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 Table of Contents  
Year : 2021  |  Volume : 41  |  Issue : 1  |  Page : 29-37

The bHLH transcription factor E protein negatively regulates endoreplication in the salivary gland cells

1 Department of Medicine, National Defense Medical Center, Taipei, Taiwan
2 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
3 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

Date of Submission11-May-2020
Date of Decision22-Jun-2020
Date of Acceptance02-Jul-2020
Date of Web Publication15-Aug-2020

Correspondence Address:
Dr. Lan-Hsin Wang
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmedsci.jmedsci_128_20

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Background: Endoreplication is a variant cell cycle which generates massive DNA replication with no features of mitosis. In addition to abnormal occurrence of endoreplication in cancer cells, it is often found in plants and many different animal organs, such as liver, placenta, and Drosophila larval tissues. In treatment with anti-mitotic drugs, it has been shown that cancer cells may undergo endoreplication to escape apoptosis. However, the underlying mechanisms of endoreplication in normal and pathological circumstances remain obscure. Methods: The regulation and function of most physiological processes are highly conserved between the fruit fly Drosophila melanogaster and mammals. In addition, using Drosophila as a research model can largely reduce genetic redundancy issues and provide a suitable way to observe cell autonomy. To address the aforementioned questions, we use the Drosophila as an animal model to study the function of fundamental regulators in endoreplication. Results: In the present study, we demonstrated that high levels of bHLH transcription factor E protein are capable of inhibiting endoreplication in larval salivary glands. The negative regulation of E protein in endoreplication depends on the dysregulation of cell cycle regulators, including E2f1 and its target genes Cyclin E and PCNA. However, the endoreplication defects caused by E protein overexpression are independent of the Hippo tumor suppressor pathway. Conclusions: Our results reveal that endoreplication can be prevented by high levels of E protein through disrupting the oscillations of cell cycle regulators.

Keywords: Endoreplication, hippo pathway, cell cycle, polyploidy, Drosophila

How to cite this article:
Ho CW, Chung YC, Chiu YL, Wang LH. The bHLH transcription factor E protein negatively regulates endoreplication in the salivary gland cells. J Med Sci 2021;41:29-37

How to cite this URL:
Ho CW, Chung YC, Chiu YL, Wang LH. The bHLH transcription factor E protein negatively regulates endoreplication in the salivary gland cells. J Med Sci [serial online] 2021 [cited 2021 Apr 16];41:29-37. Available from: https://www.jmedscindmc.com/text.asp?2021/41/1/29/292360

  Introduction Top

Polyploid cells, which contain multiples of the diploid sets of chromosomes, have been found in normal developmental program and can be achieved through variant cell cycle progression.[1],[2] However, high ploidy formation also leads to pathological processes as well as ageing. Division of 4N cells (i.e., 2N in each daughter cell) is precisely controlled by the cell cycle checkpoint. When 4N cells bypass this checkpoint, it will increase genomic instability and trigger tumorigenesis in the early phase of cancer progression. Cellular senescence induced by deregulated expression of oncogene or tumor suppressor gene is often associated with polyploidization.[3],[4] It has also been shown that cancer cell senescence can in turn act as a barrier against tumor progression.[5] Thus, understanding the mechanistic basis of cellular polyploidy in normal development may provide insights for developing anticancer therapy.

Endoreplication represents a variant cell cycle that generates a polyploid genome by multiple rounds of DNA replication without cell divisions, thus providing an important aspect to study the physiological roles of polyploidy. Repeated endoreplication of the genome during G-S endocycles results in high DNA content and large cell size. Endocycle consists of only S phase and G phase without M phase. It has been proposed that the function of endocycle is to promote protein synthesis, thus achieving growth and development of an organism.[2],[6] Endoreplication occurs in many larval tissues during Drosophila development, including salivary glands, gut, fat body, malpighian tubules, and trachea.[7] Furthermore, it is commonly found in plants and many different animal tissues, such as liver, muscle, and placenta.[2],[7],[8]

