Embryonic Environmental Niche Reprograms Somatic Cells to Express Pluripotency Markers and Participate in Adult Chimaeras

The phenomenon of the reprogramming of terminally differentiated cells can be achieved by various means, like somatic cell nuclear transfer, cell fusion with a pluripotent cell, or the introduction of pluripotency genes. Here, we present the evidence that somatic cells can attain the expression of pluripotency markers after their introduction into early embryos. Mouse embryonic fibroblasts introduced between blastomeres of cleaving embryos, within two days of in vitro culture, express transcription factors specific to blastocyst lineages, including pluripotency factors. Analysis of donor tissue marker DNA has revealed that the progeny of introduced cells are found in somatic tissues of foetuses and adult chimaeras, providing evidence for cell reprogramming. Analysis of ploidy has shown that in the chimaeras, the progeny of introduced cells are either diploid or tetraploid, the latter indicating cell fusion. The presence of donor DNA in diploid cells from chimaeric embryos proved that the non-fused progeny of introduced fibroblasts persisted in chimaeras, which is evidence of reprogramming by embryonic niche. When adult somatic (cumulus) cells were introduced into early cleavage embryos, the extent of integration was limited and only cell fusion-mediated reprogramming was observed. These results show that both cell fusion and cell interactions with the embryonic niche reprogrammed somatic cells towards pluripotency.

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65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab65 ◽

2008 ◽

Vol 20 (1) ◽

pp. 113

Author(s):

H. M. Zhou ◽

B. S. Li ◽

L. J. Zhang

Keyword(s):

Somatic Cell ◽

Somatic Cells ◽

Calf Serum ◽

Cumulus Cells ◽

Developmental Competence ◽

Developmental Potential ◽

Reconstructed Embryos ◽

Electrical Pulses ◽

Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

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31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab31 ◽

2006 ◽

Vol 18 (2) ◽

pp. 124 ◽

Cited By ~ 1

Author(s):

F. Du ◽

J. Xu ◽

S. Gao ◽

L. Y. Sung ◽

D. Stone ◽

...

Keyword(s):

Somatic Cell ◽

Cell Fusion ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Cumulus Cells ◽

Full Term ◽

Fusion Method ◽

Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

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210 CO-CULTURE WITH AUTOLOGOUS CUMULUS CELLS SUPPORTS THE INDIVIDUAL DEVELOPMENT OF SINGLY IN VITRO-MATURED AND FERTILIZED BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab210 ◽

2011 ◽

Vol 23 (1) ◽

pp. 204

Author(s):

I. G. F. Goovaerts ◽

J. L. M. R. Leroy ◽

E. Merckx ◽

S. Andries ◽

P. E. J. Bols

Keyword(s):

Somatic Cells ◽

Cumulus Cells ◽

Individual Development ◽

Control Individual ◽

In Vitro Production ◽

Group Culture ◽

The Individual ◽

Vitro Production ◽

Hatching Rates

The ability to produce embryos singly in vitro (in vitro production, IVP) would be a useful tool for many purposes. Without the interfering effects of other developing or degenerating oocytes or embryos, such an individual IVP system is the tool of choice for studies on oocyte quality and oocyte–embryo metabolism. Unfortunately, individual IVP in most cases leads to unsatisfactorily low blastocyst rates. Earlier work showed that individual culture of zygotes on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos, as obtained following regular group culture (Goovaerts et al. 2009 Theriogenology 71, 729–738). However, co-culture with somatic cells is often criticised because of the undefined culture conditions and for sanitary reasons. In the cited study, CC for monolayer production were obtained from a different batch of ovaries. Our specific aim was to use CC from the zygote itself (autologous CC). Grade I COC (n = 660) were collected from slaughterhouse ovaries and randomly assigned to 2 treatments (5 replicates): a completely individual ‘single-oocyte’ IVP protocol, or routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was stripped and cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2), to which the autologous stripped CC were added. Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were determined 2, 8, and 10 days post-fertilization, respectively. Possible effects of the individual and group cultures were evaluated with binary logistic regression (SPSS 15.0, SPSS Inc., Chicago, IL). No interactions between replicate and treatment were found (P > 0.05). Although a blastocyst rate of 15.1% was obtained using single IVP, the general efficacy of the single-embryo production system was lower when compared with group culture (Table 1). In conclusion, although developmental competence was impaired using individual IVP, co-culture with autologous cumulus cells can be useful in specific experimental setups in which the influence of other oocytes or embryos or heterologous somatic cells is unacceptable. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro production (IVP)

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Cell fusion induced by lysolecithin and concanavalin A in Drosophila melanogaster somatic cells cultured in vitro

Experimental Cell Research ◽

10.1016/0014-4827(76)90166-x ◽

1976 ◽

Vol 100 (2) ◽

pp. 399-404 ◽

Cited By ~ 13

Author(s):

Carlotta Halfer ◽

Lucia Petrella

Keyword(s):

