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This is a protocol to detect HIV-1 reverse transcription products in cytoplasmic and nuclear fractions of cells infected with VSV-G-pseudotyped envelope-defective HIV-1. This protocol can also be extended to HIV-1 with regular envelope.

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Cell Fractionation and Quantitative Analysis of HIV-1 Reverse Transcription in Target Cells
靶细胞中HIV-1 逆转录的细胞分级分离和量化分析

微生物学 > 微生物遗传学 > RNA > qRT-PCR
作者: Vaibhav B Shah
Vaibhav B ShahAffiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, USA
For correspondence: shahvaibhav10@gmail.com
Bio-protocol author page: a482
 and Christopher Aiken
Christopher AikenAffiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, USA
Bio-protocol author page: a932
Vol 3, Iss 20, 10/20/2013, 4702 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.949

[Abstract] This is a protocol to detect HIV-1 reverse transcription products in cytoplasmic and nuclear fractions of cells infected with VSV-G-pseudotyped envelope-defective HIV-1. This protocol can also be extended to HIV-1 with regular envelope.

Keywords: HIV-1(HIV-1), Reverse Transcription(反转录), Cytoplasm(细胞质), Nucleus(核), Quantitative PCR(定量PCR)

[Abstract] This is a protocol to detect HIV-1 reverse transcription products in cytoplasmic and nuclear fractions of cells infected with VSV-G-pseudotyped envelope-defective HIV-1. This protocol can also be extended to HIV-1 with regular envelope.

Materials and Reagents

  1. HEK 293T cells
  2. HeLa cells
  3. Dulbecco’s Modified Eagle Medium (DMEM) (Mediatech, Cellgro®, catalog number: 10-013-CV )
  4. R9-ΔE plasmid ((Zhou and Aiken, 2001), an HIV-1 proviral DNA clone created by introducing a frameshift mutation in envelope of the wild-type infectious R9 clone. Virions produced by this clone are non-infectious but can be made infectious by pseudotyping with envelopes from VSV or other viruses)
  5. pHCMV-G (VSV-G) plasmid ((Yee et al., 1994), a retrovirus-derived plasmid in which the retroviral envelope glycoprotein is replaced with glycoprotein from vesicular stomatitis virus [VSV]).
  6. p24 ELISA kit (in-house)
  7. Phosphate-buffered saline (PBS) (Mediatech, Cellgro®, catalog number: 21-0310-CV )
  8. VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE) virus particles
  9. Efavirenz (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
    NIH, catalog number: 11680 )
  10. DNase I (Roche, catalog number: 10104159001 )
  11. 0.25% Trypsin/2.21 mM EDTA (Mediatech, Cellgro®, catalog number: 25-053-CI )
  12. Triton X-100 (Mallinckrodt, catalog number: 9002-93-1 )
  13. DNeasy blood and tissue kit (QIAGEN, catalog number: 69506 )
  14. cOmplete, Mini, EDTA-free protease-inhibitor cocktail tablet (Roche, catalog number: 11836170001 )
  15. 4 to 20% Polyacrylamide gradient Tris-glycine gels (Bio-Rad)
  16. Nitrocellulose membrane (General Electric Company)
  17. Mouse monoclonal anti-GAPDH antibody (Santa Cruz, catalog number: sc-47724 )
  18. Mouse monoclonal anti-LaminB1 antibody (Life Technologies, catalog number: 33-2000 )
  19. SYBR green (ABI, catalog number : 4309155 )
  20. DpnI (New England Biolabs, catalog number: R0176L )
  21. DTT
  22. Yeast tRNA (Roche, catalog number : 10109541001 )
  23. Forward primer MH531 (5’-TGTGTGCCCGTCTGTTGTGT-3’)
  24. Reverse primer MH532 (5’-GAGTCCTGCGTCGAGAGAGC-3’)
  25. DNase/RNase-free water
  26. SDS-PAGE sample buffer
  27. Sodium deoxycholate (Sigma-Aldrich, catalog number: 30970 )
  28. N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES) (Sigma-Aldrich, catalog number: B4554 )
  29. Hypotonic buffer (see Recipes)
  30. Radioimmunoprecipitation buffer (see Recipes)
  31. 2x BES-buffered saline (BBS) (see Recipes)

