER, Physiologie et Ecophysiologie des Mollusques Marins, Laboratoire de Biologie et Biotechnologies Marines, Caen, France
Glycoside hydrolase family 18 (GH18) is a
phylogenet-ically conserved group of proteins present in
eukaryo-tes, prokaryotes and viruses The GH18 family is
characterized by a Glyco_18 domain adopting an
(a⁄ b)8 triose phosphate isomerase-barrel structure that
consists of a specific arrangement of eight parallel
b-strands, forming the barrel core, surrounded by eight
a-helices [1] This family classification, based only on
similarities in amino acid sequences, groups together
chitinases and proteins devoid of catalytic activity due
to the substitution of a critical amino acid in the
cata-lytic centre This latter singular class of proteins, called
chitinase-like proteins (CLPs), has been identified only
recently in plants [2], mammals [3], insects [4] and
mol-luscs [5] CLPs have been implicated in many
biologi-cal processes, such as control of nodulation [2] and
growth⁄ differentiation balance during development in plants [6] Insect CLPs such as imaginal disc growth factors represent the first proliferating polypeptides reported from invertebrates [7] These mitogenic growth factors cooperate with insulin to stimulate pro-liferation, polarization and mobility of imaginal disc cells in vitro Imaginal disc growth factors may also constitute morphogenetic factors controlling embryonic and larval development, and could stimulate the cell growth required for wound healing [8,9] In mammals, CLPs such as YM1⁄ 2 and YKL-40 (40 kDa mamma-lian protein with the N-terminus YKL) [also known as human cartilage glycoprotein-39 (HC-gp39) in humans] are considered to be cytokines [10,11] involved in tis-sue remodelling during pathological conditions [12,13] Recently, the first lophotrochozoan CLP was identified
Keywords
chitinase-like protein; Crassostrea gigas;
immunity; lectin; mollusk
Correspondence
P Favrel, Universite´ de Caen
Basse-Normandie, IBFA, UMR M100 IFREMER,
Physiologie et Ecophysiologie des
Mollusques Marins, 14032 Caen cedex,
France
Fax: +33 231565346
Tel: +33 231565361
E-mail: pascal.favrel@unicaen.fr
(Received 22 February 2007, revised
10 May 2007, accepted 23 May 2007)
doi:10.1111/j.1742-4658.2007.05898.x
Chitinase-like proteins have been identified in insects and mammals as non-enzymatic members of the glycoside hydrolase family 18 Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes However, the precise physiologi-cal roles of Cg-Clp1 in oysters remain unknown Here, we report the clo-ning and the characterization of a new chitinase-like protein (Cg-Clp2) from the oyster Crassostrea gigas Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling Both Clp1- and Cg-Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity
Abbreviations
Cg-Clp1 ⁄ 2, Crassostrea gigas chitinase-like protein 1 ⁄ 2; CLP, chitinase-like protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GH, glycoside hydrolase; HC-gp39, human cartilage glycoprotein-39 (also called YKL-40); LPS, lipopolysaccharide; YKL-40, 40 kDa
mammalian protein with the N-terminus YKL.