The initiation of DNA replication is controlled by a system that prevents re-replication. The DNA damage checkpoints and the spindle assembly checkpoint maintain the integrity of the diploid genome. Endoreplication results from a dysregulation of cell cycle, including the inactivation of Cyclins B and/or A. Cyclin E persists during endoreplication S phases, thereby making cells accumulate rounds of DNA replication and growth without division. Hence, the limiting factor that controls G1/S transition and S phase progression, Cyclin E and its designated kinase partner Cdk2, have been documented as a key regulator of Drosophila endocycles. Prior to the initiation of endoreplication S phases, cyclin E gene is transcribed and is then required for the endocycle progression.[9] By contrast, overexpression of Cyclin E inhibits endocycling in Drosophila,[10],[11] indicating that oscillations in Cyclin E activity could be essential for successive endocycle progression.

E proteins and ID proteins are transcription factors that were first discovered through immunoglobulin gene regulation and now known to regulate cell proliferation and differentiation in many tissues.[12],[13],[14] E proteins can function as homodimers that act to arrest the cell cycle and promote senescence as transcriptional activators of cyclin-dependent kinase inhibitors (CDKIs), including p16, p21, p27, and p57.[15],[16],[17] ID proteins act oppositely as transcriptional inhibitors when they dimerize with E proteins or basic helix-loop-helix proteins.[18] During cell cycle progression, ID proteins can indirectly antagonize retinoblastoma protein (RB) and activate E2F-mediated transcription through dimerization with E proteins. Alternatively, a direct interaction between RB and ID2 allows promotion of cell cycle progression. The connection between E/ID proteins and cancer has been well-established.[19],[20],[21],[22],[23] Mutations of E proteins have been extensively studied in cancer such as leukemia and lymphoma.[19],[24] A major discovery concerning E/ID protein function in growth control through activating the tumor suppressor Hippo pathway has been proposed by studying Drosophila, in which there is only one E protein Daughterless (Da) and one ID protein Extramacrochaetae (Emc).[25] It has been reported that the growth defect caused by abnormal E/ID protein levels mediates through transcriptional regulation of cdc25 phosphatase, thus regulating G2/M transition in the cell cycle.[26] In this study, we provide a novel insight connecting high E protein levels with endocycling impairment, which acts through the G1-S transition regulators E2f1 and Cyclin E in the cell cycle. Moreover, we are the first to reveal that the regulation of Drosophila E protein in endoreplication is independent of the Hippo pathway.

  Methods Top

Drosophila genetics

The Gal4/UAS system was used for overexpression of transgenes.[27] Clonal overexpression was achieved using Flp-out Gal4 (Act > CD2 > Gal4 UAS-GFP.[28] eyg-Gal4[29] and ptc-GAL4[30] drivers were used for overexpression or knocked-down experiments. UAS-E protein,[25] UAS-E protein-E protein,[25] UAS-Hpo (kindly provided by Prof. Johnston L at Columbia University[31]), UAS-Cyclin E (BDSC BL#4781), UAS-Yki (BDSC BL#28816) transgenes were used to overexpress the corresponding gene products. UAS-Yki RNAi (BDSC BL#31965) transgene was used to knock down yki expression. BDSC stocks are from Bloomington Stock Center. The summary of fly genetics is shown in [Table 1]. Flp-on expression clones were generated by crossing UAS-lines to hs-FLP 122; Act5C>CD2 >Gal4, UAS-GFP. Fly culture and cross were performed according to standard procedures at 25°C except E protein overexpression experiments driven by ptc-Gal4 were performed at 20°C to minimize lethality.
Table 1: Fly genetics

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Immunohistochemistry and imaging

Salivary glands were dissected from late third instar larval stage and fixed in PBS solution with 4% paraformaldehyde. Immunostaining is then conducted following the previous protocol. 32] Primary antibody used is anti-E2f1 (1:100 dilution; kindly provided by Prof. Orr-Weaver TL at Massachusetts Institute of Technology), anti-Drosophila E protein (1:200 dilution), followed by Cy2, Cy3-, or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch). DAPI staining was performed to visualize DNA content. Confocal imaging was performed using Zeiss LSM 880 microscopy at Neuroscience Program of Academia Sinica (NPAS). The quantification of nuclear size is performed using NIH Image J. As comparison, images with the same magnification and pixels were quantified. By using Image J, area of nuclei is measured. The unit of area is represented as arbitrary unit.

