c3c19d1d9b6c3aecb5b2943e352ceb87 ijms-23-14868.pdf 2d22d670f9eb9a963cd1a6a9f2afaafcd94ecb28 ijms-23-14868.pdf a3db49ffb84b9485aef33aa023bccd241e01ca2c069b351d860d7b100c001aae ijms-23-14868.pdf Title: Deciphering the Genetic Crosstalk between Microglia and Oligodendrocyte Precursor Cells during Demyelination and Remyelination Using Transcriptomic Data Subject: Demyelinating disorders show impaired remyelination due to failure in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature myelin-forming oligodendrocytes, a process driven by microglia–OPC crosstalk. Through conducting a transcriptomic analysis of microarray studies on the demyelination–remyelination cuprizone model and using human samples of multiple sclerosis (MS), we identified molecules involved in this crosstalk. Differentially expressed genes (DEGs) of specific regions/cell types were detected in GEO transcriptomic raw data after cuprizone treatment and in MS samples, followed by functional analysis with GO terms and WikiPathways. Additionally, microglia–OPC crosstalk between microglia ligands, OPC receptors and target genes was examined with the NicheNet model. We identified 108 and 166 DEGs in the demyelinated corpus callosum (CC) at 2 and 4 weeks of cuprizone treatment; 427 and 355 DEGs in the remyelinated (4 weeks of cuprizone treatment + 14 days of normal diet) compared to 2- and 4-week demyelinated CC; 252 DEGs in MS samples and 2730 and 12 DEGs in OPC and microglia of 4-week demyelinated CC. At this time point, we found 95 common DEGs in the CC and OPCs, and one common DEG in microglia and OPCs, mostly associated with myelin and lipid metabolism. Crosstalk analysis identified 47 microglia ligands, 43 OPC receptors and 115 OPC target genes, all differentially expressed in cuprizone-treated samples and associated with myelination. Our differential expression pipeline identified demyelination/remyelination transcriptomic biomarkers in studies using diverse platforms and cell types/tissues. Cellular crosstalk analysis yielded novel markers of microglia ligands, OPC receptors and target genes. Keywords: corpus callosum; cuprizone; demyelination; oligodendrocytes; microglia Author: Jennifer Enrich-Bengoa, Gemma Manich, Irene R. Dégano and Alex Perálvarez-Marín Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Nov 28 09:26:21 2022 CET ModDate: Mon Nov 28 09:43:32 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3809670 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 LWJOET+VnURWPalladioL Type 1 Custom yes yes no 15 0 JDZZCO+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 QIVWCS+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 OZTILL+URWPalladioL-Ital 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 FXXUVH+CMSY10 Type 1 Builtin yes yes no 71 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 82 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 85 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 88 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 102 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 105 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 108 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 122 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 125 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 128 0 MGFFKE+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 131 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 146 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 149 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 152 0 MGFFKE+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 155 0 QDCWHF+EURM10 Type 1 Builtin yes yes no 162 