Download 1. Introduction

1. Introduction
Discovered centuries ago, regeneration is a biological phenomenon that attract even
the common people and it is a fascinating field in novel biology that aim to solve the
impossible lost structure to regain again. The field may promise to implement the progress of
the success in near future upto the clinical setup. Regeneration is a biological phenomenon in
which the lost organ or tissues are restored to its nearly identical structures with the help of
associated cells, tissues and organs. This remarkable reparative process includes the
recognition and recapitulation of missing structures, with the functional integration between
the newly formed and pre-existing tissues in order to direct physiological and structural
alterations. Furthermore, the process of regeneration involving wound healing as an initiative
process which is accomplished by cellular proliferation at the site of amputation. Signaling
initiators are necessary to trace out the injury and the participating cells in regeneration are
guided by the cellular signals to the wound healing site and once the regeneration are
completed with the help of memory of the lost structure, specific process are required to
report the success of the regeneration which finally end with signal termination.
Regeneration ability is a wide spread among the animal kingdom from simple to
complex animal (Gabor and Hotchkiss, 1979). The specialized cells that are involved in the
regeneration are known as stem or progenitor cells. At the time of tissue repair mechanism
the stem or progenitor cells get activated and they start to proliferate and further they
differentiated to accomplish the lost structure. Numerous pathways are involved in stem cell
activation, the most well known pathways are Wnt, BMP, Notch, hedgehox, TGF. The
regeneration ability within the individuals varies with age, as we age the mutation are
deposited within the stem cells and it decline the regeneration capability that ultimately drive
to many age related symptoms. The proliferation of stem or progenitor cell is a double edged
sword. The controlled stem cell proliferation results in successful regeneration. On the other
hand, if the regeneration is not controlled it puts the tissues at risk of hyper proliferative
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diseases, the most deadly of which is cancer. This risk is controlled by many tumorsuppressor mechanisms, the most of which is p21 mediated tumor suppressor. This
mechanism particularly recognizes misbehaving cells thereby eliminating the potential cancer
cells by means of apoptosis or prevent their proliferation, by halting the cell division cycle.
The proper functioning of the stem cell affords longevity and on the other side it might risk
the animal with a life-threatening disease, the most of which is cancer.
1.1. Animal models for Regeneration studies:
Zebra fish has the regeneration ability and renews the myocardium vigorously and
restricts scar formation (Poss et al., 2002; Raya et al., 2003). In Enchytraeus japonensis,
regeneration was controlled by the nervous system (Yoshida-Noro et al., 2000) and, it was
reported that axon regeneration in Caenorhabditis elegan reveals that regeneration of several
neuron types including motor and sensory neurons, after injury (Ghosh-Roy and Chisholm,
2010). The Salamander can regenerate its limb, tail, upper and lower jaws, ocular tissues such
as the lens and retina, the intestine, and small sections of the heart (Eguchi, 1998; Ghosh et
al., 1994; Oberpriller and Oberpriller, 1974; O'Steen and Walker, 1962). The process behind
regeneration from planaria to amphibian limb regeneration was the formation of regeneration
blastema, a proliferative mass of undifferentiated progenitor cells from which new
differentiated cells arise (Reddien and Alvarado, 2004; Brockes and Kumar, 2005). In
addition, it was reported that in most of the oligocheate worms, neoblast has a common
morphological characteristics of undifferentiated cell types, such a high nucleocytoplasmic
ratio, a large nucleus with a large nucleolus and a basophilic cytoplasm (Randolph, 1897;
Krecker, 1923; Turner, 1934; Turner, 1935; Bilello and Potswald, 1974). Transplantation of
blastema taken from the amputated limb to the anterior chamber of the eye or a tunnel bored
in the connective tissue of the dorsal fin leads to formation of normal regeneration (Pietsch
and Webber, 1965; Stocum, 1968). It was reported that cells keeps the memory of their tissue
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origin during axolotl limb regeneration (Kragl et al., 2009). The complete knowledge
underneath regeneration was unexplored, in order to boosting up and to understand the basic
molecular and cellular processes governing regeneration the present study was carried out
and identified that the earthworm, Eudrilus eugeniae and Perionyx excavatus as a novel
model system to study the regeneration and stem cell biology. In addition, animal system
noted for regeneration studies were Hydra, Caenorhabditis elegans, Schmidtea mediterranea
and Enchytraeus japonensis, but earthworm is the highest evolutionary species capable of
regeneration ability.