Equipment

  1. 10 cm cell culture dish
  2. 0.45-μm-pore-size syringe filters (Thermo Fisher Scientific, catalog number: 190-2545 )
  3. 0.20- μm-pore-size syringe filters (Thermo Fisher Scientific, catalog number: 190-2520 )
  4. 1.5 ml screw-cap tube
  5. Tabletop centrifuge (Thermo Fisher Scientific, Sorvall®)
  6. Tabletop refrigerated centrifuge (Thermo Fisher Scientific)
  7. Mx-3000p thermocycler (Stratagene)
  8. CO2 incubator

Procedure

  1. Production of VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE clone) virus particles (Aiken, 1998)
    1. Culture 293T cells in DMEM containing 10% v/v fetal bovine serum (FBS) and supplemented with antibiotics [Penicillin (100 IU/ml) and Streptomycin (100 μg/ml)] at 37 °C, 5% CO2.
    2. Detach cells from a nearly confluent culture dish with the help of 0.25% Trypsin/2.21 mM EDTA and seed 2 x 106 cells in 9 ml medium per 100 mm culture dish and incubate at 37 °C.
    3. Transfect of 293T cells next day using the calcium phosphate-BBS method (Chen and Okoyama, 1987).
      1. Mix 15 μg of R9-ΔE and 5 μg of pHCMV-G (VSV-G) plasmids in a tube.
      2. Add 0.2 μm filtered water to the tube to make up the volume to 450 μl.
      3. Add 50 μl of 2.5 M CaCl2 to the tube.
      4. Add 500 μl of 2x BBS to the tube dropwise.
      5. Gently mix the contents of the tube by pipetting few times.
      6. Incubate the tube at room temperature for 20 to 30 min.
      7. Add the mixture to 293T cells with gentle swirling and incubate cells at 35 °C and 3% CO2.
    4. Aspirate media from the transfected dish ~16 h after transfection, wash cells with 5 ml PBS, replenish with 5 ml of fresh cell culture media and incubate at 37 °C, 5% CO2.
    5. Two days after transfection, harvest culture supernatant containing virus particles, centrifuge at 1,500 x g for 5 min to pellet cells and debris.
    6. Filter the supernatant through 0.45-μm-pore-size syringe filters, aliquot and freeze at -80 °C.

  2. Infection of HeLa cells with VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE)
    1. Plate HeLa cells at a density of 1.5 x 105 cells/well in 12-well plates (1 ml total culture volume per well).
    2. 24 hours later treat virus inocula with DNase I (20 μg/ml) plus MgCl2 (10 mM) and incubate in a water bath at 37 °C for 1 h.
    3. Infect cells with DNase I-treated inocula equivalent to 15 ng of p24 (determined by p24 ELISA using in-house kit (Wehrly and Chesebro, 1997)).
    4. Perform parallel infection in the presence of efavirenz (1 μM) to define the residual plasmid DNA levels carried over from transfection.
    5. Incubate infected cells at 37 °C for 8 h.
      Note: One can also analyse time course of reverse transcription by harvesting infected cells at different time intervals after infection.

