Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells

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In this protocol we describe a nonradiolabelled labelling of GPI anchor in Candida albicans. The method uses a fluorescent probe to bind specifically to GPI anchors so that the level of GPI-anchored proteins at the cell surface can be measured. The labelling does not need permeabilization of cells and can be carried out in vivo.

Keywords: FLAER(FLAER), GPI anchor(GPI锚点), Candida albicans(白色念珠菌)


The GPI (glycosylphosphatidylinositol) anchor is a post translational modification occurring in the endoplasmic reticulum (ER). The preformed GPI anchor is attached in the lumen of the ER to the C-terminus of specific proteins that carry a GPI anchor attachment signal sequence. These proteins are subsequently transported (and extensively further modified) by the secretory pathway to the cell surface where the proteins are present anchored to the extracellular leaflet of the plasma membrane or covalently linked to the cell wall in organisms that have a wall. A variety of proteins get GPI anchored proteins in eukaryotes, for example, hydrolytic enzymes, cell surface adhesion molecules or receptor proteins (Orlean and Menon, 2007). In fungal pathogens, such as Candida albicans, many of the adhesins involved in host recognition and adherence, as well as several pathogenesis and virulence factors are GPI anchored proteins; besides, several GPI-anchored proteins in Candida albicans have proteolytic activity (Richard and Plaine, 2007; Nobile et al., 2008). The pathway is essential in Candida albicans and downregulating it or targeting it could be a useful strategy to combat Candida infections. The protocol used for assessing GPI anchor levels in Candida albicans is elaborated here.

Fluorescently Labelled Aerolysin (FLAER) is a fluorescein labelled inactive derivative of aerolysin, a Gram-negative bacterial toxin, which binds to GPI-anchored proteins in the cell membrane of the eukaryotic host and forms pores (Howard et al., 1987; Parker et al., 1994). In FLAER, proaerolysin is tagged with Alexa Fluor 488 dye which can bind to GPI anchors of eukaryotic cells without any harm to the cell (Sutherland et al., 2007). FLAER is also clinically used to detect GPI anchor levels in Paroxysmal Nocturnal Hemoglobinuria (PNH) cells, a disease caused by somatic mutations in PIGA (a subunit of glycosylphosphatidylinositol-N-acetylglucosamine transferase enzyme complex) in mammals (Brodsky et al., 2000). Once stained with FLAER, the fluorescence of cells can be monitored/quantified under a confocal fluorescence microscope in the GFP channel and the data used as a measure of GPI anchored proteins on the cell surface. In a previous study, we monitored the levels of GPI-anchored proteins on the cell surface of a GPI biosynthetic mutant, Cagpi14, in Candida albicans by using FLAER (Singh et al., 2016). CaGPI14 encodes for the catalytic subunit of the first mannosyltransferase of the GPI-anchor biosynthesis pathway. Deficiency in CaGpi14 severely affects cell growth and wall integrity in the organism although low levels of expression appear to be sufficient to make the cells viable. Its homolog in S. cerevisiae is essential (Kim et al., 2007). The protocol for FLAER labelling used to assess GPI anchor levels in this mutant of Candida albicans is described here.