1.2. Regeneration variation among animals:
The regeneration ability to replace the lost body parts in animal varies greatly
(Vorontsova et al., 1960; Goss, 1969; Brusca and Brusca, 2003) and understanding the
differences in regeneration ability is a key question in biology (Goss, 1969; Morgan, 1901;
Elder, 1979; Reichman, 1984). In 1901, T.H. Morgan, documented two modes of animal
regeneration: morphallaxis and epimorphosis. In the case of epimorphosis, regeneration
occurs by the proliferation of cells at the amputational site. Epimorphosis occurs mainly via
de-differentiation and subsequent re-differentiation of cells, or by the proliferation of reserve
population of stem cells. Examples, limb regeneration in amphibians (Brockes et al., 2001)
and compensatory regeneration of liver in mammals (Stocum, 2004; Taub, 2004). In case of a
morphallactic form of regeneration, upon amputational responses the existing nearby cells
reorganize its structure to form the newly organized structure
without the need of
proliferation of cells eg., hydra and planarians (Bode, 1973; Bosch and David, 1987;
Newmark and Alvarado, 2000). Enchytraeus japonensis is the fragmenting terrestrial
segmental worm, (enchytraeid Oligochaeta, Annelida, Lophotrochozoa, Protostomia) which
is evolutionarily close to earthworm (Nakamura, 1993). The worm has the capacity to recover
a complete individual from a fragment of the body by a combination of epimorphic mode of
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the head and tail regeneration and the morphallactic transformation to retain the proper body
proportions (Nakamura, 1993; Schmelz et al., 2000), and it has only small number of
neoblast. The ability of regeneration varies among earthworms. Eisenia fetida (Moment,
1949) and P.excavatus have the capacity to regenerate both anterior and posterior segment;
Capitella sp. II and Mediomastus sp., regenerate only the posterior segment, and they fail to
regenerate anterior parts (Bely, 2006). The mechanism determining the differences in the
ability of earthworm segments regeneration is still obscure. Amputation at the anterior
segments completely removes mouth, brain, and other germs cell associated organs and it is
remarkable to note that ingestion of nutrition is not possible upon anterior segments
amputation till the time of mouth parts is regenerated completely. The regeneration of the lost
organs entails stem cells amplification, their migration and more mitosis of differentiated
cells for organogenesis by synthesis and activation of growth factors and signalling molecules
(Baraniak and McDevitt, 2010; Konig et al., 2011; Sharma and
Snedeker, 2012).
Chondroitin sulfates are required for fibroblast growth factor-2-dependent proliferation and
maintenance in neural stem cells (Sirko et al., 2010); stem cell factor (SCF) (Cottler-Fox et
al., 2003) and TGF beta 1 (Tang et al., 2009) are required for migration of stem cell. Other
than signalling molecules, growth factors and micronutrients are essential for successful
regeneration. For instance, it has been reported that riboflavin is important for the earthworm,
E.eugeniae regeneration (Johnson et al., 2011) and the significance of vitamin A in
regeneration has been revealed (Maden, 1983a; Maden, 1983b). The micronutrients cannot
synthesize by any animal but they should obtain from either plant or microbes.
1.3. Advantages of earthworm as a model system for regeneration studies:
The hydra, the flat worm Planaria is being used as model animals for the regeneration
studies around the world (Morgan, 1898; Randolph, 1897).