Quantitative reverse transcription polymerase chain reaction

The salivary glands from eyg-Gal4 (control) and eyg-Gal4>UAS-E protein flies were dissected in cold PBS. Total RNA was extracted using the Zymo Research Micro RNA isolation kit and DNA-Free RNA kit (Zymo Research). First Strand cDNA Synthesis Kit for reverse transcription polymerase chain reaction (Roche) was used to generate cDNAs from the extracted total RNA (1 μg) using random hexamer oligo (dT) primers. The real-time quantitative PCR was performed using the ABI 7900HT Detection System (with SYBR Green Master PCR Mix, ABI) to measure mRNA levels for three times. The relative amount of specific mRNAs was calculated after normalization to the rp49 transcript.

  Results Top

Drosophila E protein led to endocycling defects as homodimer

In Drosophila larva, salivary gland cells undergo endoreplication and give rise to polyploid [Figure 1]a, thus making it a good model to study the regulatory mechanisms of polyploidy. The previous study has reported that high levels of Drosophila E protein (Daughterless, Da) can disrupt both mitotic growth of imaginal discs and endoreplication of the salivary glands.[25] To detail the underlying mechanism by which E protein regulates endoreplication, E protein was overexpressed by Gal4/UAS system.[27] Two Gal4 lines under the control of different gene promoters (eyg-Gal4 and ptc-Gal4) were used to direct the expression of E protein to the salivary gland cells (abbreviated as eyg >E protein and ptc > E protein, respectively). The size of nuclei and salivary glands was drastically decreased in both eyg >E protein and ptc > E protein animals [Figure 1]c, [Figure 1]e and [Figure 1]g when compared with eyg-Gal4 or ptc-Gal4 control [Figure 1]b and [Figure 1]d. To further determine whether the size reduction of nuclei is a cell autonomous effect of E protein overexpression, clonal expression of E protein was performed. The nuclear size remained smaller than the neighboring control cells [Figure 1]h, suggesting E protein overexpression-mediated reduction of nuclear size is a cell autonomous effect in salivary gland. Intriguingly, the number of nuclei in E protein overexpression glands was similar to ptc-Gal4 control gland [Figure 1]i. Taken together, these results indicate that endoreplication in salivary gland cells is impaired by high levels of E protein. E protein is an important regulator required for cellular differentiation by forming dimer with various tissue-specific bHLH proteins.[12],[13] However, there is no known bHLH partner of E protein in the Drosophila salivary gland. It has been shown that E protein can form homodimer to bind to DNA,[12],[33],[34] raising the possibility that E protein might affect endoreplication as homodimer. To test this hypothesis, a E protein homodimer was overexpressed using ptc-GAL4 (abbreviated as ptc > E protein homodimer). As expected, the size of glands and nuclei was significantly reduced in ptc > E protein homodimer salivary glands [Figure 1]f and [Figure 1]g. This result suggests that high levels of E protein might be forced to homodimerize and then cause endocycling defects in larval salivary gland.
Figure 1: Drosophila E protein negatively regulates polypoid and salivary gland development. (a) The schematic diagram of a pair of Drosophila salivary glands in the third instar larval stage. The inset shows enlarge of salivary gland structure. The salivary glands consist of three cell types, including secretory cells (salivary gland, marked by blue color), the imaginal ring cells (marked by green color), and duct cells (salivary duct, marked by magenta color). Note that salivary gland and salivary duct are polypoid while the imaginal ring cells are diploid. Therefore, the nuclei of imaginal ring cells are much smaller than gland and duct cells. (b and d) Salivary glands of eyg-Gal4 or ptc-Gal4. (c and e and f) The indicated transgenic lines under the control of eyg-Gal4 (c) or ptc-Gal4 (e and f). E protein (green) staining represents the overexpression of E protein (c) when compared to the control (b). DAPI (blue) marks DNA (d-f). (g) The quantification of nuclear size for (d-f). Fifteen nuclei of each genotype are analyzed. Arbitrary unit denotes arbitrary unit. Area of nuclei is measured in microns using NIH Image J. Mean ± standard error of the mean is shown. (h) Salivary glands containing E protein overexpressing clones (act-Gal4 > GFP + E protein, GFP positive, green) and stained with Ex-lacZ (red). Ex-lacZ is a nuclear lacZ reporter. Note that GFP-positive nucleus (E protein overexpression) is smaller than GFP-negative nuclei. (i) The quantification of nuclear number in salivary glands. The number of nuclei (visualized by DAPI staining) is analyzed from 10 salivary glands of ptc-Gal4 and ptc > E protein, respectively. Note that each image for nuclear number counting are merged z-stacks. Mean ± standard error of the mean is shown. The scale bar is 50 μm

