be4ad64401113d56e3b0e33111a9950a antioxidants-a2020v9n12p1202.pdf df8eb5efb90b0d166a747c9415934f94cd7bdaee antioxidants-a2020v9n12p1202.pdf 19ecf67ef00d51979fff82dd53eecb008edbe4156f2edcca5fca9b721af4288e antioxidants-a2020v9n12p1202.pdf Title: Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation Subject: Death-associated protein kinase 1 (DAPK1) is a pleiotropic hub of a number of networked distributed intracellular processes. Among them, DAPK1 is known to interact with the excitotoxicity driver NMDA receptor (NMDAR), and in sudden pathophysiological conditions of the brain, e.g., stroke, several lines of evidence link DAPK1 with the transduction of glutamate-induced events that determine neuronal fate. In turn, DAPK1 expression and activity are known to be affected by the redox status of the cell. To delineate specific and differential neuronal DAPK1 interactors in stroke-like conditions in vitro, we exposed primary cultures of rat cortical neurons to oxygen/glucose deprivation (OGD), a condition that increases reactive oxygen species (ROS) and lipid peroxides. OGD or control samples were co-immunoprecipitated separately, trypsin-digested, and proteins in the interactome identified by high-resolution LC-MS/MS. Data were processed and curated using bioinformatics tools. OGD increased total DAPK1 protein levels, cleavage into shorter isoforms, and dephosphorylation to render the active DAPK1 form. The DAPK1 interactome comprises some 600 proteins, mostly involving binding, catalytic and structural molecular functions. OGD up-regulated 190 and down-regulated 192 candidate DAPK1-interacting proteins. Some differentially up-regulated interactors related to NMDAR were validated by WB. In addition, a novel differential DAPK1 partner, LRRFIP1, was further confirmed by reverse Co-IP. Furthermore, LRRFIP1 levels were increased by pro-oxidant conditions such as ODG or the ferroptosis inducer erastin. The present study identifies novel partners of DAPK1, such as LRRFIP1, which are suitable as targets for neuroprotection. Keywords: LRRFIP1; DAPK1; neuron; OGD; MCAO; NMDA; ROS; ferroptosis Author: Núria DeGregorio-Rocasolano, Verónica Guirao, Jovita Ponce, Marc Melià-Sorolla, Alicia Aliena-Valero, Alexia García-Serran, Juan B. Salom, Antoni Dávalos, Octavi Martí-Sistac and Teresa Gasull Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Mon Nov 30 07:45:35 2020 CET ModDate: Tue Dec 1 10:06:18 2020 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4753784 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LNZVRW+Pxsy Type 1 Builtin yes yes no 10 0 MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 14 0 JHHTBC+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 SDEZFQ+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 BPHJLT+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 OFLXAT+Rpxr Type 1 Builtin yes yes no 32 0 CCDETI+VnURWPalladioL-Bold Type 1 Custom yes yes no 36 0 QTLELQ+Pxbsy Type 1 Builtin yes yes no 40 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 48 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 70 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 74 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 78 0 THGEKM+Rpxsc Type 1 Builtin yes yes no 85 0 YICMPF+Rpcxr Type 1 Builtin yes yes no 92 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 96 0 JNLIIF+Rpxi Type 1 Builtin yes yes no 103 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 120 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 124 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 128 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 132 