  3. Cell fractionation of HIV-1 infected HeLa cells
    1. After incubation for desired time, aspirate culture media and wash cells once with PBS.
    2. Dislodge adherent cells by incubation with 500 μl of 0.25% Trypsin-EDTA at 37 °C for 2 min.
    3. Collect trypsinized cells in a 1.5 ml screw-cap tube. Centrifuge at 300 x g for 5 min to pellet cells.
    4. Lyse cell pellets in 200 μl of hypotonic buffer containing 0.1% Triton-X-100 and incubate on ice for 15 min.
      Note: Concentration of Triton-X-100 was optimized for HeLa cells. The concentration of Trition X-100 represents the lowest concentration at which about 95% of the cells counted under the microscope had intact nuclei but no plasma membrane.
    5. Centrifuge at 17,000 x g for 5 min at 4 °C and collect the supernatant as cytoplasmic fraction.
    6. Wash the nuclear pellet with 1 ml hypotonic buffer without Triton-X-100 thrice. After each wash centrifuge at 17,000 x g for 5 min at 4 °C to pellet the nuclei and aspirate off supernatant.
    7. Isolate DNA from nuclear pellet using DNeasy blood and tissue kit as per manufacturer’s protocol. Elute DNA in the last step in a fresh collection tube using 100 μl DNase/RNase-free water. Eluted DNA can be stored at -80 °C or used directly to perform qPCR.
    8. In parallel, prepare whole-cell, cytoplasmic and nuclear lysates from uninfected cells to check for cytoplasmic contamination of nuclear fractions.
    9. To prepare whole cell lysate, lyse cells in radioimmunoprecipitation (RIPA) buffer (follow steps C2-C5 except the use of RIPA buffer instead of hypotonic buffer). Add equal volume of 2x SDS-PAGE sample buffer for gel electrophoresis and heat at 95 °C in a heat block for 5 min.
    10. Prepare cytoplasmic lysate as described above (steps C2 to C5). Add equal volume of 2x SDS-PAGE sample buffer for gel electrophoresis and heat at 95 °C in a heat block for 5 min.
    11. To prepare nuclear lysate, follow steps C2 to C6, and then lyse the nuclear pellet in 1x SDS-PAGE sample buffer. Heat at 95 °C in a heat block for 5 min. Resolve equal volumes of whole cell, cytoplasmic and nuclear lysates on a 4-20% polyacrylamide gradient Tris-glycine gel.
    12. Transfer resolved proteins onto a nitrocellulose membrane.
    13. Block the membrane with 5% non-fat milk solution in PBS and probe with anti-GAPDH and anti-LaminB1 antibodies (concentrations recommended by manufacturer) followed by appropriate secondary antibodies (concentrations recommended by manufacturer) as cytoplasmic and nuclear markers respectively.

  4. SYBR green-based Quantitative PCR for quantitation of viral reverse transcription products
    1. Treat isolated DNA from step C7 with DpnI (17 μl DNA + 2 μl buffer + 1 μl of DpnI-20 units) by incubation at 37 °C for 1 to 2 h. Inactivate DpnI by incubation at 80 °C for 20 min.
    2. Quantitation of viral reverse transcription products.
      1. Prepare reaction mixture by mixing DNA (5 μl), PCR mix containing SYBR green (12.5 μl), forward primer (150 nM), reverse primer (150 nM) and tRNA (1 μg/μl) containing DNase/RNase-free water up to 25 μl.
      2. Prepare standards ranging from 10 to 109 copies/reaction of R9-ΔE plasmid. Dilutions of standards should be made in 1 μg/μl tRNA-containing water.
      3. Set PCR reaction using the following thermal profile:

Recipes

  1. Hypotonic buffer
    10 mM Tris pH 8.0
    10 mM KCl
    1.5 mM MgCl2
    1 mM DTT
    Protease inhibitor cocktail (one tablet per 10 ml of buffer)
  2. Radioimmunoprecipitation buffer
    50 mM Tris pH 7.5
    1% Triton-X-100
    250 mM NaCl
    5 mM EDTA
    0.1% SDS
    1% sodium deoxycholate
    Protease inhibitors cocktail (one tablet per 10 ml of buffer)
  3. 2x BES-buffered saline (BBS)
    50 mM BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid)
    1.5 mM Na2HPO4
    280 mM NaCl
    pH 6.95

Acknowledgments

This protocol is adapted from Shah et al (2013). This protocol was supported by NIH grant AI076121 to C.A.