When the earthworm is
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compared with other lower animals, earthworm is the best system to study the regeneration
because of the following reasons: 1. The worm E.eugeniae has ability to regenerate the lost
segments and organs; such as brain, heart, prostate, reproductive organs and many more in
two weeks of time and hence, for the molecular aspect of stem cell research, the earthworm
system is a wonderful resource; 2. Major part of its genome sequencing has been completed
by Expressed Sequence Tag project (http://www.earthworms.org); 3. Economical and easy to
maintain the earthworm in laboratory; 4. Higher growth rate as little as 5 weeks to reach
maturity (Rodriguez and Lapeire, 1992) and attains 12mg body weight per day; 5. It is
extremely fertile (Dominguez et al., 2001), and 6. It can tolerate extreme temperature
differences (ranges from 15-30°C) (Dominguez et al., 2001; Viljoen and Reinecke, 1992).
Many genes which are associated with the stem cell biology and the regeneration has been
reported (Salo et al., 2009). Among the reported genes, Spk 1, HOXA9 and HOXA10 are a
few examples. Those genes have been reported to have a role in the stem cell and
regeneration biology.
The interested model animals for the present study are the earthworms, E.eugeniae
and P.excavatus. Inorder to understand the regeneration variation that is observed between
the two species of the worm the present study was carried out. The systematic position of the
earthworm, E.eugeniae belongs to the kingdom - Animalia; Phylum - Annelida; Class Clitellata; Subclass - Oligochaeta; Order - Haplotaxida; Family - Eudrilidae; Genus Eudrilus and Species - eugeniae.
1.4. Taxonomy (systematic position) of earthworm, E.eugeniae:
Kingdom
:
Animalia
Phylum
:
Annelida
Class
:
Clitellata
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Subclass
:
Oligochaeta
Order
:
Haplotaxidae
Family
:
Eudrilidae
Genus
:
Eudrilus
Species
:
eugeniae
1.5. Taxonomy (systematic position) of earthworm, P.excavatus:
Kingdom
:
Animalia
Phylum
:
Annelida
Class
:
Clitellata
Subclass
:
Oligochaeta
Order
:
Haplotaxidae
Family
:
Megascolecidae
Genus
:
Perionyx
Species
:
excavatus
1.6. Importance of the earthworm in the field of science:
Earthworms are hermaphrodites (both male and female reproductive organs are
present in the each worm) and it has blood, circulatory system, developed nervous system,
digestive system and coelomic cavity, which is filled with coelomic fluid. Earthworm is a
beneficial organism and commonly called as “the farmers friend” by most of the authors
(Morowati, 2000) and it has the following beneficial roles such as soil fertility by increase
water holding capacity, improving and maintaining soil fertility, convert organic waste into
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manure (Edwards and Lofty, 1969; McCredie and Parker, 1992), drug and vitamins source, as
a natural detoxicant (Paoletti et al., 1991) and used as a bait for fish market (Ghosh, 2004)
and in addition it was also reported that the earthworm, E.eugineae plays a vital role in solid
waste management in the form of vermin-compost (Datar et al.,
1997). In contrast,
earthworm is also used for toxicological studies, it was reported that exposure of copper and
cadmium has a direct effect on reproduction, cocoon production and survival rate of
earthworm.
In addition to the above application, regeneration studies were also reported in the
following earthworms such as Eisenia andrei, Eisenia fetida, Lumbricus rubellus
(Blakemore, 1999a; Gates, 1972; Blakemore, 1998; Slims and Gerard, 1985), Ptychodera
flava (Rychel and Swalla, 2008) and Enchytraeus japonensis (Takeo et al., 2008). But there
was no report on regeneration studies in E.eugineae.
1.7. Biology of the earthworms, Eudrilus eugeniae and Perionyx excavatus:
The body segments of the earthworms are divided into three parts, namely i.) Preclitellar; ii) Clitellum and iii) Post-clitellar segments. The Pre-clitellum segments (1st-12th
segment in both E.eugeniae and P.excavatus) consist of all vital organs such as prostomium,
mouth, brain, heart, seminal vesicle, testis and a part of the ovary.
Clitellum region
(segments from 13th to 18th and 13th to 17th in E.eugeniae and P.excavatus, respectively) has a
major role in the reproduction. Post- clitellum region (segment 19th to last and 18th to last in
E.eugeniae and P.excavatus, respectively) has a pair of prostate gland, intestine and anus.
