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RESEARCH |
1 Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia, 2 Department of Paediatrics, University of Melbourne, Australia and 3 DAKO Australia, 12 Lord Street, Botany, NSW 2019, Australia
Correspondence should be addressed to K J Fowler; Email: kerry.fowler{at}mcri.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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-satellite DNA in man or minor satellite in mouse via a 17 bp sequence known as the CENP-B box motif. These dual properties of CENP-B suggest that this protein organises the assembly of repetitive DNA at the primary constriction, facilitating centromere/kinetochore formation (Yoda et al. 1992). The role of CENP-B in centromere biology has been controversial for many years, early studies in cell culture suggesting that it is an indispensable protein (Bernat et al. 1990, 1991). Recent studies in fission yeast support this and suggest that two to three CENP-B homologues are required to establish a functional centromere (Baum & Clarke 2000, Irelan et al. 2001). On the other hand, there are many independent lines of evidence to show that CENP-B is expendable for mammalian centromere formation (Earnshaw et al. 1989, Sunkel & Coelho 1995, Goldberg et al. 1996, Choo 1998). While gene knockout experiments in mice have indicated that Cenpb is non-essential for mitosis and meiosis (Hudson et al. 1998, Kapoor et al. 1998, Perez-Castro et al. 1998, Tomascik-Cheeseman et al. 2002), it has been shown to be crucial for proper uterine development and function (Fowler et al. 2000).
Cenpb-null mice generated from early-generation heterozygous intercrosses on three different genetic backgrounds, known as R1, W9.5 and C57, displayed a marked reduction in uterine weight at 10 weeks old and demonstrated an age-dependent reproductive dysfunction that was more severe in the C57BL/6 background. Breeding studies with 8–15-week-old C57 Cenpb-null female mice resulted in slow or difficult delivery of pups, whereas the R1 and W9.5 Cenpb-null females bred normally for the first three to four litters before developing pregnancy problems by the age of 9 months. Many overdue females sickened, and on autopsy dead intact or resorbing foeti and pyometra were found (Fowler et al. 2000).
When Cenpb-null early-generation mice were compared with age-matched wildtype female mice, no difference in ovarian tissues, progesterone and ß-oestradiol levels or embryo number was detected. However, histological analysis of uterine tissue from 10-week-old C57 and 6–9-month-old R1 Cenpb-null females revealed abnormal luminal and glandular epithelium, fewer endometrial glands, and increased leucocyte infiltration, haemorrhage and infection. Furthermore, in situ hybridisation using a specific Cenpb-antisense probe detected high levels of Cenpb expression in the epithelial lining of the uterine lumen and endometrial glands of normal mice (Fowler et al. 2000). In this study we further explore the unexpected role of Cenpb in mouse uterine development and function. Our specific objective was to determine whether interbreeding Cenpb-null mice over successive generations would result in the exacerbation of the Cenpb-null phenotype, and, if so, what the underlying causes might be.
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Materials and Methods |
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Ethics of experimentation
This study was conducted in accordance with Australian code of practice for the care and use of animals for scientific purposes and under approval of the Royal Children’s Hospital Animal Ethics Committee (project numbers A388 and A450).
Examination of mice for chronic infectious agents
Serum samples from breeding colonies of mice were sent to the Murine Virus Monitoring Service, South Australia, for routine health testing every 6 months over the past 3 years. The sample size was statistically representative of mice in the colony and showed an absence of mouse hepatitis virus, rotavirus and parvovirus. In addition, the Department of Microbiology and Infectious Diseases, Royal Children’s Hospital and Cerberus Sciences, South Australia, performed supplementary testing for known mouse uterine pathogens. Serum and uterine swabs were collected from 18-week-old G2 Cenpb-wildtype (n = 2), -null (n = 2) and -heterozygote (n = 4) virgin female mice and 10–11-week-old G3 Cenpb-wildtype (n = 2) and -null (n = 2) virgin female mice. Mice tested negative for minute virus of mice, mouse parvovirus, Mycoplasma pulmonis, Sendai virus, Chlamydia, Pasteurella pneumotropica and Salmonella enteritidis. No other bacterial pathogen of consequence was detected.