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Hippo pathway is dispensable for E protein-mediated endoreplication defects

In mitotic diploid cells, previous studies have demonstrated that the growth defects caused by E protein overexpression mediate through affecting Hippo signaling pathway and G2/M transition.[25],[26] Because endocycles do not have G2/M phases, we first determined whether the endoreplication defect caused by E protein overexpression acts through the Hippo signaling pathway. To address this, we checked whether E protein-overexpressing salivary glands can be modified when the gene dosage of Hippo pathway components is reduced. The large tumor suppressor 1 (LATS1 in mammals, Wts in Drosophila) kinase is the core kinase of the tumor suppressor Hippo pathway in mammals and Drosophila. However, the reduced size of salivary gland was not rescued in heterozygotes of wts mutants [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d. Similar results were observed when we overexpressed E protein in heterozygous mutant background of other Hippo pathway components such as hpo and ex (data not shown). To further investigate whether Hippo pathway is involved in endoreplication, the Hpo kinase or Yorkie (Yki/YAP in mammals; the core transcriptional coactivator of Hippo pathway) was overexpressed to determine whether endoreplication can be disrupted in salivary glands. Overexpression of hpo or yki did not obviously affect the nuclear size in larval salivary glands [Figure 2]e, [Figure 2]f, [Figure 2]g, [Figure 2]h, [Figure 2]i. Similarly, knockdown of yki did not affect nuclear size [Figure 2]g and [Figure 2]i. These observations suggest that reducing the levels of Hippo signaling cannot rescue E protein-overexpressing effect in the polyploid cells.
Figure 2: Hippo pathway is not implicated in E protein-mediated endoreplication defects. (a-c) Salivary glands of ptc-Gal4 (a), E protein homodimer under the control of ptc-Gal4 (b), and E protein homodimer under the control of ptc-Gal4 in heterozygous wts mutant background (c). (d) The quantification of nuclear size for (a-c). Fifteen nuclei from (a-c) are analyzed. (e-h) Salivary glands of Gal4 only and the indicated transgenic lines under the control of ptc-Gal4. (i) The quantification of nuclear size for genotypes of (e-h). For each genotype, three nuclei are selected from five salivary glands. Fifteen nuclei of each genotype are analyzed. Mean ± standard error of the mean is shown. The scale bar is 50 μm