The external morphology of the earthworm, E.eugeniae is reddish brown in colour,
cylindrical in shape and metamerically segmented. The average length of the earthworm
varies from 12-18 cm and weight ranges from 1-2 gram. The arrangement of setae is
lumbricine type, 8 setae per each segment which serves as a locomatory organ. The anterior
part of the worm starts with the prostomium which is epilobus in nature. On the ventrolateral
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surface, the body segments 8, 9, 10, 11 and 12 shows the presence of spermathecal openings.
Exactly on the 12 segment, a pair of female genital openings is present. Segments 13-18
forms the unsegmented collar like structure called the clitellum. Clitellum is the important
segment for the release of cocoons and it has three different secretory cells namely 1. Cocoon
secreting cells; 2. Mucocytes which produces the mucus for mating and 3. Albumin secreting
cells, (Adiyodi et al., 1937). In the 16 segment, the pair of male genital openings is present. A
pair of well defined prostate gland starts from 18th segment and it ends in 24th segment of the
worm. The posterior parts of the worm have an intestine caeca to culture microbial gut
symbionts and the last segment of the worm ends with the anus.
The external morphology of the earthworm, P.excavatus is dark red in colour and the
body is dorso-ventrally flattened. The length of the worm varies from 5-18 cm with segments
ranges from 115-178. The anterior segment of the worm starts with Prostomium (epilobous).
Setae perichaetine, ranges from 40-54 with narrow mid-dorsal gaps. First dorsal pore is
present at 4/5 or 5/6. Clitellum covers from 13-17th segment. Two pairs of Spermathecal
pores in 7/8 and 8/9 which are large, very obvious, closely paired. Female pore single in 14th
segment. Male pores closely paired in 18th segment, in deep clefts in a common depressed but
tumid field. Gizzard absent or vestigial in 6th segment. Intestine starts from 18th segment and
the intestinal caeca are absent. Spermathecae two pairs in 8th and 9th segment. Large paired
ovaries are located in 13th segment. Testes two pairs each pair in segment 10 and 11. Seminal
vesicles in 9-12th segment. Prostate glands paired in 18th segment, large, round and racemose
type (Gates, 1927).
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1.8. Prostate cancer:
Prostate cancer (PCa) or carcinoma of the prostate (CaP) is one of the most prominent
cancers affecting the human male population around the world. A man has a 1/5 chance of
developing PCa during his lifetime (Feuer, 1997). The incidence of prostate cancer is rising
each year and in the UK more than 30,000 men are diagnosed with prostate cancer every year
(Gibbs et al., 2000). In the US, prostate cancer is the most common cancer in men and over
200,000 new cases were estimated to occur in 2007 (Jemal et al., 2007). Therefore, early,
definitive, and sensitive diagnosis of PCa is needed to initiate therapy to avoid the worsening
development of the disease.
The better knowledge about the factors that controlling the cell growth and
understanding the associated mechanism are necessary for developing the effective therapies
in regenerative medicine and cancer. Historically, lots of literature is supporting the link
between the cancer and tissue regeneration process, proposing regeneration is nothing but it is
the source of cancer and a method to inhibit tumorigenesis.
The non senescent phenotype of cancer resembles that of stem cells in many ways. An
fascinating hypothesis is that the genetic damage and the epigenetic changes that are
accumulated within a stem cell may involved in producing the cancer phenotype, and that
types of cells are essential and key to develop and maintenance of the cancer (Reya et al.,
2001; Tu et al., 2002; Taipale and Beachy, 2001)
1.9. Current Status of Biomarkers for Prostate Cancer:
To data tumour markers for prostate cancer are well defined. Some of which are blood
based markers and even others are prostate tissue based markers. The most predominant
biomarkers that revolutionized the detection process of prostate cancer are blood based
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marker, Prostate serum antigen (PSA) (Lundwall and Lilja, 1987; Oesterling, 1991). Other
than this prostein (Xu et al., 2001; Kalos et al., 2004), prostate stem cell antigen (PSCA) (Gu
et al., 2000), prostate-specific membrane antigen (PSMA) (Kawakami and Nakayama, 1997;
Su et al., 1995; Israeli et al., 1993), prostatic acid phosphatase (PAP) (Solin et al., 1990) and
transient receptor potential p8 (trp-p8) (Tsavaler et al., 2001) are well defined. One of the
major drawbacks of prostate tissue based markers are it needs a biopsy for detection and to
monitor the prostate cancer before and after the treatment regimen. Inorder to select the
precise treatment the sample of biopsy is necessary and that put the patient at uncomfortable
condition.