Trang 2from the oyster Crassostrea gigas [5] Interestingly, this
protein, named C gigas chitinase-like protein 1
(Cg-Clp1) was found to be involved in the control of
growth and remodelling processes in a manner similar
to its YKL-40 mammalian counterpart These findings
argue for an early evolutionary origin and a high
con-servation of this class of proteins at both the structural
and functional levels Given the multiplicity of CLPs
in humans and insects [14], we hypothesized that
homologues of the previously characterized Cg-Clp1
remain to be found in C gigas
In this article, we report the characterization of a
new CLP, named Cg-Clp2, from the oyster C gigas
The tissue distribution and temporal pattern of
expres-sion of the gene encoding Cg-Clp2 during oyster
devel-opment were established by real-time PCR and in situ
hybridization In addition, the involvement of both
Cg-Clp2 and the previously identified Cg-Clp1 in
oys-ter immune defence was established
Results
Isolation and sequence analysis of Cg-Clp2
full-length cDNA
RT-PCR with degenerate primers whose design was
based on the conserved amino acid sequences of the
catalytic domain of members of the GH18 family
resulted in the amplification of an expected 147 bp
sequence Cloning and sequencing of this fragment
revealed an ORF showing amino acid sequence
simi-larity to members of the GH18 family Subsequently,
specific primers deduced from this 147 bp sequence
were used to perform 5¢- and 3¢-RACE-PCR to obtain
the full-length cDNA This experimental strategy has
been applied successfully in former studies, leading to
the identification of the two first C gigas members of
the GH18 family, Cg-Clp1 (AJ971241) [5] and the
chi-tinase Cg-Chit (AJ971238) [15] The complete 1697 bp
cDNA (AJ971235) revealed an ORF of 1425 bp,
start-ing with an ATG at position 117 and endstart-ing with a
TAA at position 1542 This ORF encodes a protein
named C gigas chitinase-like 2, composed of 475
amino acids with a putative N-terminal 19 amino acid
signal peptide (Fig 1) Cg-Clp2 contains one potential
recognition site for N-linked oligosaccharide [16] and
two potential recognition sites for O-linked
oligosac-charide [17] (http://www.cbs.dtu.dk/services) (Fig 1)
Cg-Clp2 sequence identity with other proteins
Optimal alignment of Cg-Clp2 with Cg-Clp1 and other
GH18 family members revealed regions of significant
identity, especially in the Glyco_18 domain The glu-tamate residue known to be critical for chitinase activ-ity [18] is substituted by a glutamine, suggesting that this protein lacks chitinolytic activity, as was shown previously for recombinant Cg-Clp1 [5] and other CLPs [19] Following the Glyco_18 domain, Cg-Clp2 displays an additional 90 amino acid C-terminal sequence of unknown function (Fig 1) Hence, the overall structure of Cg-Clp2 is similar to that of Cg-Clp1
Expression of Cg-Clp2 transcripts during development and in adult tissues
To gain insights into possible physiological functions
of Cg-Clp2, determination of its tissue distribution and temporal pattern of expression during development was performed by real time RT-PCR (Fig 2A) Cg-Clp2 transcripts were mainly expressed during larval metamorphosis, in the mantle edge and the digestive gland During the reproductive cycle, expression was high in gonads during the postspawning period but not in stage I, when gonial multiplication starts [20]
To investigate which types of cell were responsible for Cg-Clp2 expression in the mantle edge, in situ hybrid-ization experiments were performed (Fig 2B) Tran-scripts were detected in both epithelial and conjunctive cells of the mantle
Cg-Clp1 and Cg-Clp2 mRNA levels are increased
in haemocytes after bacterial LPS challenges
As the two mammalian CLPs, YM1 and YKL-40, were recently categorized as immune cytokines [10,11], the possible involvement of Cg-Clp1 and Cg-Clp2 in oyster immunity was investigated Gene expression was analysed by real-time RT-PCR in haemocytes at differ-ent times after injection of bacterial lipopolysaccharide (LPS) into the posterior adductor muscle and after an
in vitroLPS challenge
A marked increase in Cg-Clp1 expression was observed in vivo 9 h and 12 h after LPS injection (Fig 3A) relative to the respective controls Cg-Clp1 was also transcriptionally stimulated in vitro, 6 h and
12 h after LPS addition, in comparison to
unstimulat-ed control haemocytes (Fig 3B) However, this upreg-ulation was substantially lower than that observed for
in vivo challenge In contrast, Cg-Clp2 expression was not affected by in vivo LPS challenge (data not shown) but, as compared to unstimulated haemocytes, was stimulated in vitro 2 h after LPS addition (Fig 3C) Surprisingly, the Cg-Clp2 expression level was also significantly enhanced in adherent nonstimulated
Trang 3Fig.
