Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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tion in the relative peak intensity for lower concentrations can be caused by reorientation of NAADP molecules with respect to the gold nanoparticles’ surfaces in the SERS sensor. 31,32 The correlation between the SERS spectra of cells treated with acetylcholine and that of pure NAADP was conducted using principal component analysis (PCA). 33 Principal component analysis is a technique which minimizes the dimensionality of the analyzed data array and permits assessment of the degree of correlation between large data sets. It is one of the most widely used methods in chemometrics, and it has been demonstrated to be efficient for analyzing SERS data. 34-36 According to the PCA results (Figure 5b), the control data acquired on untreated cells form a cluster which is denoted as “A”, away from the data of the treated cells, denoted as “B”. This confirms that the SERS sensor employed here distinguishes between the cells producing different amounts of NAADP. Furthermore, there is a clear correlation between the SERS spectra of the treated cells and that of the aqueous solution of 100 µM NAADP. This concentration is within the range of the expected induced NAADP concentration increase, according to the protocol that was used in this work. While this concentration (32) Barhoumi, A.; Zhang, D. M.; Halas, N. J. J. Am. Chem. Soc. 2008, 130, 14040–14041. (33) Jolliffe, I. T. Principal Component Analysis, 2nd ed.; Springer: New York, 2002. (34) Eliasson, C.; Loren, A.; Engelbrektsson, J.; Josefson, M.; Abrahamsson, J.; Abrahamsson, K. Spectrochim. Acta, Part A 2005, 61, 755–760. (35) Pearman, W. F.; Fountain, A. W. Appl. Spectrosc. 2006, 60, 356–365. (36) Hedegaard, M.; Krafft, C.; Ditzel, H. J.; Johansen, L. E.; Hassing, S.; Popp, J. R. Anal. Chem. 2010, 82, 2797–2802. 6774 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 is higher than the one which can be detected with enzymatic and radioreceptor binding assays, there is a significant advantage of time efficiency and accessibility of the SERS-based method. CONCLUSION Label-free NAADP detection and quantification in cell extracts is enabled by SERS, which permits the rapid detection of NAADP with a 100 µM concentration without any special sample purification or labeling. Importantly, this concentration does not represent a limit for SERS sensing of second calcium messengers. We were able to successfully detect 10 nM concentrations of NAADP in aqueous solution, which is on the order of basal levels of NAADP in cells, suggesting that intracellular SERS detection of the calcium messengers is possible. ACKNOWLEDGMENT This work was supported by a W. M. Keck Foundation grant to establish the W. M. Keck Institute for Attofluidic Nanotube- Based Probes at Drexel University, by the Pennsylvania Nanotechnology Institute (NTI) through Ben Franklin Technology Partners of Southeastern Pennsylvania, and by NIH Grants HL 90804 and HL 90804-01A2S1 to E.B. Raman spectroscopy analysis and scanning electron microscopy were conducted at the Centralized Research Facilities (CRF) at Drexel University. Received for review March 2, 2010. Accepted July 2, 2010. AC100563T
Anal. Chem. 2010, 82, 6775–6781 Highly Sensitive Fluorescent Method for the Detection of Cholesterol Aldehydes Formed by Ozone and Singlet Molecular Oxygen Fernando V. Mansano, Rafaella M. A. Kazaoka, Graziella E. Ronsein, Fernanda M. Prado, Thiago C. Genaro-Mattos, Miriam Uemi, Paolo Di Mascio, and Sayuri Miyamoto* Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP26077, CEP 05513-970, São Paulo, SP, Brazil Cholesterol oxidation gives rise to a mixture of oxidized products. Different types of products are generated according to the reactive species being involved. Recently, attention has been focused on two cholesterol aldehydes, 3�-hydroxy-5�-hydroxy-B-norcholestane-6�-carboxyaldehyde (1a) and 3�-hydroxy-5-oxo-5,6-secocholestan-6-al (1b). These aldehydes can be generated by ozone-, as well as by singlet molecular oxygen-mediated cholesterol oxidation. It has been suggested that 1b is preferentially formed by ozone and 1a is preferentially formed by singlet molecular oxygen. In this study we describe the use of 1-pyrenebutyric hydrazine (PBH) as a fluorescent probe for the detection of cholesterol aldehydes. The