It has been shown that serum prostate-specific antigen (PSA) is the most reliable
tumor marker to detect PCa at the early stage and to monitor the recurrence of the disease
after treatment (Benson et al., 1992; Bradford et al., 2006; Brawer, 1999; Stephan et al.,
2006). Beyond that the quality of the present bio-marker (PSA) is still lacking the diagnostic
specificity. It was noted that more than half of the men with a PSA value over 4.0 ng/ml are
negative on initial biopsy (Stamey, 2001). In addition, it was also identified that PSA level
was also elevated in obesity or increased blood lipid level individuals (Larre et al., 2007;
Murtola et al., 2007). The protein is secreted in elevated amount during aging also. Hence it
hinders the screening of the cancer in the prostate.
Hence, identification of effective diagnosis tool is needed for screening the patients
having prostate cancer. The outcome of the biomarkers are either produced from the
cancerous tissue or by the body in response to the tumor. Prostate-specific G-protein coupled
receptor (PSGR) is a member of the G-protein coupled odorant receptor family, and is highly
expressed in prostate cancer cells compared with normal prostate cells (Xu et al., 2000; Yuan
et al., 2001; Weng et al., 2005), suggesting that PSGR may be targeted for the development
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of novel immunotherapeutic strategies against prostate cancer. The effort made by Weng et
al., 2005, in understanding the expression of PSGR in normal and prostate cancer patients
came with the interesting results of over expression of PSGR protein up to 10 folds higher
than the normal patients in the prostate gland. Since it is a prostate tissue based marker it
needs a biopsy of prostate tissue before and after the treatment regimen.
So far no promising diagnostic procedure for detecting prostate cancer in a human
therefore lot of researches are needed and effective model system also necessary to solve this
problem. Since the cancer is considered as an impaired regeneration, the knowledge obtained
in the normal process of prostate regeneration can be hand fully useful in understanding the
prostate cancer related issues.
Here we
discuss the prostate regeneration model, the invertebrate earthworm,
E.eugeniae in light of cancer regulation as well as the E.eugeniae emerge as a molecular and
genetic model system in which recent insights begin to molecularly dissect cancer and
regeneration in adult tissues. In this system, E.eugeniae the earthworm is able to regenerate
the lost or injured prostate gland which is implied in the diagnosis and understanding the
molecular biology of prostate cancer yet the lack of molecular biology tools to work with
these E.eugeniae coupled with their large and unsequenced genome constitute difficulties in
their use as animal models.
The prostate regeneration in earthworm consists of two phases, one is the wound
healing phase which begins immediately after amputation. The second phase is called the
redevelopment phase, in this phase the earthworm starts to regenerate the lost prostate gland.
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1.10. Animal models for prostate cancer:
An animal model for prostate cancer helps to understand the underlying mechanisms
that are behind the prostate cancer. For instant 72% of lewis rat and 27% of Wistar rats
develop prostatisis on administration of 17β-estradiol (Naslund et al., 1988). Still in realistic
the rarity of tumour formation, phenotypic variation, long latency periods, and lack of
metastases, makes the probability of using them as models as difficult. Other than this
transgenic mouse eg: prostate ovalbumin expressing transgenic mice (POEAT-3) were widely
used to study the acute prostatisis. Transgenic rat with p53 gene knockout and generating
knockout rats by transposon mutagenesis in spermatogonial stem cells is recently reported.
Three articles have recently been published describing different methods of generating
knockout rats (Geurts et al., 2009; Izsvák et al., 2010). The approach of genetically
engineered rat model could increase in the coming years (Hamra, 2010).