Genomic scan of Cenpb-null mice on C57BL/6 background
Tail DNAs from Cenpb-heterozygous mice on a C57BL/6 background (C57+/-) (Fowler et al. 2000) were sent to the Jackson Laboratory’s Speed Congenic Service (Bar Harbor, ME, USA). Simple sequence length polymorphisms (SSLP) analysis using 108 markers that are polymorphic between C57 and 129 DNAs were used to determine the amount of residual 129 DNA surrounding the targeted Cenpb locus.
Monitoring of mice for reproductive performance
Breeding pairs were set up and females were examined for vaginal plugs, pregnancy and number of pups born by routine mouse husbandry procedures (Fowler et al. 2000).
Organ weighing, histology and immunohistochemical analysis of endometrial gland cells
Organ weights (mg) were determined by weighing wet uteri and ovaries (Fowler et al. 2000). After weighing, mouse uteri were formalin-fixed, and a 2-3 mm piece of tissue was dissected from the mid-region of randomly selected left or right uterine horns and paraffin-embedded, and 5 µm transverse sections were cut onto silanised slides and deparaffinised. Following antigen retrieval using DAKO Target Retrieval Solution, pH6 (S1699; DAKO, Carpinteria, CA, USA), in a water bath for 20 min at 95–99 °C, the slides were cooled for 20 min and equilibrated in Tris-buffered saline with Tween 20. After endogenous peroxidase activity was quenched using peroxidase blocking reagent (DAKO S2001) for 5 min, polyclonal anti-pro-gesterone receptor antibody (DAKO A0098), monoclonal anti-oestrogen receptor antibody (DAKO M7047) and anti-pan cytokeratin antibody (DAKO M3515) (Moll et al. 1982) were used at room temperature at dilutions of 1/50, 1/50 and 1/200 respectively.
Monoclonal antibodies were stained according to manufacturer directions using the DAKO Animal Research Kit (K3954), which uses a modified avidin-biotin/peroxidase technique. Polyclonal antibody staining was carried out using the DAKO EnVision + /HRP (K4003), which uses an enzyme-labelled polymer technique. Immunolabelled cells were visualised with 3,3'-diaminobenzidine (K3468, DAKO) for 5 min and counterstained with Mayer’s haematoxylin. All staining was performed using a DAKO Auto-stainer.
Assessment of endometrial gland density
To assess the number of glands in the uterine endometrial layer 2, haematoxylin and eosin-stained full-face transverse 5 µm sections from the mid-region of randomly selected left or right uterine horns were examined microscopically. All visible glands were counted regardless of size, and the average was recorded for each section.
Photomicrographs
Images of stained sections were captured using an Olym-pus BX50 microscope equipped with a 10 x eyepiece and 40 x objective, and a CCD camera linked to an IBM computer with a TCI-pro program. Digital images were assembled with PhotoShop, Version 6.0.
Statistical analyses
Values are presented as average±S.D. P values were calculated using Student’s t-test. P values of less than 0.05 were regarded as significant.
Ovarian transplantation studies
Mice were anaesthetised with Avertin (tribromoethanol and amyl alcohol) (Hogan et al. 1994), and both ovaries were removed and replaced with one-half ovary per side, as described by Sztein et al.(1998), Mice were set up as breeding pairs with C57BL/6 males 10 days after surgery and monitored daily.
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) due to fewer litters being born (Table 1B). Comparison between R1+/+ and R1+/- breeding pairs showed no significant difference except for G3 intercrosses, where less pups and litters were produced by the R1+/+ pairs than the R1+/- breeding pairs (Table 1A and B). A similar breeding record was observed for the W9.5-/- pairs when compared with W9.5+/+ matings (Table 2A and B). In addition, when reproductive performance was compared between G1 breeding pairs and G2, G3 and G4 breeding pairs, a general trend towards deteriorating reproductive outcome through successive generations in terms of numbers of pups and litters was observed, with the difference being particularly significant when G1 and G4 W9.5-/- mice were compared (P 
0.005).