Drosophila Melanogaster ◽

Cell Fusion ◽

Concanavalin A ◽

Somatic Cells ◽

Cells Cultured

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NEDD4-like ubiquitin ligase 2 protein (NEDL2) in porcine spermatozoa, oocytes, and preimplantation embryos and its role in oocyte fertilization†

Biology of Reproduction ◽

10.1093/biolre/ioaa186 ◽

2020 ◽

Author(s):

Jiude Mao ◽

Michal Zigo ◽

Dalen Zuidema ◽

Miriam Sutovsky ◽

Peter Sutovsky

Keyword(s):

Ubiquitin Ligase ◽

In Vitro Maturation ◽

Somatic Cells ◽

Cumulus Cells ◽

High Mass ◽

Preimplantation Embryos ◽

Oocyte Fertilization ◽

Sperm Dna ◽

Porcine Spermatozoa

Abstract The ubiquitin-proteasome system plays diverse regulatory and homeostatic roles in mammalian reproduction. Ubiquitin ligases are the substrate-specific mediators of ubiquitin-binding to its substrate proteins. The NEDD4-like ubiquitin ligase 2 (aliases NEDL2, HECW2) is a HECT-type ubiquitin ligase that contains one N-terminal HECW ubiquitin ligase domain, one C-terminal HECT ubiquitin ligase domain, one C2 domain, and two WW protein-protein interaction modules. Beyond its predicted ubiquitin-ligase activity, its cellular functions are largely unknown. Current studies were designed to investigate the content and distribution of NEDL2 in porcine spermatozoa, oocytes, zygotes, and early preimplantation embryos, and in cumulus cells before and after in vitro maturation with oocytes, and fibroblast cells as positive control by western blot and immunocytochemistry, and to examine its roles during oocyte fertilization. Multiple isoforms of NEDL2 were identified by WB. One at approximately 52 kDa was detected only in the germinal vesicle (GV) stage and metaphase II oocytes, and in early preimplantation embryos. Other isoforms were high mass bands at 91, 136, and 155 kDa, which were only detected in somatic cells. Interestingly, ejaculated spermatozoa prominently displayed the same 52 kDa band as oocytes; they also had two minor bands of 74 and 129 kDa, which were not detected in somatic cells or oocytes. By immunofluorescence, NEDL2 showed a diffused cytoplasmic localization in all cell types and accumulated in distinct foci in the germinal vesicles (GVs) of immature oocytes, in maternal and paternal pronuclei of zygotes and nuclei of embryo blastomeres and somatic cells. In blastocysts, the labeling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast, indicating higher NEDL2 content in the ICM cells than in trophectoderm. NEDL2 abundance was 10 times higher in post-maturation oocyte-surrounding cumulus cells than that of cumulus cells before in vitro maturation with hormones, indicating that NEDL2 may have a unique role in cumulus cells after ovulation. Microinjection of anti-NEDL2 antibody into oocyte before IVF did not affect the percentage of oocytes fertilized, percentage of oocytes cleaved, or blastocyst formation. However, the anti-NEDL2 antibody decreased the number of pronuclei, accelerated the formation of nuclear precursor bodies at 6 h postfertilization, inhibited sperm DNA decondensation, and resulted in more fertilized oocytes without male pronuclear formation. In summary, NEDL2 may play a key role during fertilization, especially during sperm DNA decondensation.

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75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab75 ◽

2005 ◽

Vol 17 (2) ◽

pp. 187 ◽

Cited By ~ 1

Author(s):

F. Yang ◽

B. Kessler ◽

S. Ewerling ◽

E. Wolf ◽

V. Zakhartchenko

Keyword(s):

Nuclear Transfer ◽

Cultured Cells ◽

Somatic Cells ◽

Cumulus Cells ◽

Cell Stage ◽

Electric Pulses ◽

Donor Cells ◽

Fetal Fibroblasts

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

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065. THE MAMMALIAN OOCYTE: FROM BENCH TO CLINIC

Reproduction Fertility and Development ◽

10.1071/srb09abs065 ◽

2009 ◽

Vol 21 (9) ◽

pp. 18

Author(s):

R. B. Gilchrist

Keyword(s):