Trang 4haemocytes as compared to freshly harvested
circula-ting cells
Discussion
In the present study, we identified a second oyster
CLP named Cg-Clp2 Comparative sequence analyses
with other GH18 family members show that Cg-Clp2
displays the same protein organization as the
previ-ously identified Cg-Clp1, with a Glyco_18 domain (in
a catalytically inactive form [5]) followed by an addi-tional C-terminal sequence of about 90 amino acids of unknown function The high degree of identity of the Cg-Clp1 and Cg-Clp2 Glyco_18 domains (84% iden-tity) argues for a conservation of the tertiary structure and associated biochemical properties (such as chitin binding) Evidence for a high level of conservation of the tertiary structure of CLPs during evolution is also supported by the observation that both Cg-Clp1 and its closest mammalian homologue YKL-40 present
A
B
Fig 2 Expression of Cg-Clp2 mRNAs in adult tissues and during development measured by real-time quantitative RT-PCR (A) Each value is the mean + SE of three pools of four animals (tissues) or the mean of one pool of embryos or larva from one spawn Expression levels are related to 100 copies of GAPDH (B) Localization of Cg-Clp2 mRNA expression in the mantle edge investigated by in situ hybridization Arrows indicate hybridization signals.
Trang 5similar biological activities on mammalian
chondro-cytes [5] As YKL-40 is only composed of the sole
Glyco_18 domain, the C-terminal tail of C gigas CLPs
may not noticeably contribute to the structure and the
function of these proteins Interestingly, Cg-Clp1 and
Cg-Clp2 C-terminal regions share relatively low levels
of sequence identity (46%), probably as the result of a lower pressure of selection during evolution Neverthe-less, these discrepancies may also account for slightly distinct biochemical properties
Analysis of mRNA distribution during development and in adult tissues shows that Cg-Clp2 is expressed
A
B
C
Fig 3 Real time quantitative RT-PCR analysis of Cg-Clp1 and Cg-Clp2 mRNA expression in haemocytes following bacter-ial LPS challenges In vivo experiment: time-dependent effect of LPS (100 lg) injection
on Cg-Clp1 expression (A) Results are means + SE of at least three oysters.
In vitro experiment: time-dependent effect
of LPS addition (final concentration
13 lgÆmL)1) to cell culture medium on Cg-Clp1 (B) and Cg-Clp2 (C) expression Results are means + SE of three wells Statistical analysis of the results was per-formed with Student’s t-test (*P < 0.05;
**P < 0.02).
Trang 6during metamorphosis, in the mantle edge and
post-spawning gonads Metamorphosis represents the
ulti-mate stage of oyster development, and is characterized
by the degeneration of larval tissues, such as the velum
and the foot, and the remodelling of larval tissues to
produce adult tissues (i.e the development of the gills
and the production of an adult shell), which is
accom-panied by significant growth of the soft body parts
[21] The mantle edge governs shell formation and
body growth by the secretion of shell organic matrix
and by cell proliferation As Cg-Clp2 appears to be
expressed in both epithelial and conjunctive cell types
of the mantle edge, this protein could orchestrate the
synthesis of extracellular components and⁄ or the
pro-liferation of mantle cells, as was proposed for Cg-Clp1
[5] The postspawning gonad is characterized by the
resorption of gonadic tubules and the rebuilding of
storage tissues [22] The expression of Cg-Clp2 during
this particular period is somewhat reminiscent of the
finding that certain mammalian CLPs such as CLP-1
and MGP40 are specifically expressed during
mam-mary gland involution [23,24] Considering Cg-Clp2
patterns of expression, this protein could be involved
in tissue growth and remodelling, as was formerly
postulated for Cg-Clp1 [5]
Messenger RNAs encoding Cg-Clp1 and Cg-Clp2
were upregulated in haemocytes after stimulation
with bacterial LPS This supports a role for Cg-Clp1
and Cg-Clp2 in defence against Gram-negative