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), litters (P = 1.0; Fig. 1B) and pups per litter (P = 0.890; Fig. 1C). However, the R1-/- G4 females, when examined against the R1+/+ G4 females, produced significantly fewer pups (P < 0.001; Fig. 1A), litters (P = 0.005; Fig. 1B) and pups per litter (P = 0.038; Fig. 1C).
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), they were not smaller than 10-week-old R1-/- and W9.5-/- G1 uteri from the original heterozygous crosses (R1-/- G1: 79.7 ± 27.8 mg (n = 7) vs R1-/- G4: 55.9 mg (n = 1), P = ND; W9.5-/- G1: 82.5 ± 19.0 mg (n = 5) vs W9.5-/- G4: 70.8±17.2 (n = 4), P = 0.376) (Fowler et al. 2000), suggesting that breeding through successive generations did not have an effect on the size of the uterus in the null mice.
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; Table 4A and C). In contrast, 10-week-old W9.5-/- G4 mice (n = 3) and R1-/- G4 mice (n = 4) of the equivalent generation derived from heterozygous breeding pairs did not display a significant reduction in the number of endometrial glands when compared with littermate W9.5+/+ (n = 3) and R1+/+ (n = 4) G4 mice respectively (Fig. 2C and D; Table 4B and D).
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–H). When 24- and 32–36-week-old G4 R1-/- from heterozygous breeding (Table 4G and H) were compared with 10-week-old G4 R1-/- (Table 4D), the number of endometrial glands had declined with ageing (10 vs 24 weeks, P = 0.053; 10 vs 32 weeks, P = 0.037). On the other hand, the homozygous breeding regime resulted in R1-/- G4 mice with a low number of endometrial glands at 10 weeks old that remained low with age (Table 4C, E and F). However, a reverse trend was observed in the wildtype R1 mice from the homozygous breeding strategy, in which the number of endometrial glands actually increased with age (Table 4C, E and F; 10 vs 24 weeks, P < 0.001; 10 vs 32 weeks, P = 0.009). Although the wildtype mice from the R1+/+ heterozygous breeding programmes had increased their gland number with age, it was not significant (Table 4D, G and H; 10 vs 24 weeks, P = 0.070; 10 vs 32 weeks, P = 0.129).
Abnormal luminal epithelium in uteri of 24-week-old R1-/- G4 mice
Besides a reduction in endometrial gland number, uteri from 24-week-old R1-/- G4 mice from heterozygous or homozygous breeding programmes (n = 7 total) were observed to have ulcerated (n = 2) or disorganised (n = 1) luminal epithelium or oedema in the endometrium (n = 1, Fig. 2F and H), whereas no obvious abnormality was noted in the uteri of control R1+/+ mice (n = 10 total; Fig. 2E and G). Given the similarity of these observations to those seen in our previous study, we re-examined the haematoxylin and eosin slides of uteri of 24-week-old G1 mice from the original heterozygous crosses (Fowler et al. 2000). Counting of endometrial glands revealed a reduction in the average number of glands in R1-/- G1 mice (15.8 ± 0.7; n = 5) when compared with R1+/+ G1 mice (50.1± 35.1; n = 7), but the difference was not statistically significant (P = 0.080).
Greatly reduced number of endometrial glands in 3-, 4-, 6-, 8-, 10- and 24-week-old C57-/- G1 mice
The observed reduction in the number of endometrial glands of R1-/- and W9.5-/- G4 mice and the more severe breeding problems of Cenpb-null and -wildtype mice on the C57BL/6 background (Fowler et al. 2000) prompted us to examine uterine sections of C57-/- and C57+/+ mice at 3, 4, 6, 8, 10 and 24 weeks of age at G1. C57-/- females were found to have a significant reduction in gland number when compared with age-matched C57+/+ (Table 5A–F), suggesting that the endometrial glands failed to develop properly during the pre- and post-pubertal period on this genetic background.