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 176 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 179 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 182 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 196 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 199 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 202 0 MGFDPI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 226 0 MGFDPK+PalatinoLinotype TrueType WinAnsi yes yes no 229 0 MGFDPM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 232 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-29 02:29:02 CEST RepresentationInformation: ijms-23-14868.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-06-23 15:00:37 CEST Size: 3809670 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 494 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: Identification of Demyelination and Remyelination Marker Genes in the Cuprizone Model Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Differentially Expressed Genes (DEGs) in the CC Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: DEGs in Microglia Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: DEGs in OPCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Common DEGs in the CC, Microglia and OPCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Identification of Common Demyelination Marker Genes in Multiple Sclerosis Samples and in Samples from the Cuprizone Mice Model Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Identification of Remyelination Marker Genes in the CC in the Cuprizone Model Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Cell Type Composition in CC Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Microglia Oligodendrocyte Interaction in Cuprizone-Induced Demyelination Remyelination: Potential Receptor Ligand Pairs to Promote Remyelination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Children: Item: Title: Data Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Identification of Demyelination/Remyelination Biomarkers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Cell Type Analysis in CC Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Microglia and OPC Crosstalk Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Info: Title: Deciphering the Genetic Crosstalk between Microglia and Oligodendrocyte Precursor Cells during Demyelination and Remyelination Using Transcriptomic Data Author: Jennifer Enrich-Bengoa, Gemma Manich, Irene R. Dégano and Alex Perálvarez-Marín Subject: Demyelinating disorders show impaired remyelination due to failure in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature myelin-forming oligodendrocytes, a process driven by microglia OPC crosstalk. Through conducting a transcriptomic analysis of microarray studies on the demyelination remyelination cuprizone model and using human samples of multiple sclerosis (MS), we identified molecules involved in this crosstalk. Differentially expressed genes (DEGs) of specific regions/cell types were detected in GEO transcriptomic raw data after cuprizone treatment and in MS samples, followed by functional analysis with GO terms and WikiPathways. Additionally, microglia OPC crosstalk between microglia ligands, OPC receptors and target genes was examined with the NicheNet model. We identified 108 and 166 DEGs in the demyelinated corpus callosum (CC) at 2 and 4 weeks of cuprizone treatment; 427 and 355 DEGs in the remyelinated (4 weeks of cuprizone treatment + 14 days of normal diet) compared to 2- and 4-week demyelinated CC; 252 DEGs in MS samples and 2730 and 12 DEGs in OPC and microglia of 4-week demyelinated CC. At this time point, we found 