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Moreover, it has been reported that LATS1 (Wts in Drosophila) is essential for the regulation of actin polymerization in mitosis and loss of LATS1 induces multinucleate phenotype and cause cytokinesis failure.[35] This promoted us to examine whether cytokinesis failure exists in E protein-overexpressing salivary glands. We then checked phospho-histone H3 (pH 3) staining in E protein-overexpressing salivary glands. Endocycles do not have G2/M phases so that salivary gland cells should not have any feature of mitosis such as pH 3 staining. Indeed, no pH 3-positive labeling was detected in E protein-overexpressing glands [Figure 3]. This finding suggests that E protein overexpression-mediated endoreplication defect is not resulted from cytokinesis failure. Taken together, our data indicate that the endoreplication defect caused by E protein overexpression is not acting through the Hippo pathway.
Figure 3: E protein-mediated endoreplication defects do not have mitotic feature. (a) The schematic showing the difference of cell cycle progression between mitotic cell cycle and endocycle (endoreplication). Note that phospho-H3 is a mitotic marker. (b-b”) The salivary glands of ptc-Gal4 (indicated by yellow color) and E protein homodimer driven by ptc-Gal4 (indicated by green color) are shown in the same image. DAPI marks DNA (b). Note that certain fat body cells surrounding the salivary glands are indicated by white arrows. (b') Phospho-H3 staining of (b). The merged image of DAPI and phospho-H3 is shown in (b”). The scale bar is 50 μm

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E protein mediates cell cycle-dependent mechanism to regulate endoreplication

It is well known that the main regulator to ensure the oscillations of endocycle is the Cyclin E protein, whose periodic production enables the S phase initiation through activating Cdk2.[6] Oscillations of Cyclin E require multiple positive and negative feedback regulatory loops. The dimeric E2F1/DP transcription factors are thought to be important for endocycle progression because they control the periodic transcription of Cyclin E at G-S phases.[36] Interestingly, protein levels of E2f1 were elevated in mosaic salivary glands containing E protein-overexpressing cells [Figure 4]a. We then examined whether the effects of high E protein levels on endoreplication can be rescued by reducing E2f1 gene dosage in E protein-overexpressing salivary gland cells. As expected, the size of glands and nuclei was partially rescued in ptc > E protein, E2f1+/- flies [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e. E2F1-Cyclin E-positive feedback loop rises Cyclin E/Cdk2 activity to phosphorylate the transcriptional repressor RB protein, thereby activating the transcription of cyclin E and other E2F1 target genes such as PCNA (Proliferating Cell Nuclear Antigen; essential for DNA replication). The Cyclin E/Cdk2 activity is in turn inhibited by CDKIs.[6],[37]
Figure 4: E2f1 is essential for E protein-mediated endoreplication defects. (a) Salivary glands containing E protein overexpressing clones (act-GAL4 > GFP + E protein, GFP positive, green, a') and stained with E2f1 antibody (red, a”). (b-d) The salivary glands of ptc-Gal4 (b), E protein driven by ptc-Gal4 (c), and E protein driven by ptc-Gal4 in heterozygous E2f1 mutant background (d). Note that removing one copy of E2f1 can partially rescue the size of nuclei and salivary gland in ptc > E protein flies. (e) The quantification of nuclear size for (b-d). Fifteen nuclei from (b-d) are analyzed. Mean ± standard error of the mean is shown. The scale bar is 50 μm

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To address whether the elevated E2f1 protein activates the transcription of E2f1 target genes such as cyclin E and PCNA, we checked the expression levels of the related cell cycle regulators in E protein-overexpressing salivary glands. The mRNA levels of cyclin E and PCNA were increased while E2f1 and Rbf1 mRNAs were not changed in E protein-overexpressing salivary glands when compared with the Gal4 only control [eyg > E protein vs. eyg-Gal4 in [Figure 5]a. These results suggest that high levels of E protein may sustain protein levels of E2f1 rather than affecting E2f1 transcription. Hence, in E protein-overexpressing endocycling cells, E2f1 protein levels remain high and then activate the transcription of E2f1 target genes. Indeed, the size of nuclei and salivary glands was reduced when the E2f1 target gene cyclin E was driven by the ptc-Gal4 [Figure 5]b, [Figure 5]c, [Figure 5]d. When E2f1 is reduced, the expression of its target genes would be reduced, thus allowing the attenuation of endoreplication defects. In sum, our results imply that E2f1 accumulation is a critical factor for high E protein-mediated endoreplication defects.
Figure 5: S phase regulators are affected in E protein-mediated endoreplication defects. (a) A graph comparing cyclin E, PCNA, E2f1, and Rbf1 mRNA levels in eyg-Gal4 and E protein-overexpressing salivary glands, as measured by quantitative reverse transcription polymerase chain reaction. Note that cyclin E and PCNA are increased in E protein-overexpressing glands. (b and c) The salivary glands of ptc-Gal4 and cyclin E transgene driven by ptc-Gal4, respectively. Similar to overexpression of E protein, overexpression of Cyclin E reduces the size of nuclei and salivary gland. (d) The quantification of nuclear size for (b and c). Fifteen nuclei from (b and c) are analyzed. Mean ± standard error of the mean is shown. The scale bar is 50 μm