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Immunohistochemical analysis of endometrial gland cells
It was noted when counting the haematoxylin and eosin-stained glands in the uterine endometrium of Cenpb-null mice that some glands had markedly reduced lumens as compared with the glands of wildtype mice (Fig. 3A and B). To ensure that all glands were being scored on the haematoxylin and eosin sections, a number of slides were stained with anti-pan cytokeratin antibody, which marks epithelial cells (Moll et al. 1982) (Fig. 3C and D). The counting results were comparable, indicating that the assessment on haematoxylin and eosin sections was accurate. To examine whether the glandular epithelium layer of R1-/- G4 mice had retained oestrogen and progesterone receptors, sections of uterus from 32–36-week-old, non-mated mice were stained with anti-oestrogen receptor and anti-progesterone receptor antibodies. Positive-staining nuclei at apparently comparable density were present in the endometrial glands and stroma of R1-/- and R1+/+ G4 mice, suggesting that functional oestrogen (Fig. 3E and F) and progesterone (Fig. 3G and H) receptors were present in the R1-/- mice.
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Ovarian transplantation fails to rescue the poor breeding performance of Cenpb-null mice
Further evidence that the ovary was not responsible for the poor breeding performance of young adult Cenpb-null mice was provided by the failure of ovarian transplantation to rescue the phenotype. R1+/+ and R1-/- G4 mice 7–9 weeks old were transplanted with ovaries from 4-week-old R1+/+ G4 mice, as described by Sztein et al.(1998) The wildtype ovaries failed to give rise to normal numbers of litters and pups in the R1-/- females. The R1-/- females (n = 5) averaged just one litter of 2.0±1.6 pups per litter, whereas transplanted R1+/+ mice (n = 6) were able to generate, on average, 5.8±1.0 litters with 4.8±2.3 pups per litter. When the transplanted mice were 9 months old, they were culled, and the number of endometrial glands per transverse uterine section was assessed. The R1+/+ mice had 36.0±19.3 glands per transverse section, whereas the R1-/- mice had 6.0±5.6 glands (P = 0.043). Furthermore, when 5-week-old R1+/+ G4 mice (n = 2) were transplanted with ovaries from 32-week-old R1-/- G4 mice, the mice were able to produce an average of 5.5±0.7 litters with 4.4±2.3 pups per litter.
In addition, four virgin, 24-week-old C57+/- G1 females underwent ovarian transplantation with 4-week-old C57BL/6 ovaries. One female failed to become pregnant and two females developed birthing problems with their first litter similar to their non-transplanted counterparts.
Genome scan analysis of C57+/- mice
DNA microsatellite markers that detect polymorphisms between 129 and C57BL/6 DNAs were used to determine the amount of remaining 129-derived DNA in Cenpb-null mice on C57BL/6 background. Genome scan analysis revealed a residual 129 region encompassing approximately 41 cM around the targeted Cenpb locus (73 cM position) on chromosome 2 (mouse genome database, MGD). The closest C57 markers were located at the 43 and 84 cM positions, 129 markers being present at 52 and 69 cM.
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The significant reduction in the number of endometrial glands of 10-week-old R1-/- and W9.5-/- G4 mice (Table 4A and C) is intriguing. It would appear that Cenpb-null G4 females carried and reared by a Cenpb-heterozygote R1 or W9.5 dam surrounded by heterozygous and wildtype littermates were not quite as susceptible to significant decreases in endometrial gland number at 10 weeks old (Table 4B and D). However, by 24 weeks of age, any possible parental R1 or W9.5 heterozygote effect had dissipated, as Cenpb-null G4 mice from both homozygous and heterozygous breeding pairs were noted to have significantly lower numbers of endometrial glands (Table 4E and G).