References

  1. Aiken, C. (1998). Mechanistic independence of Nef and cyclophilin A enhancement of human immunodeficiency virus type 1 infectivity. Virology 248(1): 139-147.
  2. Chen, C. and Okayama, H. (1987). High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7(8): 2745-2752.
  3. Shah, V. B., Shi, J., Hout, D. R., Oztop, I., Krishnan, L., Ahn, J., Shotwell, M. S., Engelman, A. and Aiken, C. (2013). The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating. J Virol 87(1): 422-432.
  4. Wehrly, K. and Chesebro, B. (1997). p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods 12(4): 288-293.
  5. Yee, J. K., Friedmann, T. and Burns, J. C. (1994). Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol 43 Pt A: 99-112. 
  6. Zhou, J. and Aiken, C. (2001). Nef enhances human immunodeficiency virus type 1 infectivity resulting from intervirion fusion: evidence supporting a role for Nef at the virion envelope. J Virol 75(13): 5851-5859.

材料和试剂

  1. HEK 293T细胞
  2. HeLa细胞
  3. Dulbecco改良的Eagle培养基(DMEM)(Mediatech,Cellgro ,目录号:10-013-CV)
  4. R9-ΔE质粒(Zhou和Aiken,2001),通过在野生型感染性R9克隆的包膜中引入移码突变而产生的HIV-1原病毒DNA克隆。由该克隆产生的病毒体是非感染性的,但可以制备 通过用来自VSV或其他病毒的信封假型包装而感染)
  5. pHCMV-G(VSV-G)质粒(来自逆转录病毒的质粒,其中逆转录病毒包膜糖蛋白被来自水泡性口炎病毒[VSV]的糖蛋白替换)。
  6. p24 ELISA试剂盒(内部)
  7. 磷酸盐缓冲盐水(PBS)(Mediatech,Cellgro ,目录号:21-0310-CV)
  8. VSV-G-假型包膜缺陷型HIV-1(R9-ΔE)病毒颗粒
  9. Efavirenz(NIH AIDS Research and Reference Reagent Program,Division of AIDS,NIAID,
    NIH,目录号:11680)
  10. DNase I(Roche,目录号:10104159001)
  11. 0.25%胰蛋白酶/2.21mM EDTA(Mediatech,Cellgro ,目录号:25-053-CI)
  12. Triton X-100(Mallinckrodt,目录号:9002-93-1)
  13. DNeasy血液和组织试剂盒(QIAGEN,目录号:69506)
  14. cOmplete,Mini,无EDTA的蛋白酶抑制剂混合物片剂(Roche,目录号:11836170001)
  15. 4至20%聚丙烯酰胺梯度Tris-甘氨酸凝胶(Bio-Rad)
  16. 硝化纤维素膜(General Electric Company)
  17. 小鼠单克隆抗GAPDH抗体(Santa Cruz,目录号:sc-47724)
  18. 小鼠单克隆抗LaminB1抗体(Life Technologies,目录号:33-2000)
  19. SYBR green(ABI,目录号:4309155)
  20. DpnI(New England Biolabs,目录号:R0176L)
  21. DTT
  22. 酵母tRNA(Roche,目录号:10109541001)
  23. 正向引物MH531(5'-TGTGTGCCCGTCTGTTGTGT-3')
  24. 反向引物MH532(5'-GAGTCCTGCGTCGAGAGAGC-3')
  25. DNase/RNase-free水
  26. SDS-PAGE样品缓冲液
  27. 脱氧胆酸钠(Sigma-Aldrich,目录号:30970)
  28. N,N' - 双[2-羟乙基] -2-氨基乙磺酸(BES)(Sigma-Aldrich,目录号:B4554)
  29. 低密度缓冲区(参见配方)
  30. 放射免疫沉淀缓冲液(参见配方)
  31. 2x BES缓冲盐水(BBS)(参见Recipes)

设备

  1. 10厘米细胞培养皿
  2. 0.45-μm孔径注射器过滤器(Thermo Fisher Scientific,目录号:190-2545)
  3. 0.20-μm孔径注射器过滤器(Thermo Fisher Scientific,目录号:190-2520)
  4. 1.5 ml螺旋盖管
  5. 台式离心机(Thermo Fisher Scientific,Sorvall )
  6. 台式冷冻离心机(Thermo Fisher Scientific)
  7. Mx-3000p热循环仪(Stratagene)
  8. CO <2>孵化器