Materials and Reagents

  1. Pipette tips
  2. Sterile plastic toothpick
  3. Culture tubes (50 ml)
  4. 1.5 ml micro-centrifuge tubes
  5. Glass slides
  6. Cover slips
  7. Candida albicans strains CAF2-1 (URA3/ura3::imm434 IRO1/iro1::imm434) and conditional null mutant of CaGPI14 (CAF2-1::Cagpi14/pMET3-CaGPI14), encoding the catalytic subunit of the first mannosyltransferase in the GPI biosynthetic pathway, for this study
  8. Synthetic defined (SD) medium with Uridine+Cys-Met- dropout mix was made in the lab for this study since the strains were uridine auxotrophs. Cys/Met was used to repress CaGPI14 expression (all L-amino acids were obtained from Sisco Research Laboratory). This was possible because the conditional mutant strain had the only surviving allele of CaGPI14 placed under the control of the MET3 promoter, which permits gene expression in the absence of Cys/Met and represses its expression in the presence of Cys/Met
  9. Liquid FLAER (25 µg/0.5 ml) from Pinewood Scientific Services Inc. (FL1S-C)
  10. 50% glycerol (Merck, catalog number: 1.07051.0521 )
  11. D-glucose (Fisher Scientific, catalog number: D16-500 )
  12. Yeast nitrogen base (HiMedia Laboratories, catalog number: M878 )
  13. Cysteine (Sisco Research Laboratory, catalog number: 034890 )
  14. Methionine (Sisco Research Laboratory, catalog number: 134869 )
  15. Sodium chloride (NaCl) (Fisher Scientific, catalog number: 15915 )
  16. Potassium chloride (KCl) (Merck, catalog number: 1049360500 )
  17. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-3 )
  18. Potassium phosphate monobasic (KH2PO4) (Merck, catalog number: 1.93205.0521 )
  19. Hydrochloric acid (HCl)
  20. SD Uri+Cys-Met-/SD Uri+Cys+Met+ broth (100 ml) (see Recipes)
  21. Phosphate buffered saline (PBS; pH 7.5) (see Recipes)


  1. Pipettes
    100-1,000 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642090 )
    20-200 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642080 )
    2-20 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642060 )
  2. Vortex mixer (REMI GROUP, model: CM-101 )
  3. Shaker incubator (Set at either 30 °C or 20 °C according to requirement) (Labtech, model: LSI-5002M )
  4. Centrifuge (Plasto Craft Industries, model: Micro-spin R-V/FM )
  5. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: MultiskanTM GO Microplate Spectrophotometer , catalog number: 51119300)
  6. Andor spinning Disk confocal microscope
  7. Autoclave


  1. Andor iQ2.7 (for image capturing)
  2. GIMP 2.8.18 software (for quantification)
  3. Origin 0.5 software


  1. Primary culture of all the strains is set up in 10 ml of SD Uri+Cys-Met- medium using a tip of a stock culture picked up with a sterile plastic toothpick. The cultures are allowed to grow at 30 °C overnight in shaking condition at 220 rpm.
  2. Secondary cultures are set up with 2% of overnight grown cultures (200 μl of overnight grown culture in 10 ml) in fresh appropriate medium (CAF2-1 in SD Uri+Cys-Met- and conditional null of CaGPI14 in SD Uri+Cys-Met- [permissive] as well as in SD Uri+Cys+Met+ [repressive] media) at 30 °C with shaking (220 rpm).
  3. After 5 h of growth (log phase) the cells are spun down by centrifugation at 3,000 x g for 5 min at room temperature (30 °C). Cells in log phase are preferred since we observed extensive cell clumping in the Cagpi14 mutant cells at late stages of growth and in stationary phase. It is also important to point out that in saturated cultures, cell death processes may be initiated and this in turn alters cell wall and membrane stability, affecting the staining of the cells. To avoid errors arising from these problems, log phase cells are preferred.
  4. The cell pellets are washed with PBS by centrifugation at 3,000 x g and resuspended in 10 ml of PBS.
  5. Equal numbers of cells in each case, corresponding to OD600 nm ~0.6, are taken and resuspended in a total volume of 200 µl PBS in 1.5 ml micro-centrifuge tubes.
  6. FLAER (10 μl of 50 μg/ml i.e., 0.5 μg) is added to the 200 μl cell suspension (1:20 dilution).
  7. After mild vortexing at ~500 rpm, the cell suspension is incubated for 1 h in a shaker-incubator (70 rpm) at 20 °C in the dark.
  8. The cells are then spun down at 3,000 x g for 3 min and the cell pellets washed thrice with PBS by pipetting.
  9. For microscopic analysis cell pellets are resuspended in 40 µl of 50% glycerol (v/v in water).
  10. 5 µl of cell suspensions are spotted on glass slides and covered with cover slips.
  11. Fluorescent images of the cells are captured using a confocal fluorescence microscope with an Alexa 488 filter with 0.4 sec and 0.2 sec exposure time for fluorescence and DIC images, respectively. Camera EM gain is 100 with camera exposure units of 1 for both type of images. A representative image for the obtained results is shown in Figure 1.