Further support for endometrial gland development being influenced pre- and postpubertally can be gleaned from the Cenpb-null mice on a C57BL/6 background. On this background the uterine phenotype of Cenpb-null ice has been reported as being more dramatic (Fowler et al. 2000). Disorganisation of luminal and glandular epithelium was observed in the uteri of 10-week-old C57-/- G1 mice (n = 4), 50% displaying ulcerated luminal epithelia when compared with C57+/+ G1 mice (n = 3) that exhibited normal epithelia. Besides abnormal uterine epithelium, a reduction in the number of endometrial glands in 10-week-old C57-/- G1 mice was previously noted, but not quantified (Fowler et al. 2000). In the current study, 3-week-old C57-/- G1 mice carried by a C57+/- dam were found to have a 90% reduction in their endometrial gland number (Table 5A) that remained low up until at least 24 weeks old (Table 5B–F).
As well as abnormal uterine epithelium, our previous studies have shown that the majority of 8–15-week-old C57-/- G1 breeding females developed birthing problems (Fowler et al. 2000). In the present study pregnancy problems and impaired gland development were observed in 24-week-old heterozygote C57 G1 females. Histologically, 24-week-old C57+/- displayed a similar profile of endometrial gland scarcity.
Genome scan analysis of C57+/- DNA revealed a 41 cM region of residual embryonic stem cell-derived 129 DNA. Previously, Kapoor et al.(1998) had proposed that the phenotypic differences seen in their mice and ours might be due to neighbouring gene effect. Of interest are genes known as progestogen-dependent endometrial protein (Paep) (Chan et al. 1994) and cyclin-dependent kinase-1 (Cdc25b) (Lock et al. 1996) that have been previously assigned to the 73 cM position on mouse chromosome 2 in the region of Cenpb (Jackson Laboratory MGD). With respect to Paep, neither the NCBI nor Celera mouse databases have located Paep in the mouse genome, although PAEP has been confirmed in the human genome (GDB: 6276819). In regard to Cdc25b, knockout mouse studies have revealed a female sterility problem that is specifically due to Cdc25b-/- oocytes being arrested during meiosis, rather than a uterine problem (Lincoln et al. 2002). Furthermore, our previous work describing the generation and normal phenotype of a targeted control mouse that carried the internal ribosome entry site (IRES)-neomycin selectable marker element in the Cenpb gene, but not the translational frameshift mutation, suggests that the observed phenotype in Cenpb-null mice is due to Cenpb-gene disruption rather than a nearby gene effect induced by the targeting construct (Fowler et al. 2000).
The lack of endometrial glands in the Cenpb-null mice resembles the phenotype observed in sheep that were treated neonatally with synthetic progesterone known as norgestomet (Gray et al. 2001). Keeping the ovine uterus in a progesterone-rich postnatal environment suppressed epithelial oestrogen receptor- protein expression and altered the expression of normal paracrine cues for the development and proliferation of endometrial glands from the luminal epithelium (Gray et al. 2000). This resulted in a permanent ablation of endometrial glands and subsequent implantation failure and loss of reproductive fitness in treated ewes transplanted with normal blastocysts. Unlike the Gray studies, we have found no evidence of abnormal hormone levels (Fowler et al. 2000) or loss of oestrogen receptor expression contributing to the lack of endometrial glands observed in Cenpb-null mice. However, future studies on the expression levels of other growth factors and/or receptors involved in endometrial gland formation or uterine receptivity (Gray et al. 2000, 2001, Cheng et al. 2001) may help shed light on the uterine phenotype of Cenpb-null mice.
Interestingly, some aspects of the phenotype of G1 Cenpb-null mice (Hudson et al. 1998, Fowler et al. 2000) are reminiscent of telomerse-deficient mice (mTR-/-) following intermating over a number of generations (Lee et al. 1998, Herrera et al. 1999b). Generation 4 mTR-/- mice on a mixed C57BL6/129Sv background were found to have significantly smaller litters than G1 mTR-/- mice, whereas generation 6 intercrosses exhibited infertility due to a number of factors, including a possible compromise in uterine structure and function (Lee et al. 1998, Herrera et al. 1999a). The worsening phenotype of the mTR-/- mice was linked to a reduction in telomere-length and chromosomal abnormalities. This phenotype was exacerbated when mTR-/- mice were generated on a C57BL/6 background with mice being fertile only to the third generation (Lee et al. 1998, Herrera et al. 1999b). These observations suggest that Cenpb may be essential for short- and long-term maintenance of uterine integrity over a number of generations.