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 149 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 153 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 157 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 170 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 174 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 178 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 182 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 196 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 200 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 204 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 208 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 224 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 228 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 232 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 243 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 247 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 251 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 263 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 267 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 271 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 275 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 291 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 295 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 299 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 04:23:50 CEST RepresentationInformation: antioxidants-a2020v9n12p1202.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:39:58 CEST Size: 4753784 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 559 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Primary Culture of Cortical Neurons Destination: subsection.2.1 Item: Title: Oxygen and Glucose Deprivation (OGD) Destination: subsection.2.2 Item: Title: Treatments Destination: subsection.2.3 Item: Title: Assessment of Neuronal Viability Destination: subsection.2.4 Item: Title: Co-Immunoprecipitation (Co-IP) Procedure to Identify Protein Interactors by LC-MS/MS Destination: subsection.2.5 Item: Title: Co-Immunoprecipitation Assays Destination: subsection.2.6 Item: Title: Western Blot Destination: subsection.2.7 Item: Title: Immunocytochemistry Destination: subsection.2.8 Item: Title: Transient Right Focal Cerebral Ischemia Destination: subsection.2.9 Item: Title: Statistical Analysis Destination: subsection.2.10 Item: Title: Antibodies Destination: subsection.2.11 Item: Title: Results Destination: section.3 Children: Item: Title: Effects of OGD on Neuronal Death, and on DAPK1 Expression, Dephosphorylation, and Cleavage Destination: subsection.3.1 Item: Title: Main Effects of OGD on the Neuronal DAPK1 Interactome Destination: subsection.3.2 Item: Title: Several DAPK1 Interactors Are CTD-NR2B Binding Partners Destination: subsection.3.3 Item: Title: The New Protein Candidate LRRFIP1 Is Part of the DAPK1 Protein Complex during Ischemia In Vivo and Excitotoxicity In Vitro Destination: subsection.3.4 Item: Title: Colocalization of NR2B and DAPK1 with LRRFIP1 Destination: subsection.3.5 Item: Title: The Ferroptosis Inducer Erastin Increases Neuronal LRRFIP1 Levels Preceding Loss of Neuronal Viability Destination: subsection.3.6 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation Author: Núria DeGregorio-Rocasolano, Verónica Guirao, Jovita Ponce, Marc Melià-Sorolla, Alicia Aliena-Valero, Alexia García-Serran, Juan B. Salom, Antoni Dávalos, Octavi Martí-Sistac and Teresa Gasull Subject: Death-associated protein kinase 1 (DAPK1) is a pleiotropic hub of a number of networked distributed intracellular processes. Among them, DAPK1 is known