bac-teria in response to LPS Nevertheless, it was
recently reported that commercial preparations of
LPS are often contaminated with peptidoglycan,
which actually constitutes the true
immunostimula-tory component in Drosophila [25] Thus, we cannot
rule out the possibility that a similar situation occurs
in C gigas
The fact that Cg-Clp1 (and most likely Cg-Clp2) is
known to bind tightly and specifically to chitin [5]
strongly supports a role of this lectin in the immune
response to chitinous pathogens, such as fungi and
nematodes, as was postulated for its mammalian
homologue HC-gp39 [11] Because bacteria do not
contain chitin, enhanced expression of Cg-Clp1 and
Cg-Clp2 in response to either LPS or peptidoglycan
stimulation might be considered as a general
nonspe-cific response of the organism to foreign invaders On
the other hand, both LPS and peptidoglycan harbour
GlcNAc, the constituent of chitin, in their molecular
structure A possibility is that Cg-Clp1 and Cg-Clp2
bind to bacteria via these cell wall components; if this
is so, the resulting overexpression of these lectins
should be considered as a specific immune response to
bacteria
The fact that Cg-Clp1 stimulates the proliferation and regulates the synthesis of extracellular matrix com-ponents of mammalian chondrocytes [5] endorses the possibility that Cg-Clp1 promotes cell (haemocyte) proliferation and⁄ or tissue repair, both processes occurring during immune responses [26,27] Such a role was also suggested for insect imaginal disc growth factors [8,9,28] As was observed for its murine homologue (YM-1), which behaves as a chemotactic cytokine that recruits cells to sites of inflammation and promotes eosinophilia around larvae of nematode parasites [10], mediation of immune cell (haemocytes) migration or aggregation might also represent a potential function for Cg-Clp1 Because Cg-Clp1 and Cg-Clp2 are two close paralogues sharing a very sim-ilar structure, the several roles predicted for Cg-Clp1
in immunity may also be relevant for Cg-Clp2 Interestingly, haemocyte adhesion to the culture plastic dish induces on its own a strong increase in Cg-Clp2 transcript expression, whereas no effect was detected for Cg-Clp1 Such a surprising result was previously observed for the oyster chitinase Cg-Chit [15] This
in vitro assay somehow mimics haemocyte conversion from circulating cells to cells that interact with and adhere to each other or to a foreign target surface, as
is observed for encapsulation [29] These ‘activated haemocytes’ may become immunologically competent cells capable of producing acute phase immune effec-tors, as was recently reported for Manduca sexta plas-matocytes, which express only the specific lectin
‘lacunin’ upon adhering to a foreign surface [30] This would explain why stimulation of Cg-Clp2 transcript expression is effective under in vitro but not in vivo conditions, when only circulating cells are harvested for gene quantification On the contrary, the partial failure of the in vitro cell culture conditions to elicit LPS stimulation of Cg-Clp1 gene expression may be due to the absence of pertinent haemolymph circulating factors in these experimental conditions Indeed, such extracellular molecules could be necessary for bacterial recognition as the first step in a process leading to an increase in Cg-Clp1 transcript quantity This hypothesis is in agreement with the observation that Drosophila host defence against Gram-negative bacteria may involve the secretion in the haemolymph
of a pattern recognition receptor [31,32] Alternatively, one could postulate that Cg-Clp1 is expressed mainly
in nonadhering haemocytes
Our results with C gigas Cg-Clp1 and Cg-Clp2 suggest strongly that these proteins fulfil an important function as immunity regulators and⁄ or effectors in molluscs The structural similarities shared by these two protein isoforms suggest they have similar
Trang 7biochemical mechanisms In contrast, their discrete
responses to bacterial challenge hint at distinctive