By virtue of its constitutive centromere-binding property, any attempt at deciphering a role of Cenpb has to date been directed at some aspects of chromosome segregation behaviour. Furthermore, if such a role is functionally important, then any perturbation of the protein function would be expected to result in a missegregation outcome throughout all actively dividing tissues in the mice, as gene knockouts of other key centromere-binding proteins have shown (Kalitsis et al. 1998, 2000, Dobles et al. 2000, Uren et al. 2000, Babu et al. 2003). It is therefore surprising that the phenotype seen in our Cenpb-null mice appears to be confined to the uterine tissues. While it is true that uterine tissue has a high expression level of Cenpb mRNA in mouse (Fowler et al. 2000), other tissues with similar Cenpb expression, such as mouse testes and intestinal epithelium (K J Fowler, unpublished observations), are not affected functionally by the null mutation, suggesting that high mRNA alone is insufficient to explain the observed uterine phenotype.
Studies in the fission yeast Schizosaccharomyces pombe have identified three CENP-B homologues Adp1, Cbh1 and Cbh2 (Baum & Clarke 2000, Irelan et al. 2001). Knockout experiments on these genes have shown that the ablation of one alone causes only a mild phenotype, whereas deletion of two of these genes, Adp1 and Cbh1 or Adp1 and Cbh2, results in severe defects in chromosomal segregation and cell growth (Baum & Clarke 2000, Irelan et al. 2001). These studies indicate the functional redundancy of the Adp1, Cbh1 and Cbh2 proteins. Although at present no CENP-B functional homologue(s) have been identified in mammals, including mice, it is conceivable that such homologue(s) exist, and this would explain the general non-essential nature of Cenpb. It is further possible that any such redundant protein(s) may not be able to replace fully the functions of Cenpb, and that the uterine tissue is particularly sensitive to such functional discrepancy. Our observation that the uterine phenotype becomes progressively more severe with increase in both age of the mice and generation time appears consistent with the speculation that the inadequacies of the putative CENP-B homologues could be exacerbated through increasing numbers of both mitotic and meiotic passages.
How does Cenpb exert the observed uterine effect? Although Cenpb is related to transposase by sequence homology, it has lost its transposase activity (Kipling & Warburton 1997), and, as such, any role it might have is unlikely to be directly linked to transposase. As indicated both in the present and our earlier study (Fowler et al. 2000), the contrasting severity of the uterine phenotype in different mouse strains is another example where the roles of modifier genes are strongly implicated in phenotypic presentation (Banbury Conference Consortium 1997, Threadgill et al. 1995, Herrera et al. 1999a). However, what these modifier genes in Cenpb-null mice are remains unknown. A potentially useful future effort to resolve what role Cenpb might play in uterine morphogenesis and, more generally, in mitotic and meiotic cell divisions, may be in attempts to identify its functional homologue(s) in mammals, as, if these exist, they could provide the missing pieces to the puzzle.
In summary, the observed decrease in endometrial glands in the Cenpb-null mouse leads to a lack of reproductive capability, indicating that a certain number of endometrial glands are required to establish successful pregnancies. Of note is the fact that this decrease in endometrial gland development is caused by the targeted knockout of a gene, the function of which is known to be specific to the centromere. Furthermore, the decreasing endometrial phenotype is exacerbated through successive generations of breeding. The underlying mechanisms responsible for these observations are at present unclear. These mice offer a model for studying these mechanisms as well as possible genetic and physiological requirements for endometrial glands during pregnancy.
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References |
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