to interact with the excitotoxicity driver NMDA receptor (NMDAR), and in sudden pathophysiological conditions of the brain, e.g., stroke, several lines of evidence link DAPK1 with the transduction of glutamate-induced events that determine neuronal fate. In turn, DAPK1 expression and activity are known to be affected by the redox status of the cell. To delineate specific and differential neuronal DAPK1 interactors in stroke-like conditions in vitro, we exposed primary cultures of rat cortical neurons to oxygen/glucose deprivation (OGD), a condition that increases reactive oxygen species (ROS) and lipid peroxides. OGD or control samples were co-immunoprecipitated separately, trypsin-digested, and proteins in the interactome identified by high-resolution LC-MS/MS. Data were processed and curated using bioinformatics tools. OGD increased total DAPK1 protein levels, cleavage into shorter isoforms, and dephosphorylation to render the active DAPK1 form. The DAPK1 interactome comprises some 600 proteins, mostly involving binding, catalytic and structural molecular functions. OGD up-regulated 190 and down-regulated 192 candidate DAPK1-interacting proteins. Some differentially up-regulated interactors related to NMDAR were validated by WB. In addition, a novel differential DAPK1 partner, LRRFIP1, was further confirmed by reverse Co-IP. Furthermore, LRRFIP1 levels were increased by pro-oxidant conditions such as ODG or the ferroptosis inducer erastin. The present study identifies novel partners of DAPK1, such as LRRFIP1, which are suitable as targets for neuroprotection. Keywords: LRRFIP1; DAPK1; neuron; OGD; MCAO; NMDA; ROS; ferroptosis Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Mon Nov 30 07:45:35 CET 2020 ModDate: Tue Dec 01 10:06:18 CET 2020 ID: 0x0be937d26eb84d240f849f1f6cc11b8d, 0x0be937d26eb84d240f849f1f6cc11b8d Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OFLXAT+Rpxr FontSubset: true FirstChar: 11 LastChar: 62 FontDescriptor: FontName: OFLXAT+Rpxr Flags: Symbolic FontBBox: -40, -283, 904, 728 FontFile: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: JHHTBC+URWPalladioL-Ital FontSubset: true FirstChar: 40 LastChar: 122 FontDescriptor: FontName: JHHTBC+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CCDETI+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: CCDETI+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: THGEKM+Rpxsc FontSubset: true FirstChar: 100 LastChar: 108 FontDescriptor: FontName: THGEKM+Rpxsc Flags: Symbolic FontBBox: -37, -172, 852, 718 FontFile: true Font: BaseFont: JNLIIF+Rpxi FontSubset: true FirstChar: 11 LastChar: 47 FontDescriptor: FontName: JNLIIF+Rpxi Flags: Symbolic FontBBox: -162, -280, 856, 733 FontFile: true Font: BaseFont: QTLELQ+Pxbsy FontSubset: true FirstChar: 121 LastChar: 121 FontDescriptor: FontName: QTLELQ+Pxbsy Flags: Symbolic FontBBox: 5, -978, 1382, 913 FontFile: true Font: BaseFont: SDEZFQ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 121 FontDescriptor: FontName: SDEZFQ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LNZVRW+Pxsy FontSubset: true FirstChar: 0 LastChar: 121 FontDescriptor: FontName: LNZVRW+Pxsy Flags: Symbolic FontBBox: -21, -994, 1292, 912 FontFile: true Font: BaseFont: BPHJLT+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: BPHJLT+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YICMPF+Rpcxr FontSubset: true FirstChar: 151 LastChar: 174 FontDescriptor: FontName: YICMPF+Rpcxr Flags: Symbolic FontBBox: -212, -286, 1344, 973 FontFile: true Font: BaseFont: MWHIBA+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: MWHIBA+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 