formation of the fluorescent adduct between 1a with PBH was confirmed by HPLC-MS/MS. The fluorescence spectra of PBH did not change upon binding to the aldehyde. Moreover, the derivatization was also effective in the absence of an acidified medium, which is critical to avoid the formation of cholesterol aldehydes through Hock cleavage of 5r-hydroperoxycholesterol. In conclusion, PBH can be used as an efficient fluorescent probe for the detection/quantification of cholesterol aldehydes in biological samples. Its analysis by HPLC coupled to a fluorescent detector provides a sensitive and specific way to quantify cholesterol aldehydes in the low femtomol range. Cholesterol (cholest-5-en-3�-ol) is a neutral lipid found in the cellular membranes of mammals. 1 Cholesterol is susceptible to oxidation mediated by enzymatic and nonenzymatic mechanisms. 2-5 The nonenzymatic oxidation can be mediated by reactive oxygen species. 2 Several oxidized products of cholesterol have been characterized including hydroperoxides, epoxides, and aldehydes. 2,6,7 These oxysterols have been detected in biological tissues and their formation has been associated to neurodegenerative and cardio- * To whom correspondence should be addressed. Phone: (55) (11) 3091- 3810 (x261). Fax: (55) (11) 3815-5579. E-mail: miyamoto@iq.usp.br. (1) Maxfield, F. R.; Tabas, I. Nature 2005, 438, 612–621. (2) Brown, A. J.; Jessup, W. Atherosclerosis 1998, 142, 1–28. (3) Björkhem, I.; Diczfalusy, U. Arterioscler. Thromb. Vasc. Biol. 2002, 22, 734–742. (4) Garenc, C.; Julien, P.; Levy, E. Free Radical Res. 2010, 44, 47–73. (5) Schroepfer, G. J., Jr. Physiol. Rev. 2000, 80, 361–554. (6) Smith, L. L. Lipids 1996, 31, 453–487. (7) Girotti, A. W. J. Photochem. Photobiol. B 1992, 13, 105–118. vascular diseases. 2-5 Recently, attention has been focused on cholesterol aldehydes that can be formed by the oxidation of cholesterol by ozone 8 and singlet molecular oxygen. 9,10 The ozonation of cholesterol produces several oxidized products, in special two aldehydes (Figure 1), the cholesterol 5,6secosterols,3�-hydroxy-5�-hydroxy-B-norcholestane-6�-carboxyaldehyde (1a) and 3�-hydroxy-5-oxo-5,6-secocholestan-6-al (1b). 11,12 Wentworth and co-workers showed the presence of 1a and 1b in atherosclerotic plaques 8 and LDL oxidized with several oxidants. 13 These aldehydes have been also detected in neurodegenerative diseases, like Lewy body dementia 14 and Alzheimer disease. 15 The role of 1a and 1b in the pathogenesis of cardiovascular and neurodegenerative diseases has been investigated. In vitro studies have shown that cholesterol aldehydes can covalently modify proteins, as well as, accelerate their aggregation, as in the case of amyloid �-peptide formation 15-17 and R-synuclein 14 . Further studies have shown that covalent modification of apo-B by 1b causes this protein to misfold, rendering the LDL particle more susceptible to macrophage uptake. 18 Moreover, some reports have also shown that cholesterol aldehydes can induce apoptosis in macrophages and cardiomyoblasts. 19,20 The induction of apoptosis in cardi- (8) Wentworth, P., Jr.; Nieva, J.; Takeuchi, C.; Galve, R.; Wentworth, A. D.; Dilley, R. B.; DeLaria, G. A.; Saven, A.; Babior, B. M.; Janda, K. D.; Eschenmoser, A.; Lerner, R. A. Science 2003, 302, 1053–1056. (9) Brinkhorst, J.; Nara, S. J.; Pratt, D. A. J. Am. Chem. Soc. 2008, 130, 12224– 12225. (10) Uemi, M.; Ronsein, G. E.; Miyamoto, S.; Medeiros, M. H. G.; Di Mascio, P. Chem. Res. Toxicol. 2009, 22, 875–884. (11) Gumulka, J.; Smith, L. L. J. Am. Chem. Soc. 1983, 105, 1972–1979. (12) Jaworski, K.; Smith, L. L. J. Org. Chem. 1988, 53, 545–554. (13) Wentworth, A. D.; Song, B. D.; Nieva, J.; Shafton, A.; Tripurenani, S.; Wentworth, P. Chem. Commun. 2009, 3098–3100. (14) Bosco, D. A.; Fowler, D. M.; Zhang, Q.; Nieva, J.; Powers, E. T.; Wentworth, P.; Lerner, R. A.; Kelly, J. W. Nat. Chem. Biol. 2006, 2, 249–253. (15) Zhang, Q.; Powers, E. T.; Nieva, J.; Huff, M. E.; Dendle, M. A.; Bieschke, J.; Glabe, C. G.; Eschenmoser, A.; Wentworth, P., Jr.; Lerner, R. A.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4752–4757. (16) Scheinost, J. C.; Wang, H.; Boldt, G. E.; Offer, J.; Wentworth, P. Alzheimer Dis. 2008, 47, 3919–3922. (17) Usui, K.; Hulleman, J. D.; Paulsson, J. F.; Siegel, S. J.; Powers, E. T.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18563–18568. (18) Takeuchi, C.; Galve, R.; Nieva, J.; Witter, D. P.; Wentworth, A. D.; Troseth, R. P.; Lerner, R. A.; Wentworth, P. Biochemistry 2006, 45, 7162–7170. (19) Sathishkumar, K.; Gao, X.; Raghavamenon, A. C.; Parinandi, N.; Pryor, W. A.; Uppu, R. M. Free Radical Biol. Med. 2009, 47, 548–558. (20) Gao, X.; Raghavamenon, A. C.; D’Auvergne, O.; Uppu, R. M. Biochem. Biophys. Res. Commun. 2009, 389, 382–387. 10.1021/ac1006427 © 2010 American Chemical Society 6775 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/21/2010