physiological functions in immunity
Experimental procedures
Animals
Adult C gigas oysters were purchased from a local oyster
farm (Saint Vaast La Hougue, France) The embryonic and
larval stages were produced in the IFREMER shellfish
laboratory of Argenton (France)
RNA purification, reverse transcription, cloning
and sequencing
Total RNA was isolated from the oyster mantle edge using
Tri-Reagent (Sigma-Aldrich, St Louis, MO, USA)
accord-ing to the manufacturer’s instructions mRNAs were
isola-ted using oligodT coupled to magnetic beads as described
by the manufacturer (Dynal, Invitrogen, Carlsbad, CA,
USA) Reverse transcription was carried out using
oli-go(dT)17as primer, 1 lg of mantle edge mRNA, and 200 U
of Moloney murine leukaemia virus reverse transcriptase
(Promega, Madison, WI, USA) cDNAs were used as
tem-plates for PCR amplifications using two degenerated
prim-ers designed to anneal to conserved consensus regions of
GH18 family members (chitinases and CLPs) from different
bilaterian species The sense primer corresponding to the
LK(I⁄ M)L(F ⁄ L)(S ⁄ T ⁄ R ⁄ C ⁄ W)VGG amino acid
seque-nce was 5¢-CTN AAR ATN CTN YTN WSN GTN GGN
GG-3¢, whereas the antisense primer corresponding to the
FDGLDLA amino acid sequence was 5¢-GGC NAG RTC
NAG NCC RTC RAA-3¢ (Y ¼ C or T, R ¼ A or G, S ¼
C or G, W¼ A or T, N ¼ A or C or G or T) PCR was
performed in a total volume of 50 lL with 10 ng of mantle
edge cDNA in 10 mm Tris⁄ HCl (pH 9.0), containing
50 mm KCl, 0.1% Triton X-100, 0.2 mm each dNTP, 1 lm
each primer, 2.5 mm MgCl2 and 1 U of Taq DNA
poly-merase (Eurogentec, Liege, Belgium) The reaction was
cycled between 94C, 50 C and 72 C (45 s, 60 s and 90 s,
respectively), and this was followed by an extension step at
72C for 5 min After 40 cycles, a resulting 147 bp
frag-ment was isolated Full-length cDNA was generated by
5¢-and 3¢-RACE using the Marathon cDNA amplification kit
(Clontech, Takara, Mountain View, CA, USA)
Double-stranded cDNA from oyster mantle edges was ligated to
adaptors, and 25 ng of this template was used to PCR
amplify 5¢- and 3¢-RACE fragments using adaptor-specific
primers and gene-specific primers deduced from the
ini-tial 147 bp fragment sequence PCR products were
sub-cloned into pGEM-T easy vector using a TA cloning kit
(Promega), and sequenced using ABI cycle sequencing
chemistry
Real-time quantitative PCR Quantitative RT-PCR analysis was performed using the iCycler apparatus (Bio-Rad, Hercules, CA, USA) Total RNA was isolated from oocytes, embryos, larvae and adult tissues using Tri-Reagent (Sigma-Aldrich) according to the manufacturer’s instructions After treatment for 20 min at
37C with 1 U of DNase I (Sigma-Aldrich) to prevent ge-nomic DNA contamination, 1 lg of total RNA was reversed transcribed using 1 lg of random hexanucleotidic primers (Promega), 0.5 mm dNTPs and 200 U of Moloney murine leukaemia virus Reverse Transcriptase (Promega) at
37C for 1 h in the appropriate buffer The reaction was stopped by incubation at 70C for 10 min The iQ SYBR Green supermix PCR kit (Biorad) was used for real-time monitoring of amplification (5 ng of cDNA template, 40 cycles: 95C for 15 s, 60 C for 15 s) with the following primers: QsCgClp1 (5¢-CTTCCTCCGCTTCCATGA-3¢) and QaCgClp1 (5¢-CCATGAAGTCCGCGAATC-3¢); and QsCgClp2 (5¢-GCATAGCGATGTGGACGA-3¢) and QaCgClp2 (5¢-GAGGACCGAGACCGTGAA-3¢) The abbreviations ‘Qs’ and ‘Qa’ refer, respectively, to sense and antisense primers Accurate amplification of the target amplicon was checked by obtaining a melting curve Using QsGAPDH (5¢-TTCTCTTGCCCCTCTTGC-3¢) and QaGAPDH (5¢-CGCCCAATCCTTGTTGCTT-3¢), a paral-lel amplification of oyster glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CGI548886) reference tran-scripts was carried out to normalize the expression data of Cg-Clp1 and Cg-Clp2 transcripts The relative level of expression of each target gene was calculated for 100 copies
of GAPDH transcript by using the following formula:
N¼ 100 · 2(Ct GAPDH ) cycle threshold transcript of interest)
In situ hybridization