程序

  1. VSV-G假型包膜缺陷型HIV-1(R9-ΔE克隆)病毒颗粒的产生(Aiken,1998)
    1. 在37℃,5%CO 2下,将293T细胞在含有10%v/v胎牛血清(FBS)和补充有抗生素[青霉素(100IU/ml)和链霉素(100μg/。
    2. 借助0.25%胰蛋白酶/2.21mM EDTA从几乎汇合的培养皿中分离细胞,并在9ml培养基/100mm培养皿中种子2×10 6个细胞,并在37℃下孵育。
    3. 次日使用磷酸钙-BBS方法(Chen和Okoyama,1987)转染293T细胞
      1. 在管中混合15μgR9-ΔE和5μgpHCMV-G(VSV-G)质粒。
      2. 向管中加入0.2μm过滤的水,使体积达到450μl
      3. 向管中加入50μl2.5M CaCl 2。
      4. 向管中滴加500μl的2×BBS
      5. 用移液管轻轻混匀几次
      6. 在室温下孵育试管20至30分钟。
      7. 将混合物加入到293T细胞中,轻轻涡旋并在35℃和3%CO 2下孵育细胞。
    4. 从转染的皿中吸出培养基〜转染后16小时,用5ml PBS洗涤细胞,补充5ml新鲜细胞培养基,并在37℃,5%CO 2孵育。
    5. 转染两天后,收获含有病毒颗粒的培养物上清液,以1500×g离心5分钟以沉淀细胞和碎片。
    6. 通过0.45-μm孔径注射器过滤器过滤上清液,等分并在-80℃冷冻。

  2. 用VSV-G假型包膜缺陷型HIV-1感染HeLa细胞(R9-ΔE)
    1. 在12孔板中以1.5×10 5个细胞/孔的密度铺板HeLa细胞(每孔1ml总培养物体积)。
    2. 24小时后,用DNA酶I(20μg/ml)加上MgCl 2(10mM)处理病毒接种物,并在37℃的水浴中孵育1小时。
    3. 用DNA酶I处理的接种物相当于15ng的p24(通过使用内部试剂盒的p24ELISA(Wehrly和Chesebro,1997)测定)感染细胞。
    4. 在efavirenz(1μM)存在下进行平行感染,以确定转染后携带的残留质粒DNA水平。
    5. 在37℃孵育感染细胞8小时 注意:还可以通过在感染后以不同的时间间隔收获感染的细胞来分析逆转录的时间过程。

  3. HIV-1感染的HeLa细胞的细胞分级分离
    1. 孵育所需时间后,吸出培养基并用PBS洗涤细胞一次
    2. 通过与500μl的0.25%胰蛋白酶-EDTA在37℃下温育2分钟来去除贴壁细胞。
    3. 收集胰蛋白酶细胞在1.5毫升螺旋盖管。在300×g离心5分钟以沉淀细胞
    4. 溶解细胞沉淀在200μl含有0.1%Triton-X-100的低渗缓冲液中,并在冰上孵育15分钟。
      注意:对HeLa细胞优化Triton-X-100的浓度。 Trition X-100的浓度代表在显微镜下计数的约95%的细胞具有完整细胞核但没有质膜的最低浓度。
    5. 在4℃下以17,000×g离心5分钟,收集上清液作为细胞质级分。
    6. 用1ml低渗缓冲液,无Triton-X-100三次洗涤核沉淀。在每次洗涤后,在4℃下以17,000×g离心5分钟以沉淀细胞核并吸出上清液。
    7. 根据制造商的方案使用DNeasy血液和组织试剂盒分离核沉淀DNA。洗脱DNA在最后一步在新鲜的收集管使用100微升DNA酶/无RNA酶的水。洗脱的DNA可以 储存在-80°C或直接用于执行qPCR
    8. 平行地,从未感染的细胞制备全细胞,细胞质和核裂解物,以检查核级分的细胞质污染。
    9. 为了制备全细胞裂解物,在放射免疫沉淀(RIPA)缓冲液中裂解细胞(遵循步骤C2-C5,除了使用RIPA缓冲液代替低渗缓冲液)。加入等体积的2x SDS-PAGE样品缓冲液进行凝胶电泳,并在95℃在加热块中加热5分钟。
    10. 如上所述制备细胞质裂解物(步骤C2至C5)。加入等体积的2x SDS-PAGE样品缓冲液进行凝胶电泳,并在加热块中在95℃加热5分钟。
    11. 为了制备核裂解物,遵循步骤C2至C6,然后在1x SDS-PAGE样品缓冲液中裂解核沉淀。在95℃在加热块中加热5分钟。在4-20%聚丙烯酰胺梯度Tris-甘氨酸凝胶上分离等体积的全细胞,细胞质和核裂解物。
    12. 将解析的蛋白转移到硝酸纤维素膜上
    13. 用PBS中的5%脱脂奶溶液和含有抗GAPDH和抗LaminB1抗体(制造商推荐的浓度)的探针以及随后的适当的第二抗体(制造商推荐的浓度)分别作为胞质和核标记物阻断膜。