75, 757, 216, 793 Annotation: Subtype: Link Rect: 474, 757, 519, 793 Annotation: Subtype: Link Rect: 227, 640, 238, 654 Annotation: Subtype: Link Rect: 497, 640, 508, 654 Annotation: Subtype: Link Rect: 281, 627, 292, 640 Annotation: Subtype: Link Rect: 372, 627, 383, 640 Annotation: Subtype: Link Rect: 294, 615, 305, 626 Annotation: Subtype: Link Rect: 181, 38, 261, 50 Annotation: Subtype: Link Rect: 387, 38, 519, 50 Annotation: Subtype: Link Rect: 466, 401, 518, 420 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 508, 642, 515, 651 ActionDest: 310 Annotation: Subtype: Link Rect: 318, 615, 325, 624 ActionDest: 313 Annotation: Subtype: Link Rect: 445, 588, 452, 597 ActionDest: 313 Annotation: Subtype: Link Rect: 148, 412, 155, 421 ActionDest: 313 Annotation: Subtype: Link Rect: 381, 399, 388, 408 ActionDest: 313 Annotation: Subtype: Link Rect: 388, 399, 395, 408 ActionDest: 313 Annotation: Subtype: Link Rect: 220, 318, 227, 327 ActionDest: 313 Annotation: Subtype: Link Rect: 227, 318, 234, 327 ActionDest: 313 Annotation: Subtype: Link Rect: 236, 291, 243, 300 ActionDest: 313 Annotation: Subtype: Link Rect: 423, 264, 430, 273 ActionDest: 313 Annotation: Subtype: Link Rect: 400, 237, 412, 246 ActionDest: 313 Annotation: Subtype: Link Rect: 315, 209, 322, 219 ActionDest: 313 Annotation: Subtype: Link Rect: 141, 169, 153, 178 ActionDest: 313 Annotation: Subtype: Link Rect: 302, 155, 309, 165 ActionDest: 313 Annotation: Subtype: Link Rect: 310, 156, 322, 165 ActionDest: 313 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 446, 447, 458, 456 ActionDest: 313 Annotation: Subtype: Link Rect: 459, 447, 471, 456 ActionDest: 313 Annotation: Subtype: Link Rect: 288, 103, 300, 112 ActionDest: 313 Annotation: Subtype: Link Rect: 395, 103, 407, 112 ActionDest: 313 Annotation: Subtype: Link Rect: 408, 103, 420, 112 ActionDest: 313 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 484, 511, 496, 520 ActionDest: 313 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 171, 128, 183, 137 ActionDest: 313 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 299, 224, 306, 236 ActionDest: 113 Annotation: Subtype: Link Rect: 386, 197, 393, 209 ActionDest: 113 Annotation: Subtype: Link Rect: 340, 183, 347, 195 ActionDest: 113 Annotation: Subtype: Link Rect: 429, 183, 436, 195 ActionDest: 113 Annotation: Subtype: Link Rect: 327, 170, 334, 182 ActionDest: 113 Annotation: Subtype: Link Rect: 442, 156, 449, 168 ActionDest: 113 Annotation: Subtype: Link Rect: 368, 129, 375, 141 ActionDest: 113 Annotation: Subtype: Link Rect: 308, 118, 320, 127 ActionDest: 313 Page: Sequence: 7 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 230, 707, 237, 719 ActionDest: 113 Annotation: Subtype: Link Rect: 308, 669, 315, 678 ActionDest: 313 Annotation: Subtype: Link Rect: 279, 653, 287, 665 ActionDest: 143 Annotation: Subtype: Link Rect: 411, 639, 418, 651 ActionDest: 143 Annotation: Subtype: Link Rect: 446, 626, 453, 638 ActionDest: 143 Annotation: Subtype: Link Rect: 193, 491, 200, 502 ActionDest: 143 Annotation: Subtype: Link Rect: 375, 410, 495, 421 Annotation: Subtype: Link Rect: 503, 412, 515, 421 ActionDest: 313 Annotation: Subtype: Link Rect: 251, 396, 258, 408 ActionDest: 162 Annotation: Subtype: Link Rect: 347, 383, 354, 394 ActionDest: 162 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 123, 149, 130, 160 ActionDest: 113 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 492, 72, 499, 84 ActionDest: 190 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 500, 726, 507, 738 ActionDest: 190 Annotation: Subtype: Link Rect: 190, 702, 202, 711 ActionDest: 313 Annotation: Subtype: Link Rect: 