Naturally some strains of rat and dogs are developing the prostate cancer and they
serve as a valuable model for prostate cancer study (Rosol et al., 2003). Especially the dog
are closely associated and resembles to the human in the term of prostate cancer (LeRoy and
Northrup, 2009). Still there are limitation in the dog as a model system since the prostate
cancer seems not to be in control even after castration which shows that the tumor growth is
not androgen dependent (Winter et al., 2003). Other than this the practical problem are high
cost, long gestation period, and difficulty of genetic manipulation makes the dog an
unrealistic experimental model. Several strains of rats, including the Dunning, Copenhagen,
and Wistar rats, have been well characterized, and they develop a wide range of cancer
phenotypes in the prostate (Isaacs et al., 2008; Jeet et al., 2010; Pollard, 1980).
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1.11. Prostate Gland regeneration studies:
The rodent prostate involutes rapidly after castration, with apoptosis of prostate
epithelial cells observable within 48h after castration (Isaacs, 1985). Similarly, the prostate
regenerates rapidly after administration of testosterone to castrated animals with major
increases in the growth of the Epithelial compartment occurring during the first three days.
Distribution of young transit-amplifying cells may promote prostatic gland regeneration,
which usually occurs regular basis in some species. For example, seasonal variations in
testosterone levels in male squirrel monkeys, woodchucks, and the vizcacha (a South
American rodent) are well known to undergo changes in testicular volume (Baldwin et al.,
1985; Pasqualini et al., 1986; Fuentes et al., 1993). The results cannot show any parallel
measurements of prostatic size, even it is likely that prostatic involution may occur
concurrently with testicular regression. Thus, the prostate of these animals may undergo
physiological involution regeneration and the strategically located, young, transit-amplifying
cells within the ducts may facilitate the rapid regeneration of the gland. The earthworm,
E.eugeniae has a pair of well developed prostate gland located between 19-24th segment.
There is no report regarding the prostate gland regeneration studies in the earthworm.
For the present study we demonstrated the regeneration variation between two species of
earthworm, E.eugeniae and P.excavatus. Although both worms are clitellated, the
regeneration ability varies greatly. The regeneration potential of E.eugeniae has depended on
the intact Clitellum, whereas for the regeneration of P.excavatus clitellum is not important.
P.excavatus worm has the enormous regeneration ability, even the amputated 15 segments
individually can form the new worm whereas in the case of E.eugeniae the regeneration
ability is restricted within the clitellum segments and the amputated segments which have the
clitellum only have the ability to regenerate and the amputated segments those lacks the
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clitellum dies. Possibly multiple worms are possible from single individual worm upon
amputation of P.excavatus whereas only one successful worm results from the amputation of
E.eugeniae. The goal of the present study was to find out the reasons how these two species
of earthworm, E.eugeniae and P.excavatus naturally having this remarkable regeneration
variation.
The second part of the present study focused on developing the diagnostic tool for
prostate cancer using E.eugeniae as a model system. Since the earthworm E.eugeniae has
regeneration ability, the prostate gland of this worm was amputated to study their
regeneration ability. The earthworm, E.eugeniae share 80% genome similarity with the
humans and it should be used as an effective model system for experimental studies. The
earthworm, E.eugeniae have a pair of well developed prostate gland similar to human which
is an accessory gland for reproduction. So far no promising diagnostic procedure for
detecting prostate cancer in human therefore, lot of researches are needed and effective model
system also necessary to solve this problem. The present study can also focus to solve this
problem using E.eugeniae as a model system.
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1.12. Objectives:
1. To identify the stem cell niche in the earthworm, E.eugeniae and P.excavatus.
2. To characterize the differences in the ability of regeneration between the earthworm,
E.eugeniae and P.excavatus.
3. To reveal the mechanism at the cell and molecular level of regeneration between
the earthworm, E.eugeniae and P.excavatus.
4. To study the regeneration of the prostate gland in the earthworm, E.eugeniae.
5. To characterize the prostate specific proteins to be used as a diagnostic marker for
prostate cancer.
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