95 common DEGs in the CC and OPCs, and one common DEG in microglia and OPCs, mostly associated with myelin and lipid metabolism. Crosstalk analysis identified 47 microglia ligands, 43 OPC receptors and 115 OPC target genes, all differentially expressed in cuprizone-treated samples and associated with myelination. Our differential expression pipeline identified demyelination/remyelination transcriptomic biomarkers in studies using diverse platforms and cell types/tissues. Cellular crosstalk analysis yielded novel markers of microglia ligands, OPC receptors and target genes. Keywords: corpus callosum; cuprizone; demyelination; oligodendrocytes; microglia Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Nov 28 09:26:21 CET 2022 ModDate: Mon Nov 28 09:43:32 CET 2022 ID: 0x68f048d8141c34d666fbe9226a8c0723, 0x68f048d8141c34d666fbe9226a8c0723 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: QDCWHF+EURM10 FontSubset: true FirstChar: 11 LastChar: 12 FontDescriptor: FontName: QDCWHF+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: JDZZCO+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: JDZZCO+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: FXXUVH+CMSY10 FontSubset: true FirstChar: 0 LastChar: 0 FontDescriptor: FontName: FXXUVH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: QIVWCS+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: QIVWCS+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IOJCNK+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 189 FontDescriptor: FontName: IOJCNK+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OZTILL+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: OZTILL+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LWJOET+VnURWPalladioL FontSubset: true FirstChar: 177 LastChar: 226 FontDescriptor: FontName: LWJOET+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 335, 152, 344 Annotation: Subtype: Link Rect: 34, 323, 80, 332 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: 510, 642, 521, 655 Annotation: Subtype: Link Rect: 471, 107, 478, 116 ActionDest: 239 Annotation: Subtype: Link Rect: 484, 82, 491, 91 ActionDest: 239 Annotation: Subtype: Link Rect: 492, 82, 499, 91 ActionDest: 239 Annotation: Subtype: Link Rect: 143, 17, 286, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 444, 87, 463 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 493, 736, 500, 744 ActionDest: 239 Annotation: Subtype: Link Rect: 501, 735, 508, 744 ActionDest: 239 Annotation: Subtype: Link Rect: 214, 622, 221, 631 ActionDest: 242 Annotation: Subtype: Link Rect: 224, 623, 236, 631 ActionDest: 242 Annotation: Subtype: Link Rect: 253, 572, 265, 581 ActionDest: 242 Annotation: Subtype: Link Rect: 268, 572, 280, 581 ActionDest: 242 Annotation: Subtype: Link Rect: 339, 417, 346, 429 ActionDest: 68 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 334, 720, 341, 732 ActionDest: 68 Annotation: Subtype: Link Rect: 519, 670, 526, 682 ActionDest: 92 Annotation: Subtype: Link Rect: 424, 645, 431, 657 ActionDest: 112 Annotation: Subtype: Link Rect: 541, 632, 548, 644 ActionDest: 112 Annotation: Subtype: Link Rect: 474, 227, 534, 240 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 378, 209, 385, 221 ActionDest: 68 Annotation: Subtype: Link Rect: 519, 121, 526, 133 ActionDest: 68 Annotation: Subtype: Link Rect: 362, 96, 369, 108 ActionDest: 92 Annotation: Subtype: Link Rect: 253, 58, 260, 70 ActionDest: 112 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 195, 680, 202, 692 ActionDest: 136 Annotation: Subtype: Link Rect: 228, 642, 235, 654 ActionDest: 92 Annotation: Subtype: Link Rect: 422, 298, 434, 307 ActionDest: 242 Annotation: Subtype: Link Rect: 290, 195, 297, 207 ActionDest: 68 Annotation: Subtype: Link Rect: 471, 182, 478, 194 ActionDest: 112 Annotation: Subtype: Link Rect: 417, 170, 424, 182 ActionDest: 112 Annotation: Subtype: Link Rect: 200, 132, 207, 144 ActionDest: 68 Annotation: Subtype: Link Rect: 448, 82, 455, 94 ActionDest: 112 Annotation: Subtype: Link Rect: 346, 57, 353, 69 ActionDest: 112 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 203, 521, 210, 533 ActionDest: 92 Annotation: Subtype: Link Rect: 230, 521, 237, 533 ActionDest: 166 Annotation: Subtype: Link Rect: 401, 446, 408, 458 ActionDest: 92 Annotation: Subtype: Link Rect: 428, 446, 435, 458 ActionDest: 166 Annotation: Subtype: Link Rect: 528, 373, 540, 382 ActionDest: 242 Annotation: Subtype: Link Rect: 543, 373, 555, 382 ActionDest: 242 Annotation: Subtype: Link Rect: 437, 360, 449, 369 ActionDest: 242 Annotation: Subtype: Link Rect: 244, 307, 251, 319 ActionDest: 166 Annotation: Subtype: Link Rect: 286, 282, 293, 294 ActionDest: 92 Annotation: Subtype: Link Rect: 313, 282, 320, 294 ActionDest: 186 Annotation: Subtype: Link Rect: 428, 270, 435, 282 ActionDest: 186 Page: Sequence: 7 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 549, 604, 556, 616 ActionDest: 112 Annotation: Subtype: Link Rect: 212, 456, 224, 465 ActionDest: 242 Annotation: Subtype: Link Rect: 543, 444, 555, 453 ActionDest: 242 Annotation: Subtype: Link Rect: 489, 431, 501, 440 ActionDest: 242 Annotation: Subtype: Link Rect: 336, 381, 348, 390 ActionDest: 242 Annotation: Subtype: Link Rect: 349, 381, 361, 390 ActionDest: 242 Annotation: Subtype: Link Rect: 488, 343, 500, 352 ActionDest: 242 Annotation: Subtype: Link Rect: 500, 343, 512, 352 ActionDest: 242 Annotation: Subtype: Link Rect: 406, 331, 418, 340 ActionDest: 242 Annotation: Subtype: Link Rect: 466, 331, 478, 340 ActionDest: 245 Annotation: Subtype: Link Rect: 460, 293, 472, 302 ActionDest: 245 Annotation: Subtype: Link Rect: 472, 293, 484, 302 ActionDest: 245 Annotation: Subtype: Link Rect: 379, 280, 391, 290 ActionDest: 245 Annotation: Subtype: Link Rect: 420, 218, 432, 227 ActionDest: 245 Annotation: Subtype: Link Rect: 215, 130, 227, 139 ActionDest: 242 Annotation: Subtype: Link Rect: 228, 130, 240, 139 ActionDest: 242 Annotation: Subtype: Link Rect: 356, 80, 368, 89 ActionDest: 245 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 532, 685, 544, 694 ActionDest: 245 Annotation: Subtype: Link Rect: 544, 685, 556, 694 ActionDest: 245 Annotation: Subtype: Link Rect: 456, 610, 468, 619 ActionDest: 245 Annotation: Subtype: Link Rect: 468, 610, 480, 619 ActionDest: 245 Annotation: Subtype: Link Rect: 338, 597, 350, 606 ActionDest: 245 Annotation: Subtype: Link Rect: 359, 585, 371, 594 ActionDest: 245 Annotation: Subtype: Link Rect: 429, 572, 441, 581 ActionDest: 245 Annotation: Subtype: Link Rect: 442, 572, 454, 581 ActionDest: 245 Annotation: Subtype: Link Rect: 458, 560, 470, 569 ActionDest: 245 Annotation: Subtype: Link Rect: 526, 535, 538, 544 ActionDest: 245 Annotation: Subtype: Link Rect: 287, 497, 299, 506 ActionDest: 245 Annotation: Subtype: Link Rect: 232, 447, 244, 456 ActionDest: 245 Annotation: Subtype: Link Rect: 501, 447, 513, 456 ActionDest: 245 Annotation: Subtype: Link Rect: 478, 422, 490, 431 ActionDest: 245 Annotation: Subtype: Link Rect: 544, 334, 556, 343 ActionDest: 245 Annotation: Subtype: Link Rect: 478, 321, 490, 330 ActionDest: 245 Annotation: Subtype: Link Rect: 439, 283, 451, 293 ActionDest: 245 Annotation: Subtype: Link Rect: 367, 271, 379, 280 ActionDest: 245 Annotation: Subtype: Link Rect: 404, 233, 416, 242 ActionDest: 245 Annotation: Subtype: Link Rect: 544, 196, 556, 205 