    Figure 1. Confocal imaging of FLAER labelled cells. Images of wild type (CAF2-1) and Cagpi14 conditional null mutant cells under permissive (Cys-Met-) as well as repressive (Cys+Met+) conditions after incubation with FLAER as described above. Scale bars = 10 µm in each image.

Data analysis

Quantification of fluorescence intensity:

  1. Fluorescence intensity of cells can be quantified using Nikon-AR-analysis or GIMP software. For each strain fluorescence intensity of at least 50 cells are counted. For the data plotted here, quantification was done using GIMP software. Schematic representation of quantification is shown in Figure 2. P-values for statistical significance of data are calculated by using the Student’s t-test in Microsoft Excel.

    Figure 2. Schematic diagram showing quantification of fluorescence intensity using GIMP 2.8.18 software. Quantification of single fluorescent cells has been shown as example.

  2. Relative intensity for the mutant is plotted with reference to the fluorescence intensity of wild type strain (CAF2-1) taken as 100%.
  3. The data points are plotted in Origin 0.5 software (Figure 3) in form of bar graph.
  4. In the above experiment we find roughly 40% lower fluorescence in the conditional null mutant (under repressive conditions) as compared to the wild type strain, CAF2-1. Under permissive conditions, only 20% reduction in fluorescence intensity is observed relative to the wild type strain.

    Figure 3. Quantification of the relative fluorescence intensity of FLAER labelled cells. The percent fluorescence intensity of the Cagpi14 conditional null mutant cells is plotted relative to that of wild type (CAF2-1) cells (100%). P-value < 0.003 (**).


  1. Labelling should be performed with cells from a secondary culture in the log-phase of growth.
  2. Conditions that promote clumping of cells should be avoided as this introduces errors in estimation of cell numbers as well as in levels of staining.
  3. Images should be captured on the same day of the labelling.


  1. SD Uri+Cys-Met-/SD Uri+Cys+Met+ broth (100 ml)
    2 g D-glucose
    0.67 g YNB
    0.2 g Uri-Cys-Met- dropout mix (Mixture of all L-amino acids except L-cysteine, L-methionine and uridine; 1 g of each amino acid except for adenine [0.25 g] and para-aminobenzoic acid [0.1 g])
    1 M uridine solution (made in autoclaved water)
    Add above chemicals in 80 ml double distilled water and dissolve
    Add 500 μl of 1 M uridine solution to it and make up the volume to 100 ml
    For making SD Uri+Cys+Met+ medium add cysteine (5 mM) and methionine (5 mM) to SD Uri+Cys-Met- medium and autoclave
  2. Phosphate buffer saline (PBS), pH 7.5 (1 L)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolve the above chemicals in 800 ml double distilled water
    Adjust pH to 7.5 with HCl
    Make up the volume with double distilled water


The above protocol is adapted from our previous study (Singh et al., 2016). The work has been funded by a senior research fellowship from Indian Council of Medical Research (ICMR) to SLS and a research grant (No. SB/OC/CB-03A/2014) from Department of Science and Technology (DST), India to SSK.