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  Discussion Top

Understanding how our body controls cell cycle progression appears to be essential. Any errors occur may contribute to pathological states. For instance, failed mitosis results in polyploid cells with an increased number of chromosomes, thus causing an increased risk of spindle formation defects and chromosome missegregation. The unscheduled amplification of chromosomes is believed to be a critical step in the production of cancer aneuploidy. Endoreplication plays crucial roles during development and has also been implicated in human disease, such as cancer. Interestingly, mitotic cell cycle, and endoreplication are controlled by the same regulatory proteins. Cell cycle progression requires periodic activation of serine/threonine protein kinases, called cyclin-dependent kinases (CDKs). CDKs phosphorylate specific substrates to drive S phase and mitosis, the two major events of the cell division.[7],[38] The coupling of these events requires CDK activity, thereby ensuring S phase begins when mitosis is completed. Anti-mitotic drugs are frequently used in the treatment of cancer. In certain situations, endoreplication could be triggered to bypass apoptosis when cancer cells were treated with anti-mitotic drugs, thereby developing cancer cell resistance.[39] For example, it has been shown that certain p53 mutant cancer cells may survive from anti-mitotic drug treatment through a switch from mitosis to endoreplication.[39]

In this study, we characterized the functional roles and regulation of the Drosophila E protein Daughterless (Da) in endoreplication control. Our data showed that E protein overexpression negatively affects endoreplication in Drosophila larval tissues, strongly implying that overexpression of E protein has the potential to prevent cells from resistance to the treatment of anti-mitotic drugs. The previous study has shown that high levels of E protein disrupt cellular growth and proliferation through activating the Hippo tumor suppressor pathway.[25] Surprisingly, we found that endocycling defects caused by high levels of E proteins did not act through the Hippo tumor suppressor pathway but through affecting cell cycle regulators. E2f1 protein is periodic because it is degraded in S-phase nuclei.[40] In the presence of high levels of E proteins, we found that protein levels of E2f1 were accumulated. The sustained E2f1 proteins then increased transcription levels of E2f1 target genes such as cyclin E and PCNA, thus disrupting endoreplication in the salivary glands. The degradation of E2f1 is triggered by Cul4 E3 ubiquitin ligase through the PIP motif of E2f1.[40] When E2f1 is overexpressed, E2f1 proteins still can be depleted by the normal E3-dependent degradation.[41] This suggests that the accumulation of E2f1 protein could be due to the aberrant degradation of E2f1 in E protein-overexpressing cells. Hence, our investigation of how E protein regulates endoreplication may help to develop the potential therapeutic strategies for treatments of tumors that become insensitive to anti-mitotic drugs because it has been proposed that cancer cells use endoreplication to bypass elimination during mitotic catastrophe or genotoxic stress.

  Conclusions Top

This study demonstrated that the Hippo tumor suppressor pathway is dispensable for the endoreplication defects caused by high levels of E protein. This finding is critical because it is the first evidence showing that E protein-Hippo pathway is only implicated in the regulation of mitotic cell cycle but not endocycle. Moreover, this study has uncovered that the endoreplication defects triggered by high levels of E protein act through increasing E2f1 proteins, thus promoting the transcription of its downstream targets such as cyclin E and PCNA. It is known that oscillations in Cyclin E activity are important for endoreplication. The changes in cyclin E levels therefore disrupted the endocycle oscillations. Taken together, this study provided the novel findings that high levels of E protein-mediated endocycling defects act through affecting cyclin E transcription in a Hippo-independent mechanism.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1]


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