ActionDest: 248 Annotation: Subtype: Link Rect: 317, 145, 329, 154 ActionDest: 242 Annotation: Subtype: Link Rect: 329, 146, 341, 154 ActionDest: 242 Annotation: Subtype: Link Rect: 267, 108, 279, 117 ActionDest: 248 Annotation: Subtype: Link Rect: 279, 108, 291, 117 ActionDest: 248 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 194, 710, 206, 719 ActionDest: 248 Annotation: Subtype: Link Rect: 485, 710, 497, 719 ActionDest: 248 Annotation: Subtype: Link Rect: 271, 685, 283, 694 ActionDest: 248 Annotation: Subtype: Link Rect: 226, 647, 238, 657 ActionDest: 245 Annotation: Subtype: Link Rect: 302, 635, 314, 644 ActionDest: 248 Annotation: Subtype: Link Rect: 511, 635, 523, 644 ActionDest: 248 Annotation: Subtype: Link Rect: 196, 610, 208, 619 ActionDest: 248 Annotation: Subtype: Link Rect: 345, 597, 357, 606 ActionDest: 248 Annotation: Subtype: Link Rect: 544, 597, 556, 606 ActionDest: 248 Annotation: Subtype: Link Rect: 215, 560, 227, 569 ActionDest: 248 Annotation: Subtype: Link Rect: 522, 535, 534, 544 ActionDest: 248 Annotation: Subtype: Link Rect: 344, 283, 356, 293 ActionDest: 248 Annotation: Subtype: Link Rect: 359, 283, 371, 293 ActionDest: 248 Annotation: Subtype: Link Rect: 441, 259, 453, 267 ActionDest: 248 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 227, 710, 239, 719 ActionDest: 248 Annotation: Subtype: Link Rect: 242, 710, 254, 719 ActionDest: 248 Annotation: Subtype: Link Rect: 254, 710, 266, 719 ActionDest: 248 Annotation: Subtype: Link Rect: 353, 619, 360, 631 ActionDest: 216 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 253, 321, 265, 330 ActionDest: 248 Annotation: Subtype: Link Rect: 384, 296, 396, 305 ActionDest: 248 Annotation: Subtype: Link Rect: 341, 182, 353, 192 ActionDest: 248 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 536, 395, 560, 406 Annotation: Subtype: Link Rect: 165, 383, 381, 394 Annotation: Subtype: Link Rect: 466, 236, 560, 247 Annotation: Subtype: Link Rect: 165, 224, 222, 235 Annotation: Subtype: Link Rect: 104, 146, 141, 156 Annotation: Subtype: Link Rect: 147, 146, 183, 156 Annotation: Subtype: Link Rect: 95, 100, 133, 110 Annotation: Subtype: Link Rect: 468, 87, 506, 98 Annotation: Subtype: Link Rect: 512, 87, 548, 98 Annotation: Subtype: Link Rect: 408, 76, 446, 87 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 236, 722, 274, 733 Annotation: Subtype: Link Rect: 280, 722, 316, 733 Annotation: Subtype: Link Rect: 478, 699, 515, 710 Annotation: Subtype: Link Rect: 521, 699, 557, 710 Annotation: Subtype: Link Rect: 237, 664, 275, 675 Annotation: Subtype: Link Rect: 281, 664, 317, 675 Annotation: Subtype: Link Rect: 59, 642, 96, 652 Annotation: Subtype: Link Rect: 166, 619, 204, 629 Annotation: Subtype: Link Rect: 429, 606, 466, 617 Annotation: Subtype: Link Rect: 472, 606, 508, 617 Annotation: Subtype: Link Rect: 140, 573, 177, 583 Annotation: Subtype: Link Rect: 184, 573, 220, 583 Annotation: Subtype: Link Rect: 309, 549, 346, 560 Annotation: Subtype: Link Rect: 352, 549, 388, 560 Annotation: Subtype: Link Rect: 329, 526, 367, 537 Annotation: Subtype: Link Rect: 127, 504, 164, 514 Annotation: Subtype: Link Rect: 346, 480, 383, 491 Annotation: Subtype: Link Rect: 373, 445, 411, 456 Annotation: Subtype: Link Rect: 416, 422, 454, 433 Annotation: Subtype: Link Rect: 317, 399, 355, 410 Annotation: Subtype: Link Rect: 408, 376, 446, 387 Annotation: Subtype: Link Rect: 278, 342, 316, 353 Annotation: Subtype: Link Rect: 322, 342, 358, 353 Annotation: Subtype: Link Rect: 249, 319, 287, 330 Annotation: Subtype: Link Rect: 293, 319, 329, 330 Annotation: Subtype: Link Rect: 95, 285, 133, 295 Annotation: Subtype: Link Rect: 139, 285, 