  1. Brodsky, R. A., Mukhina, G. L., Li, S., Nelson, K. L., Chiurazzi, P. L., Buckley, J. T. and Borowitz, M. J. (2000). Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 114(3): 459-466.
  2. Howard, S. P., Garland, W. J., Green, M. J. and Buckley, J. T. (1987). Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila. J Bacteriol 169(6): 2869-2871.
  3. Kim, Y.U., Ashida, H., Mori, K., Maeda, Y., Hong, Y. and Kinoshita, T. (2007). Both mammalian PIG-M and PIG-X are required for growth of GPI14-disrupted yeast. J Biochem 142(1): 123-129.
  4. Nobile, C. J., Schneider, H. A., Nett, J. E., Sheppard, D. C., Filler, S. G., Andes, D. R. and Mitchell, A. P. (2008). Complementary adhesin function in C. albicans biofilm formation. Curr Biol 18(14): 1017-1024.
  5. Orlean, P. and Menon, A. K. (2007). Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids. J Lipid Res 48(5): 993-1011.
  6. Parker, M. W., Buckley, J. T., Postma, J. P., Tucker, A. D., Leonard, K., Pattus, F. and Tsernoglou, D. (1994). Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature 367(6460): 292-295.
  7. Richard, M. L. and Plaine, A. (2007). Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicans. Eukaryot Cell 6(2): 119-133.
  8. Singh, S. L., Rai, R. C., Sah, S. K. and Komath, S. S. (2016). The catalytic subunit of the first mannosyltransferase in the GPI biosynthetic pathway affects growth, cell wall integrity and hyphal morphogenesis in Candida albicans. Yeast 33(8): 365-383.
  9. Sutherland, D. R., Kuek, N., Davidson, J., Barth, D., Chang, H., Yeo, E., Bamford, S., Chin-Yee, I. and Keeney, M. (2007). Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytometry B Clin Cytom 72(3): 167-177.



背景 GPI(糖基磷脂酰肌醇)锚是在内质网(ER)中发生的翻译后修饰。预先形成的GPI锚定体连接到携带GPI锚定附着信号序列的特异性蛋白质的C末端的ER的内腔中。这些蛋白质随后通过分泌途径转移(并进一步修饰)到细胞表面,其中蛋白质锚定在质膜的细胞外小叶上或共价连接到具有壁的生物中的细胞壁。各种蛋白质在真核生物中获得GPI锚定蛋白,例如水解酶,细胞表面粘附分子或受体蛋白(Orlean和Menon,2007)。在真菌病原体中,例如白色念珠菌,参与宿主识别和粘附的许多粘附素以及几种发病机理和毒力因子是GPI锚定蛋白;此外,白色念珠菌中的几种GPI锚定蛋白具有蛋白水解活性(Richard和Plaine,2007; Nobile等人,2008)。该途径在白色念珠菌中是必不可少的,并且其下调或靶向它可能是打击念珠菌感染的有用策略。本文详细阐述了用于评估白色念珠菌中GPI锚定水平的方案。
&NBSP;荧光标记的Aerolysin(FLAER)是荧光素标记的非溶性细胞溶解素的无活性衍生物,革兰氏阴性细菌毒素,其与真核宿主的细胞膜中的GPI锚定的蛋白质结合并形成孔(Howard等, ,1987; Parker等人,1994)。在FLAER中,用Alexa Fluor 488染料标记原溶血素,其可以与真核细胞的GPI锚点结合,而不会对细胞造成任何损害(Sutherland等人,2007)。 FLAER也临床上用于检测阵发性夜间血红蛋白尿(PNH)细胞中的GPI锚定水平,这是由PIGA 中的体细胞突变引起的疾病(糖基磷脂酰肌醇-N'-乙酰基葡糖胺的亚基转移酶复合物)(Brodsky等人,2000)。一旦用FLAER染色,可以在GFP通道的共聚焦荧光显微镜下监测/定量细胞的荧光,并且将数据用作细胞表面上GPI锚定蛋白的量度。在以前的研究中,我们通过使用FLAER(Singh em)监测了白色念珠菌中GPI生物合成突变体(Cagpi14)细胞表面GPI锚定蛋白的水等,,2016)。 CaGPI14 编码GPI锚定生物合成途径的第一个甘露糖基转移酶的催化亚基。 CaGpi14缺乏严重影响生物体的细胞生长和壁完整性,尽管低水平的表达似乎足以使细胞生存。它的同系在 S。酿酒酵母是必需的(Kim等人,2007)。这里描述了用于评估这种白色念珠菌突变体GPI锚定水平的FLAER标签的方案。