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29: Figure 4. SERS analysis of NAADP co
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: Table 3. Ambient Measurement Result
Page 63 and 64: Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82:
Figure 2. Productivity of SEQUST an
Page 83 and 84:
Figure 4. High-resolution MS/MS spe
Page 85 and 86:
Figure 6. Characterization of PSMs
Page 87 and 88:
containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
Page 91 and 92:
Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
Page 97 and 98:
constant medium for separation; we
Page 99 and 100:
Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102:
Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106:
Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
Page 119 and 120:
corresponding compound if its chemi
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Figure 1. Schematic illustration of
Page 123 and 124:
Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
Page 143 and 144:
Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153 and 154:
Table 1. Chemical Structure, pKa Va
Page 155 and 156:
pH with a tilted baseline (Figure 1
Page 157 and 158:
Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
Page 161 and 162:
the multielement capabilities, the
Page 163 and 164:
RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
Page 175 and 176:
Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
Page 179 and 180:
Figure 2. With a suitable removal o
Page 181 and 182:
the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192:
trode 28 by a finite element using
Page 193 and 194:
Figure 4. Comparison between simula
Page 195 and 196:
Figure 6. Comparison between simula
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educed in the vicinity of double bo
Page 199 and 200:
Figure 2. Normalized product ion ab
Page 201 and 202:
Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204:
Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206:
Figure 1. Schematic flowchart showi
Page 207 and 208:
difference, ppm compound Table 1. I
Page 209 and 210:
isoforms, its successful use, in th
Page 211 and 212:
Figure 5. Extracted ion current ESI
Page 213 and 214:
m/z 1172.935 was observed for Ser14
Page 215 and 216:
linked products via affinity tags.
Page 217 and 218:
Scheme 2. Fragmentation Mechanism o
Page 219 and 220:
Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221 and 222:
Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224:
Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226:
Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
Page 235 and 236:
allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
Page 247 and 248:
Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
Page 251 and 252:
Table 2. Capillary Electrophoresis
Page 253 and 254:
Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
Page 257 and 258:
Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
Page 271 and 272:
Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273 and 274:
Table 1. Effect of 1 D-LC Condition
Page 275 and 276:
Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280:
luciferase (T7 control vector) was
Page 281 and 282:
Figure 3. Inhibitory effects of luc
Page 283 and 284:
Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286:
Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
Page 289 and 290:
Table 3. Film Thickness Measurement
Page 291 and 292:
Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296:
Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300:
Scheme 1. Proposed Mechanism for Fl
Page 301 and 302:
one or more drawbacks including poo
Page 303 and 304:
Figure 2. Free fluorophores do not
Page 305 and 306:
Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308:
Figure 2. Comparative analysis of d
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