  4. SYBR基于绿色的定量PCR用于病毒逆转录产物的定量
    1. 通过在37℃下孵育1至2小时,用DpnI(17μlDNA +2μl缓冲液+1μlDpnI-20单位)处理来自步骤C7的分离的DNA。通过在80℃下孵育20分钟来灭活DpnI
    2. 病毒逆转录产物的定量
      1. 通过混合DNA(5μl),含有SYBR绿(12.5μl),正向引物(150nM),反向引物(150nM)和含有DNase/RNase-free水的tRNA(1μg/μl)的PCR混合物制备反应混合物至25μl
      2. 制备R9-ΔE质粒的10至10个拷贝/反应的标准物。 标准品的稀释应在1μg/μl含tRNA的水中进行。
      3. 使用以下热曲线设置PCR反应:

食谱

  1. 低渗缓冲液
    10 mM Tris pH 8.0
    10 mM KCl
    1.5mM MgCl 2·h/v 1 mM DTT
    蛋白酶抑制剂混合物(每10ml缓冲液一片)
  2. 放射免疫沉淀缓冲液
    50mM Tris pH7.5 1%Triton-X-100 250mM NaCl 5 mM EDTA
    0.1%SDS
    1%脱氧胆酸钠 蛋白酶抑制剂混合物(每10ml缓冲液一片)
  3. 2x BES缓冲盐水(BBS)
    50mM BES(N,N'-双[2-羟乙基] -2-氨基乙磺酸)
    1.5mM Na 2 HPO 4
    280 mM NaCl pH 6.95

致谢

该协议改编自Shah等人(2013)。 该协议由NIH授权AI076121支持。

参考文献

  1. Aiken,C。(1998)。 Nef和亲环蛋白的机制独立性增强人类免疫缺陷病毒1型感染性。 V 248(1):139-147。
  2. Chen,C.and Okayama,H。(1987)。哺乳动物细胞的高效转化质粒DNA。 Mol Cell Biol 7(8):2745-2752
  3. Shah,V.B.,Shi,J.,Hout,D.R.,Oztop,I.,Krishnan,L.,Ahn,J.,Shotwell,M.S.,Engelman,A.and Aiken, 宿主蛋白质转运蛋白SR2/TNPO3和亲环素A对HIV-1去膜剂产生相反的作用。 a> J Virol 87(1):422-432。
  4. Wehrly,K。和Chesebro,B。(1997)。 p24抗原捕获测定法,用于使用易得的廉价试剂定量人类免疫缺陷病毒。 em> Methods 12(4):288-293。
  5. Yee,J.K.,Friedmann,T。和Burns,J.C。(1994)。 生成具有非常宽的宿主范围的高滴度假型逆转录病毒载体。 Methods Cell Biol 43 Pt A:99-112。 
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How to cite this protocol: Shah, V. B. and Aiken, C. (2013). Cell Fractionation and Quantitative Analysis of HIV-1 Reverse Transcription in Target Cells. Bio-protocol 3(20): e949. DOI: 10.21769/BioProtoc.949; Full Text



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