关键字:FLAER, GPI锚点, 白色念珠菌


  1. 移液器提示
  2. 无菌塑胶牙签
  3. 培养管(50ml)
  4. 1.5 ml微量离心管
  5. 玻璃幻灯片
  6. 封面
  7. 白色念珠菌株CAF2-1(URA3 / ura3 :: imm434 iro1 :: imm434)和条件用于本研究的编码GPI生物合成途径中第一个甘露糖基转移酶的催化亚基的CaGPI14(CAF2-1 :: Cagpi14 / pMET3-CaGPI14)的无效突变体
  8. 在本研究的实验室中制备了具有尿苷 Cys - Met - 的合成定义的(SD)培养基,因为菌株是尿苷营养缺陷型。 Cys / Met用于抑制CaGPI14表达(所有L-氨基酸均得自Sisco Research Laboratory)。这是可能的,因为条件突变株具有放置在MET3启动子控制下的唯一存活等位基因的CaGPI14,其允许在不存在Cys / Met的情况下进行基因表达,并且在Cys / Met
  9. 来自松林科学服务有限公司(FL1S-C)的液体FLAER(25μg/ 0.5ml)
  10. 50%甘油(Merck,目录号:1.07051.0521)
  11. D-葡萄糖(Fisher Scientific,目录号:D16-500)
  12. 酵母氮基(HiMedia Laboratories,目录号:M878)
  13. 半胱氨酸(Sisco Research Laboratory,目录号:034890)
  14. 甲硫氨酸(Sisco Research Laboratory,目录号:134869)
  15. 氯化钠(NaCl)(Fisher Scientific,目录号:15915)
  16. 氯化钾(KCl)(默克,目录号:1049360500)
  17. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S374-3)
  18. 磷酸二氢钾(KH 2 PO 4)(Merck,目录号:1.93205.0521)
  19. 盐酸(HCl)
  20. SD Uri + Cys - Met - / SD Uri + Cys + sup> + 肉汤(100ml)(参见食谱)
  21. 磷酸盐缓冲盐水(PBS; pH 7.5)(参见食谱)


  1. 移液器
    100-1,000μl(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4642090)
    20-200μl(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4642080)
    2-20μl(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4642060)
  2. 涡街搅拌机(REMI GROUP,型号:CM-101)
  3. 振荡器培养箱(根据需要设置在30°C或20°C)(Labtech,型号:LSI-5002M)
  4. 离心机(Plasto Craft Industries,型号:Micro-spin R-V / FM)
  5. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Multiskan TM GO微孔板分光光度计,目录号:51119300)
  6. 安道尔纺丝盘共焦显微镜
  7. 高压灭菌器


  1. 安道尔iQ2.7(用于图像捕获)
  2. GIMP 2.8.18软件(用于量化)
  3. Origin 0.5软件


  1. 将所有菌株的原代培养物置于10ml SD Uri Cys Met - 培养基中,使用采集的原料培养物用无菌塑料牙签。允许培养物在30℃振荡条件下以220rpm生长过夜。
  2. 在新鲜合适的培养基(CAF1-1,SD Uri Cys )中,用2%过夜培养的培养物(200μl,10ml中培养过夜培养物) > Met - > [允许]以及SD Uri + Cys + Met + [镇压]介质)在30℃下摇动(220rpm )。
  3. 在生长5小时(对数期)后,通过在室温(30℃)下以3,000×g离心5分钟将细胞离心。对照期细胞是优选的,因为我们观察到在生长晚期和稳定期的Cagpi14突变细胞中广泛的细胞聚集。同样重要的是要指出,在饱和培养物中,可能会引发细胞死亡过程,这又会改变细胞壁和膜的稳定性,影响细胞的染色。为了避免这些问题产生的错误,优先使用对数阶段单元。
  4. 用PBS以3,000×g离心清洗细胞沉淀,并重新悬浮在10ml PBS中。
  5. 在每种情况下,对应于OD 600> nm 0.6的细胞相等数目,并在1.5ml微量离心管中重悬于200μlPBS的总体积中。
  6. 将FLAER(10μl,50μg/ ml,即0.5μg)加入200μl细胞悬液(1:20稀释)中。
  7. 在约500rpm的温和涡旋后,将细胞悬浮液在20℃在黑暗中的摇动培养箱(70rpm)中孵育1小时。
  8. 然后将细胞以3,000xg离心3分钟,并通过移液将细胞沉淀用PBS洗涤三次。
  9. 对于微观分析,将细胞沉淀重悬于40μl50%甘油(v / v水溶液)中
  10. 5μl细胞悬浮液被点在玻璃片上,盖上盖子。
  11. 使用具有Alexa 488滤色器的共聚焦荧光显微镜捕获细胞的荧光图像,分别具有用于荧光和DIC图像的0.4秒和0.2秒曝光时间。相机EM增益为100,相机曝光单位为1,适用于两种类型的图像。获得结果的代表性图像如图1所示