A 1283 bp fragment corresponding to the most 3¢-end of Cg-Clp2 was subcloned in pGEMT easy This recombinant plasmid was used as a template for the synthesis of biotin-labelled sense and antisense cRNA probes according to the manufacturer’s instructions (NEN Life Sciences, PE, Wal-tham, MA, USA) Dissected C gigas mantle edges were fixed, dehydrated in an increasing alcohol series and xylene, and embedded in paraplast Seven-micrometre sections were cut and mounted on aminosilane-coated slides Sections were rehydrated, and endogenous peroxidase activity was blocked by incubating sections in 0.3% hydrogen peroxide
in methanol for 30 min at room temperature Slides were then washed and incubated in a blocking solution accord-ing to the manufacturer’s instructions Hybridization was performed overnight at 55C Biotin-labelled probes were detected using a streptavidin–horseradish peroxidase conju-gate Peroxidase activity was revealed by 3,3¢diaminobenzi-dine substrate (Sigma-Aldrich)
Trang 8Quantification of mRNA levels in haemocytes
after bacterial LPS challenge
In vivo challenge
Animals were injected with 100 lg (in 100 lL of NaCl⁄ Pi)
of Escherichia coli 026:B6 LPS (Sigma-Aldrich) into the
posterior adductor muscle, through a hole drilled in the
shell NaCl⁄ Pi-injected oysters were used as controls After
injection, animals were placed in sea water (12C) At four
time points after LPS injection (3 h, 6 h, 9 h and 12 h),
haemolymph samples from three animals were withdrawn
from the pericardic region using a 45-gauge needle and
cen-trifuged at 1000 g for 2 min (Eppendorf 5810R centrifuge,
fixed angle rotor F45-30-11) in order to separate cells from
the haemolymph fluid
In vitro challenge
Primary haemocyte culture was performed as previously
described, with some modifications [33] Haemolymph was
recovered from the pericardic region of 90 oysters using a
45-gauge needle, and then subsequently transferred to a
sterile tube and simultaneously diluted 1 : 3 in cooled sterile
anticoagulant modified Alsever’s solution (115 mm glucose;
27 mm sodium citrate; 11.5 mm EDTA; 382 mm NaCl)
Haemocytes were rapidly plated at 4· 106
cells per 9.5 cm2 well, to which three volumes of sterile artificial sea water
were added to allow cell attachment Cultures were
main-tained at 15C in a humidified incubator (CO2-free) After
60 min of incubation, cells were washed with Hanks-199
medium modified by the addition of 250 mm NaCl, 10 mm
KCl, 25 mm MgSO4, 2.5 mm CaCl2, and 10 mm Hepes; the
final pH was 7.4, and the osmolarity was 1100 mOsmolÆL)1
Cells were then covered with fresh medium supplemented
with l-glutamine (2 mm), concanavalin A (2 mm),
strepto-mycin sulfate (76.1 IUÆmL)1) and penicillin G
(100 IUÆmL)1), and were incubated (CO2-free) at 15C
Haemocyte monolayers were then treated for 30 min with
culture medium containing bacterial LPS (1 lgÆlL)1 in
NaCl⁄ Pi, final concentration 13 lgÆmL)1) Control (medium
without LPS) haemocyte monolayers were run in parallel
After 30 min, culture media were exchanged for fresh
media Haemocytes were lysed for total RNA extraction
with Tri-Reagent (Sigma-Aldrich) at different time points
of the experiment: haemocytes in suspension, immediately
after haemocyte adhesion, 30 min, 1 h, 2 h, 3 h, 6 h and
12 h after adhesion (control haemocytes), or 30 min, 1 h,
2 h, 3 h and 6 h after addition of LPS to the medium
Statistical analysis
Results were expressed as means + SE and analysed using
Student’s t-test The significance level was set as stated in
the legend to Fig 3
Acknowledgements
This study was financially supported by the ‘Conseil Re´gional de Basse-Normandie’, the ‘Agence de l’eau Seine-Normandie’ and FEDER Presage No 4474 grant (program PROMESSE) The authors are indebted to all staff of the Argenton IFREMER experimental hatchery for the production of oyster embryos and larvae The authors thank Christophe Fleury and Emeline Furon (University of Caen) for technical assistance
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