175, 295 Annotation: Subtype: Link Rect: 492, 261, 529, 272 Annotation: Subtype: Link Rect: 519, 238, 556, 249 Annotation: Subtype: Link Rect: 315, 215, 353, 226 Annotation: Subtype: Link Rect: 165, 182, 202, 192 Annotation: Subtype: Link Rect: 372, 158, 410, 169 Annotation: Subtype: Link Rect: 291, 123, 328, 134 Annotation: Subtype: Link Rect: 86, 90, 123, 100 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 520, 722, 557, 733 Annotation: Subtype: Link Rect: 59, 711, 95, 721 Annotation: Subtype: Link Rect: 433, 687, 470, 698 Annotation: Subtype: Link Rect: 477, 687, 513, 698 Annotation: Subtype: Link Rect: 319, 664, 356, 675 Annotation: Subtype: Link Rect: 362, 664, 398, 675 Annotation: Subtype: Link Rect: 59, 630, 96, 640 Annotation: Subtype: Link Rect: 368, 606, 406, 617 Annotation: Subtype: Link Rect: 59, 573, 96, 583 Annotation: Subtype: Link Rect: 292, 537, 330, 548 Annotation: Subtype: Link Rect: 373, 514, 411, 525 Annotation: Subtype: Link Rect: 262, 491, 299, 502 Annotation: Subtype: Link Rect: 294, 469, 331, 479 Annotation: Subtype: Link Rect: 104, 435, 141, 445 Annotation: Subtype: Link Rect: 147, 435, 183, 445 Annotation: Subtype: Link Rect: 284, 400, 321, 410 Annotation: Subtype: Link Rect: 217, 376, 255, 387 Annotation: Subtype: Link Rect: 261, 376, 297, 387 Annotation: Subtype: Link Rect: 95, 343, 133, 353 Annotation: Subtype: Link Rect: 139, 343, 175, 353 Annotation: Subtype: Link Rect: 59, 308, 96, 318 Annotation: Subtype: Link Rect: 102, 308, 138, 318 Annotation: Subtype: Link Rect: 393, 284, 430, 295 Annotation: Subtype: Link Rect: 59, 251, 96, 261 Annotation: Subtype: Link Rect: 131, 216, 168, 226 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 498, 158, 536, 169 Annotation: Subtype: Link Rect: 111, 124, 148, 134 Annotation: Subtype: Link Rect: 154, 124, 190, 134 Annotation: Subtype: Link Rect: 322, 100, 360, 111 Annotation: Subtype: Link Rect: 366, 100, 402, 111 Annotation: Subtype: Link Rect: 152, 67, 189, 77 Annotation: Subtype: Link Rect: 195, 67, 231, 77 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 202, 722, 240, 733 Annotation: Subtype: Link Rect: 246, 722, 282, 733 Annotation: Subtype: Link Rect: 411, 699, 448, 710 Annotation: Subtype: Link Rect: 183, 665, 220, 675 Annotation: Subtype: Link Rect: 249, 641, 286, 652 Annotation: Subtype: Link Rect: 458, 618, 495, 629 Annotation: Subtype: Link Rect: 473, 595, 511, 606 Annotation: Subtype: Link Rect: 289, 572, 326, 583 Annotation: Subtype: Link Rect: 95, 538, 133, 548 Annotation: Subtype: Link Rect: 139, 538, 175, 548 Annotation: Subtype: Link Rect: 454, 514, 491, 525 Annotation: Subtype: Link Rect: 497, 514, 533, 525 Annotation: Subtype: Link Rect: 59, 492, 96, 502 Annotation: Subtype: Link Rect: 102, 492, 138, 502 Annotation: Subtype: Link Rect: 285, 468, 322, 479 Annotation: Subtype: Link Rect: 328, 468, 364, 479 Annotation: Subtype: Link Rect: 313, 435, 351, 445 Annotation: Subtype: Link Rect: 383, 411, 420, 422 Annotation: Subtype: Link Rect: 255, 376, 292, 387 Annotation: Subtype: Link Rect: 389, 353, 426, 364 Annotation: Subtype: Link Rect: 261, 319, 299, 330 Annotation: Subtype: Link Rect: 59, 285, 96, 295 Annotation: Subtype: Link Rect: 182, 262, 219, 272 Annotation: Subtype: Link Rect: 226, 262, 262, 272 Annotation: Subtype: Link Rect: 394, 238, 432, 249 Annotation: Subtype: Link Rect: 438, 238, 474, 249 Annotation: Subtype: Link Rect: 432, 181, 470, 192 Annotation: Subtype: Link Rect: 165, 159, 202, 169 Annotation: Subtype: Link Rect: 208, 159, 244, 169 Checksum: 9c23d9b5 Type: CRC32 Checksum: c3c19d1d9b6c3aecb5b2943e352ceb87 Type: MD5 Checksum: 2d22d670f9eb9a963cd1a6a9f2afaafcd94ecb28 Type: SHA-1