    图1. FLAER标记细胞的共焦成像野生型(CAF2-1)和Cagpi14 条件无效突变体细胞在许可下的图像(Cys - Met - )以及如上所述与FLAER孵育后的抑制(Cys + Met + )条件。每个图像中的比例尺= 10μm。



  1. 使用Nikon-AR分析或GIMP软件可以量化细胞的荧光强度。对于每个菌株,计数至少50个细胞的荧光强度。对于此处绘制的数据,使用GIMP软件进行定量。量化的示意图如图2所示。通过使用Microsoft Excel中的Student's t -test来计算数据的统计显着性的值。

    图2.使用GIMP 2.8.18软件对荧光强度进行定量的示意图单荧光细胞的定量显示为例。

  2. 参照野生型菌株(CAF2-1)的荧光强度作为100%绘制突变体的相对强度。
  3. 数据点以条形图的形式绘制在Origin 0.5软件(图3)中。
  4. 在上述实验中,与野生型菌株CAF2-1相比,在条件无效突变体(抑制条件下)中发现约40%的低荧光。在许可条件下,相对于野生型菌株,仅观察到荧光强度降低了20%

    图3. FLAER标记细胞的相对荧光强度的定量。将Cagpi14条件无效突变体细胞的荧光强度百分比相对于野生型(CAF2- 1)细胞(100%)。值 - 值= 0.003(**)。


  1. 应在生长对数阶段用来自次生培养物的细胞进行标记。
  2. 应避免促进细胞聚集的条件,因为这会导致细胞数量估计和染色水平的错误。
  3. 图像应在标签的同一天被捕获。


  1. SD Uri + Cys - Met - / SD Uri + Cys + sup> + 肉汤(100ml)
    2 g D-葡萄糖
    0.2g Uri - Met - 辍学混合物(L-半胱氨酸,L-甲硫氨酸和尿苷除外的所有L-氨基酸的混合物; 1g腺嘌呤[0.25g]和对氨基苯甲酸[0.1g]除外)各氨基酸1g 1 M尿苷溶液(在高压灭菌水中制成)
    加入500μl1 M尿苷溶液,使体积达到100 ml
    将SD Uri + Met + 将SD添加半胱氨酸(5mM)和甲硫氨酸(5mM) Cys - Met - medium和高压灭菌器
  2. 磷酸盐缓冲盐水(PBS),pH 7.5(1L)
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4
    将上述化学物质溶于800毫升双蒸水中 用HCl调节pH至7.5 用双蒸水补足体积


上述协议是从我们以前的研究(Singh等人,2016)改编而来的。这项工作由印度医学研究委员会(ICMR)到SLS的高级研究奖学金和印度科学技术部(DST)的研究资助(SB / OC / CB-03A / 2014)资助。 SSK。


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引用:Singh, S. L. and Komath, S. S. (2017). Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells. Bio-protocol 7(11): e2303. DOI: 10.21769/BioProtoc.2303.

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