0357f060cd841ac5b0d33f28a1af0169 pmc_9864670.pdf 8121d57374c09d16e35c6e7a3fc230c8adb4bad8 pmc_9864670.pdf 4c12a18cd01c0e0086ed5696a09d244872645fadbe305a0fb8ad9ca982913b63 pmc_9864670.pdf Title: Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids Subject: Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment. Keywords: biomarker; oral corticosteroids; miRNAs; individuals with severe asthma Author: Marta Gil-Martínez, Clara Lorente-Sorolla, José M. Rodrigo-Muñoz, Miguel Ángel Lendínez, Gonzalo Núñez-Moreno, Lorena de la Fuente, Pablo Mínguez, Ignacio Mahíllo-Fernández, Joaquín Sastre, Marcela Valverde-Monge, Santiago Quirce, María L. Caballero, Francisco J. González-Barcala, Ebymar Arismendi, Irina Bobolea, Antonio Valero, Xavier Muñoz, María Jesús Cruz, Carlos Martínez-Rivera, Vicente Plaza, José M. Olaguibel and Victoria del Pozo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 13 10:31:17 2023 CET ModDate: Fri Jan 13 10:35:55 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2731116 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LQMRUD+VnURWPalladioL Type 1 Custom yes yes no 10 0 CTMXWB+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 IRVMBA+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 DRYFSV+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 FJDQIS+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 BBSDEQ+EURM10 Type 1 Builtin yes yes no 74 0 UWJUYN+CMSY10 Type 1 Builtin yes yes no 78 0 QLBIEB+URWPalladioL-BoldItal Type 1 Custom yes yes no 82 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 89 0 MKAEJO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 106 0 MKAEJP+PalatinoLinotype CID TrueType Identity-H yes yes yes 109 0 MKAEKA+PalatinoLinotype TrueType WinAnsi yes yes no 115 0 MKAELC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 118 0 MKAJLF+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 121 0 MKAEJO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 149 0 MKAEJP+PalatinoLinotype CID TrueType Identity-H yes yes yes 152 0 MKAEKA+PalatinoLinotype TrueType WinAnsi yes yes no 158 0 MKAELC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 161 0 MKAJLF+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 164 0 MKAEJO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 198 0 MKAEJP+PalatinoLinotype CID TrueType Identity-H yes yes yes 201 0 MKAEKA+PalatinoLinotype TrueType WinAnsi yes yes no 207 0 MKAELC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 210 0 MKAEJO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 231 0 MKAEJP+PalatinoLinotype CID TrueType Identity-H yes yes yes 234 0 MKAEKA+PalatinoLinotype TrueType WinAnsi yes yes no 240 0 MKAELC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 243 0 MKAJLF+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 246 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-03-17 02:39:36 CET RepresentationInformation: pmc_9864670.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-26 02:11:42 CET Size: 2731116 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 452 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Characteristics of the Study Population Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: MiRNA Profile in Serum Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: MiRNAs Correlation and Signaling Pathways Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: MiRNA Profile in Lung Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: Study Subjects and Sample Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Library Preparation, MiRNAs-Seq, Bioinformatic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: RT-qPCR and Pathway Enrichment Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Info: Title: Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids Author: Marta Gil-Martínez, Clara Lorente-Sorolla, José M. Rodrigo-Muñoz, Miguel Ángel Lendínez, Gonzalo Núñez-Moreno, Lorena de la Fuente, Pablo Mínguez, Ignacio Mahíllo-Fernández, Joaquín Sastre, Marcela Valverde-Monge, Santiago Quirce, María L. Caballero, Francisco J. González-Barcala, Ebymar Arismendi, Irina Bobolea, Antonio Valero, Xavier Muñoz, María Jesús Cruz, Carlos Martínez-Rivera, Vicente Plaza, José M. Olaguibel and Victoria del Pozo Subject: Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment. Keywords: biomarker; oral corticosteroids; miRNAs; individuals with severe asthma Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 13 10:31:17 CET 2023 ModDate: Fri Jan 13 10:35:55 CET 2023 ID: 0x2dcc01dbe4755f582eafab1c832cabee, 0x2dcc01dbe4755f582eafab1c832cabee Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: QLBIEB+URWPalladioL-BoldItal FontSubset: true FirstChar: 110 LastChar: 112 FontDescriptor: FontName: QLBIEB+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IRVMBA+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 250 FontDescriptor: FontName: IRVMBA+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DRYFSV+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: DRYFSV+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: LQMRUD+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 230 FontDescriptor: FontName: LQMRUD+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: BBSDEQ+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: BBSDEQ+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: FJDQIS+VnURWPalladioL-Bold FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: FJDQIS+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: UWJUYN+CMSY10 FontSubset: true FirstChar: 0 LastChar: 21 FontDescriptor: FontName: UWJUYN+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: CTMXWB+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: CTMXWB+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 65, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 269, 77, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 144, 644, 155, 657 Annotation: Subtype: Link Rect: 397, 644, 408, 657 Annotation: Subtype: Link Rect: 165, 631, 176, 645 Annotation: Subtype: Link Rect: 370, 631, 381, 645 Annotation: Subtype: Link Rect: 515, 631, 526, 645 Annotation: Subtype: Link Rect: 290, 606, 301, 620 Annotation: Subtype: Link Rect: 216, 594, 227, 607 Annotation: Subtype: Link Rect: 354, 594, 365, 607 Annotation: Subtype: Link Rect: 446, 594, 457, 607 Annotation: Subtype: Link Rect: 264, 581, 275, 594 Annotation: Subtype: Link Rect: 139, 17, 278, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 426, 87, 445 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 301, 658, 308, 667 ActionDest: 263 Annotation: Subtype: Link Rect: 360, 632, 367, 641 ActionDest: 263 Annotation: Subtype: Link Rect: 200, 595, 207, 604 ActionDest: 263 Annotation: Subtype: Link Rect: 534, 582, 541, 591 ActionDest: 263 Annotation: Subtype: Link Rect: 238, 557, 245, 566 ActionDest: 263 Annotation: Subtype: Link Rect: 433, 532, 440, 541 ActionDest: 263 Annotation: Subtype: Link Rect: 531, 519, 538, 528 ActionDest: 263 Annotation: Subtype: Link Rect: 264, 469, 271, 478 ActionDest: 263 Annotation: Subtype: Link Rect: 272, 469, 279, 478 ActionDest: 263 Annotation: Subtype: Link Rect: 544, 431, 556, 441 ActionDest: 263 Annotation: Subtype: Link Rect: 261, 407, 273, 415 ActionDest: 263 Annotation: Subtype: Link Rect: 544, 407, 556, 415 ActionDest: 263 Annotation: Subtype: Link Rect: 525, 394, 537, 403 ActionDest: 263 Annotation: Subtype: Link Rect: 497, 381, 509, 390 ActionDest: 263 Annotation: Subtype: Link Rect: 418, 344, 430, 353 ActionDest: 263 Annotation: Subtype: Link Rect: 430, 344, 442, 353 ActionDest: 