Somatic Cells ◽

Cumulus Cells ◽

Reproductive Technologies ◽

Oocyte Quality ◽

Female Fertility ◽

Widespread Application ◽

Developmental Potential ◽

Mammalian Oocyte ◽

Wide Range

The mature mammalian oocyte is the central link between generations. It is not only responsible for the transfer of the female genome between generations, but also largely determines embryo and early fetal developmental potential. For any female, oocytes are in limited supply and are easily damaged, such that the availability of high quality or developmentally competent oocytes is a fundamental rate-limiting factor in female fertility. This is particularly relevant in Australian society today with the steadily rising age to first conception which adversely affects oocyte quality and female fertility. Yet despite years of research and clinical IVF we still have a poor understanding of the molecular and cellular processes that control oocyte quality. It is clear that oocytes acquire developmental competence in the ovarian follicle. The acquisition of competence necessitates communication between the oocyte and maternal systems, a process which endows developmental potential as the oocyte grows and matures inside the follicle. At the cellular level this is achieved by bi-directional communication between oocytes and their companion somatic cells [1]. Over the past 10 years my laboratory has focused heavily on the nature of these oocyte-somatic communication axes and their impact on oocyte quality. Over this period, our work and that of others has shaped a new paradigm in ovarian biology, which is that the oocyte is not passive in the follicle, but rather that it actively directs the differentiation of its neighbouring somatic cells into cumulus cells through the secretion of GDF9 and BMP15 growth factors [2]. In doing so, oocytes dictate the function of their neighboring cumulus cells, directing them to perform functions needed for the appropriate growth and development of the oocyte. For example, cumulus cells supply oocytes with an array of nutrients, substrates and regulatory molecules such as cAMP, many directly through gap-junctions. These communication axes establish and maintain an elaborate and intricate local oocyte-cumulus auto regulatory loop that is required to enable post-fertilisation development. A clear clinical application of this new knowledge is in Artificial Reproductive Technologies, in particular oocyte in vitro maturation (IVM) [3]. IVM biotechnologies have the capacity to capture the vast supply of oocytes in the mammalian ovary and generate mature oocytes in vitro. Generating offspring using IVM is already a clinically and commercially viable biotechnology in livestock breeding programs, particularly in cattle. IVM is a particularly attractive technology for the treatment of human infertility, as it removes the need for expensive and potentially harmful ovarian hyperstimulation protocols used in clinical IVF. However, widespread application of IVM in humans requires an increase in efficiency and further examination of safety of the technology. Recent work from my laboratory has increased IVM success rates in animals by using GDF9 and BMP15 in IVM [2, 3] and by developing a new system of “Induced-IVM” that more closely resembles the mechanisms of oocyte maturation in vivo. Most recently, the latter approach has led to substantial increases in embryo yield and pregnancy outcomes to levels equivalent to hormone-stimulated IVF [4]. The next challenge is to adapt these new approaches to clinical/field conditions to provide new opportunities for infertile women and for the development of a wide range of reproductive biotechnologies.

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Gap-junctional communication in mouse cumulus-oocyte complexes: implications for the mechanism of meiotic maturation

Reproduction ◽

10.1530/rep.0.1230041 ◽

2002 ◽

pp. 41-52 ◽

Cited By ~ 50

Author(s):

RJ Webb ◽

H Bains ◽

C Cruttwell ◽

J Carroll

Keyword(s):

Ovarian Function ◽

Ovarian Follicle ◽

Somatic Cells ◽

Cumulus Cells ◽

Meiotic Maturation ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Gap Junctional

The mechanisms underlying the hormonal stimulation of meiotic maturation are not understood. The most prevalent hypothesis is that hormone-induced maturation is stimulated by an increase in the intracellular messengers, cAMP or Ca2+. This study investigated whether Ca2+ transients in somatic cells can lead to Ca2+ transients in the oocyte, and whether hormones that stimulate meiotic maturation of mouse oocytes in vitro and in vivo stimulate an increase in intracellular Ca2+. Of a range of potential agonists of Ca2+ release, ATP and UTP were the only agents that stimulated Ca2+ release in cumulus cells. ATP-induced Ca2+ release is from intracellular stores, as the response is not blocked by chelation of extracellular Ca2+, but is inhibited by the Ca2+-ATPase inhibitor, thapsigargin. ATP and UTP are equipotent, consistent with the receptor being of the P2Y2 type. Confocal microscopy was used to show that ATP-induced Ca2+ release in cumulus cells leads to a Ca2+ increase in the oocyte. Inhibition of gap-junctional communication using carbenoxolone, as assayed by dye transfer, inhibited the diffusion of the Ca2+ signal from the cumulus cells to the oocyte. Thus, provided that a Ca2+ signal is generated in the somatic cells in response to maturation-inducing hormones, it is feasible that a Ca2+ transient is generated in the oocyte. However, FSH and EGF, both of which stimulate maturation in vitro, have no effect on Ca2+ in cumulus--oocyte complexes. Furthermore, LH, which leads to meiotic maturation in vivo, did not stimulate Ca2+ release in acutely isolated granulosa cells from preovulatory mouse follicles. These studies indicate that ATP may play a role in modulating ovarian function and that diffusion of Ca2+ signals through gap junctions may provide a means of communication between the somatic and germ cells of the ovarian follicle. However, our data are not consistent with a role for Ca2+-mediated communication in hormone-mediated induction of meiosis in mice.

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Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming

Stem Cells International ◽

10.1155/2012/414038 ◽

2012 ◽

Vol 2012 ◽

pp. 1-13 ◽

Cited By ~ 15

Author(s):

Nicholas A. Kouris ◽

Jeremy A. Schaefer ◽

Masato Hatta ◽

Brian T. Freeman ◽

Timothy J. Kamp ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Cell Fusion ◽

Somatic Cells ◽

Human Mscs ◽

Viral Fusion ◽

Fusion Products

Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cellsin vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphologyin vitro.In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMsin vivoand illustrates how viral fusion proteins might better enable such studies.

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