263 Annotation: Subtype: Link Rect: 451, 306, 463, 315 ActionDest: 263 Annotation: Subtype: Link Rect: 464, 177, 471, 189 ActionDest: 71 Annotation: Subtype: Link Rect: 492, 177, 499, 189 ActionDest: 86 Annotation: Subtype: Link Rect: 271, 126, 278, 138 ActionDest: 71 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 306, 720, 313, 732 ActionDest: 86 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 346, 543, 353, 555 ActionDest: 86 Annotation: Subtype: Link Rect: 393, 61, 400, 73 ActionDest: 86 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 199, 670, 206, 682 ActionDest: 86 Annotation: Subtype: Link Rect: 404, 657, 411, 669 ActionDest: 86 Annotation: Subtype: Link Rect: 243, 494, 250, 506 ActionDest: 129 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 356, 645, 363, 657 ActionDest: 129 Annotation: Subtype: Link Rect: 384, 582, 391, 594 ActionDest: 174 Annotation: Subtype: Link Rect: 311, 519, 318, 531 ActionDest: 174 Annotation: Subtype: Link Rect: 378, 66, 385, 78 ActionDest: 174 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 320, 695, 327, 707 ActionDest: 174 Annotation: Subtype: Link Rect: 321, 582, 328, 594 ActionDest: 221 Annotation: Subtype: Link Rect: 442, 135, 454, 144 ActionDest: 266 Annotation: Subtype: Link Rect: 455, 135, 467, 144 ActionDest: 266 Annotation: Subtype: Link Rect: 456, 97, 468, 106 ActionDest: 266 Annotation: Subtype: Link Rect: 435, 72, 447, 81 ActionDest: 263 Annotation: Subtype: Link Rect: 447, 72, 459, 81 ActionDest: 266 Annotation: Subtype: Link Rect: 459, 72, 471, 81 ActionDest: 266 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 393, 735, 405, 744 ActionDest: 266 Annotation: Subtype: Link Rect: 202, 610, 214, 619 ActionDest: 266 Annotation: Subtype: Link Rect: 280, 597, 292, 606 ActionDest: 266 Annotation: Subtype: Link Rect: 223, 572, 235, 581 ActionDest: 266 Annotation: Subtype: Link Rect: 245, 535, 257, 544 ActionDest: 263 Annotation: Subtype: Link Rect: 543, 522, 555, 531 ActionDest: 266 Annotation: Subtype: Link Rect: 529, 447, 541, 456 ActionDest: 266 Annotation: Subtype: Link Rect: 544, 447, 556, 456 ActionDest: 266 Annotation: Subtype: Link Rect: 325, 359, 337, 368 ActionDest: 266 Annotation: Subtype: Link Rect: 457, 321, 463, 330 ActionDest: 263 Annotation: Subtype: Link Rect: 512, 258, 524, 267 ActionDest: 266 Annotation: Subtype: Link Rect: 527, 258, 539, 267 ActionDest: 266 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 400, 471, 412, 480 ActionDest: 266 Annotation: Subtype: Link Rect: 357, 445, 369, 455 ActionDest: 266 Annotation: Subtype: Link Rect: 168, 292, 243, 304 Annotation: Subtype: Link Rect: 303, 131, 315, 140 ActionDest: 266 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 200, 660, 212, 669 ActionDest: 266 Annotation: Subtype: Link Rect: 328, 635, 340, 644 ActionDest: 266 Annotation: Subtype: Link Rect: 291, 623, 303, 631 ActionDest: 266 Annotation: Subtype: Link Rect: 394, 585, 406, 594 ActionDest: 266 Annotation: Subtype: Link Rect: 285, 560, 297, 569 ActionDest: 266 Annotation: Subtype: Link Rect: 340, 535, 352, 544 ActionDest: 266 Annotation: Subtype: Link Rect: 419, 535, 431, 544 ActionDest: 266 Annotation: Subtype: Link Rect: 434, 233, 446, 242 ActionDest: 269 Annotation: Subtype: Link Rect: 544, 208, 556, 217 ActionDest: 269 Annotation: Subtype: Link Rect: 531, 95, 543, 104 ActionDest: 269 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 403, 710, 415, 719 ActionDest: 269 Annotation: Subtype: Link Rect: 422, 635, 434, 644 ActionDest: 266 Annotation: Subtype: Link Rect: 536, 294, 560, 305 Annotation: Subtype: Link Rect: 165, 282, 377, 293 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 104, 478, 141, 489 Annotation: Subtype: Link Rect: 147, 478, 183, 489 Annotation: Subtype: Link Rect: 130, 455, 168, 466 Annotation: Subtype: Link Rect: 174, 455, 210, 466 Annotation: Subtype: Link Rect: 339, 420, 377, 431 Annotation: Subtype: Link Rect: 383, 420, 419, 431 Annotation: Subtype: Link Rect: 520, 409, 557, 420 Annotation: Subtype: Link Rect: 59, 398, 95, 408 Annotation: Subtype: Link Rect: 207, 374, 245, 385 Annotation: Subtype: Link Rect: 251, 374, 287, 385 Annotation: Subtype: Link Rect: 215, 351, 252, 362 Annotation: Subtype: Link Rect: 259, 351, 295, 362 Annotation: Subtype: Link Rect: 199, 328, 236, 339 Annotation: Subtype: Link Rect: 242, 328, 278, 339 Annotation: Subtype: Link Rect: 520, 316, 557, 327 Annotation: Subtype: Link Rect: 59, 306, 95, 316 Annotation: Subtype: Link Rect: 59, 271, 96, 281 Annotation: Subtype: Link Rect: 59, 237, 96, 247 Annotation: Subtype: Link Rect: 519, 213, 557, 224 Annotation: Subtype: Link Rect: 59, 202, 95, 212 Annotation: Subtype: Link Rect: 174, 179, 211, 189 Annotation: Subtype: Link Rect: 217, 179, 253, 189 Annotation: Subtype: Link Rect: 478, 167, 515, 178 Annotation: Subtype: Link Rect: 521, 167, 557, 178 Annotation: Subtype: Link Rect: 275, 144, 313, 155 Annotation: Subtype: Link Rect: 319, 144, 355, 155 Annotation: Subtype: Link Rect: 131, 110, 168, 120 Annotation: Subtype: Link Rect: 175, 110, 211, 120 Annotation: Subtype: Link Rect: 59, 87, 96, 97 Annotation: Subtype: Link Rect: 102, 87, 138, 97 Annotation: Subtype: Link Rect: 517, 63, 554, 74 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 425, 733, 462, 744 Annotation: Subtype: Link Rect: 395, 710, 432, 721 Annotation: Subtype: Link Rect: 204, 688, 242, 698 Annotation: Subtype: Link Rect: 371, 676, 408, 687 Annotation: Subtype: Link Rect: 414, 676, 450, 687 Annotation: Subtype: Link Rect: 415, 652, 452, 664 Annotation: Subtype: Link Rect: 458, 652, 494, 664 Annotation: Subtype: Link Rect: 169, 630, 207, 640 Annotation: Subtype: Link Rect: 213, 630, 249, 640 Annotation: Subtype: Link Rect: 236, 595, 273, 606 Annotation: Subtype: Link Rect: 86, 573, 124, 583 Annotation: Subtype: Link Rect: 90, 550, 128, 560 Annotation: Subtype: Link Rect: 59, 515, 96, 525 Annotation: Subtype: Link Rect: 506, 491, 544, 502 Annotation: Subtype: Link Rect: 303, 468, 340, 479 Annotation: Subtype: Link Rect: 188, 446, 226, 456 Annotation: Subtype: Link Rect: 232, 446, 268, 456 Annotation: Subtype: Link Rect: 220, 412, 258, 422 Annotation: Subtype: Link Rect: 264, 412, 300, 422 Annotation: Subtype: Link Rect: 131, 377, 168, 387 Annotation: Subtype: Link Rect: 175, 377, 211, 387 Annotation: Subtype: Link Rect: 177, 343, 215, 353 Annotation: Subtype: Link Rect: 221, 343, 257, 353 Annotation: Subtype: Link Rect: 127, 319, 165, 330 Annotation: Subtype: Link Rect: 171, 319, 207, 330 Annotation: Subtype: Link Rect: 95, 285, 133, 295 Annotation: Subtype: Link Rect: 506, 273, 544, 284 Annotation: Subtype: Link Rect: 472, 238, 510, 249 Annotation: Subtype: Link Rect: 381, 227, 478, 238 Annotation: Subtype: Link Rect: 194, 193, 231, 203 Annotation: Subtype: Link Rect: 520, 181, 557, 192 Annotation: Subtype: Link Rect: 467, 169, 505, 180 Annotation: Subtype: Link Rect: 109, 147, 146, 157 Annotation: Subtype: Link Rect: 152, 147, 188, 157 Annotation: Subtype: Link Rect: 158, 124, 195, 134 Annotation: Subtype: Link Rect: 201, 124, 237, 134 Annotation: Subtype: Link Rect: 148, 100, 260, 111 Annotation: Subtype: Link Rect: 132, 78, 170, 88 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 165, 722, 202, 733 Annotation: Subtype: Link Rect: 208, 722, 244, 733 Annotation: Subtype: Link Rect: 59, 688, 96, 698 Annotation: Subtype: Link Rect: 102, 688, 138, 698 Checksum: 482e3121 Type: CRC32 Checksum: 0357f060cd841ac5b0d33f28a1af0169 Type: MD5 Checksum: 8121d57374c09d16e35c6e7a3fc230c8adb4bad8 Type: SHA-1