491, 672, 498, 684 ActionDest: 190 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 119, 658, 131, 667 ActionDest: 313 Annotation: Subtype: Link Rect: 211, 641, 218, 653 ActionDest: 217 Annotation: Subtype: Link Rect: 291, 631, 303, 640 ActionDest: 313 Annotation: Subtype: Link Rect: 361, 614, 368, 626 ActionDest: 217 Annotation: Subtype: Link Rect: 233, 225, 240, 236 ActionDest: 237 Annotation: Subtype: Link Rect: 210, 211, 217, 223 ActionDest: 237 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 230, 202, 237, 214 ActionDest: 256 Annotation: Subtype: Link Rect: 421, 175, 428, 187 ActionDest: 256 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 490, 109, 502, 118 ActionDest: 316 Annotation: Subtype: Link Rect: 503, 109, 515, 118 ActionDest: 316 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 500, 699, 507, 711 ActionDest: 283 Annotation: Subtype: Link Rect: 202, 672, 209, 684 ActionDest: 283 Annotation: Subtype: Link Rect: 444, 645, 451, 657 ActionDest: 283 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 142, 742, 153, 752 ActionDest: 316 Annotation: Subtype: Link Rect: 154, 743, 166, 752 ActionDest: 316 Annotation: Subtype: Link Rect: 263, 743, 270, 752 ActionDest: 313 Annotation: Subtype: Link Rect: 385, 742, 392, 752 ActionDest: 313 Annotation: Subtype: Link Rect: 140, 699, 147, 711 ActionDest: 113 Annotation: Subtype: Link Rect: 474, 699, 481, 711 ActionDest: 113 Annotation: Subtype: Link Rect: 495, 702, 502, 711 ActionDest: 313 Annotation: Subtype: Link Rect: 503, 702, 515, 711 ActionDest: 313 Annotation: Subtype: Link Rect: 431, 688, 443, 698 ActionDest: 313 Annotation: Subtype: Link Rect: 443, 688, 455, 698 ActionDest: 316 Annotation: Subtype: Link Rect: 409, 672, 416, 684 ActionDest: 113 Annotation: Subtype: Link Rect: 269, 648, 281, 657 ActionDest: 316 Annotation: Subtype: Link Rect: 116, 553, 128, 562 ActionDest: 316 Annotation: Subtype: Link Rect: 166, 540, 173, 549 ActionDest: 313 Annotation: Subtype: Link Rect: 146, 497, 153, 508 ActionDest: 143 Annotation: Subtype: Link Rect: 268, 499, 280, 508 ActionDest: 313 Annotation: Subtype: Link Rect: 355, 456, 362, 468 ActionDest: 143 Annotation: Subtype: Link Rect: 328, 432, 340, 441 ActionDest: 316 Annotation: Subtype: Link Rect: 472, 388, 479, 400 ActionDest: 162 Annotation: Subtype: Link Rect: 140, 215, 152, 225 ActionDest: 316 Annotation: Subtype: Link Rect: 155, 215, 167, 225 ActionDest: 316 Annotation: Subtype: Link Rect: 488, 215, 500, 225 ActionDest: 316 Annotation: Subtype: Link Rect: 503, 215, 515, 225 ActionDest: 316 Annotation: Subtype: Link Rect: 477, 161, 484, 171 ActionDest: 313 Annotation: Subtype: Link Rect: 482, 145, 489, 157 ActionDest: 143 Annotation: Subtype: Link Rect: 224, 132, 231, 144 ActionDest: 190 Annotation: Subtype: Link Rect: 456, 121, 463, 130 ActionDest: 313 Annotation: Subtype: Link Rect: 229, 107, 241, 117 ActionDest: 316 Annotation: Subtype: Link Rect: 335, 107, 347, 117 ActionDest: 316 Annotation: Subtype: Link Rect: 105, 91, 112, 103 ActionDest: 190 Annotation: Subtype: Link Rect: 135, 94, 147, 103 ActionDest: 316 Annotation: Subtype: Link Rect: 492, 91, 499, 103 ActionDest: 190 Annotation: Subtype: Link Rect: 503, 80, 515, 90 ActionDest: 316 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 402, 726, 409, 738 ActionDest: 190 Annotation: Subtype: Link Rect: 155, 715, 167, 725 ActionDest: 313 Annotation: Subtype: Link Rect: 168, 715, 180, 725 ActionDest: 316 Annotation: Subtype: Link Rect: 180, 715, 192, 725 ActionDest: 316 Annotation: Subtype: Link Rect: 486, 688, 498, 698 ActionDest: 316 Annotation: Subtype: Link Rect: 501, 688, 513, 698 ActionDest: 319 Annotation: Subtype: Link Rect: 501, 621, 513, 630 ActionDest: 319 Annotation: Subtype: Link Rect: 385, 553, 397, 562 ActionDest: 319 Annotation: Subtype: Link Rect: 398, 553, 410, 562 ActionDest: 319 Annotation: Subtype: Link Rect: 455, 526, 467, 535 ActionDest: 313 Annotation: Subtype: Link Rect: 468, 526, 480, 535 ActionDest: 319 Annotation: Subtype: Link Rect: 491, 513, 503, 522 ActionDest: 313 Annotation: Subtype: Link Rect: 503, 513, 515, 522 ActionDest: 319 Annotation: Subtype: Link Rect: 380, 445, 392, 454 ActionDest: 319 Annotation: Subtype: Link Rect: 177, 416, 183, 427 ActionDest: 217 Annotation: Subtype: Link Rect: 288, 402, 295, 414 ActionDest: 217 Annotation: Subtype: Link Rect: 453, 361, 460, 373 ActionDest: 237 Annotation: Subtype: Link Rect: 271, 310, 283, 319 ActionDest: 319 Annotation: Subtype: Link Rect: 283, 310, 295, 319 ActionDest: 319 Annotation: Subtype: Link Rect: 147, 253, 154, 265 ActionDest: 256 Annotation: Subtype: Link Rect: 196, 242, 208, 252 ActionDest: 316 Annotation: Subtype: Link Rect: 209, 242, 221, 252 ActionDest: 319 Annotation: Subtype: Link Rect: 221, 242, 233, 252 ActionDest: 319 Annotation: Subtype: Link Rect: 179, 199, 186, 211 ActionDest: 256 Annotation: Subtype: Link Rect: 127, 118, 134, 130 ActionDest: 283 Annotation: Subtype: Link Rect: 303, 118, 310, 130 ActionDest: 283 Annotation: Subtype: Link Rect: 333, 105, 340, 117 ActionDest: 283 Annotation: Subtype: Link Rect: 439, 94, 451, 103 ActionDest: 319 Annotation: Subtype: Link Rect: 452, 94, 463, 103 ActionDest: 319 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 317, 713, 324, 725 ActionDest: 283 Annotation: Subtype: Link Rect: 503, 688, 515, 698 ActionDest: 319 Annotation: Subtype: Link Rect: 220, 618, 227, 630 ActionDest: 283 Annotation: Subtype: Link Rect: 148, 553, 160, 562 ActionDest: 319 Annotation: Subtype: Link Rect: 231, 540, 243, 549 ActionDest: 313 Annotation: Subtype: Link Rect: 333, 351, 518, 362 Annotation: Subtype: Link Rect: 480, 82, 516, 93 Annotation: Subtype: Link Rect: 100, 70, 136, 80 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 415, 714, 453, 725 Annotation: Subtype: Link Rect: 459, 714, 495, 725 Annotation: Subtype: Link Rect: 393, 688, 430, 699 Annotation: Subtype: Link Rect: 436, 688, 472, 699 Annotation: Subtype: Link Rect: 326, 650, 363, 661 Annotation: Subtype: Link Rect: 452, 624, 489, 635 Annotation: Subtype: Link Rect: 427, 598, 464, 609 Annotation: Subtype: Link Rect: 177, 560, 214, 570 Annotation: Subtype: Link Rect: 193, 521, 231, 531 Annotation: Subtype: Link Rect: 136, 483, 173, 493 Annotation: Subtype: Link Rect: 322, 443, 360, 454 Annotation: Subtype: Link Rect: 100, 405, 137, 415 Annotation: Subtype: Link Rect: 143, 405, 179, 415 Annotation: Subtype: Link Rect: 385, 378, 423, 389 Annotation: Subtype: Link Rect: 429, 378, 465, 389 Annotation: Subtype: Link Rect: 294, 340, 332, 351 Annotation: Subtype: Link Rect: 338, 340, 374, 351 Annotation: Subtype: Link Rect: 339, 288, 376, 299 Annotation: Subtype: Link Rect: 100, 250, 137, 260 Annotation: Subtype: Link Rect: 258, 224, 295, 234 Annotation: Subtype: Link Rect: 100, 173, 137, 183 Annotation: Subtype: Link Rect: 100, 134, 137, 144 Annotation: Subtype: Link Rect: 436, 107, 473, 118 Annotation: Subtype: Link Rect: 478, 69, 515, 80 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 478, 727, 516, 738 Annotation: Subtype: Link Rect: 100, 715, 136, 725 Annotation: Subtype: Link Rect: 221, 676, 258, 686 Annotation: Subtype: Link Rect: 265, 676, 301, 686 Annotation: Subtype: Link Rect: 438, 663, 475, 673 Annotation: Subtype: Link Rect: 481, 663, 516, 673 Annotation: Subtype: Link Rect: 286, 624, 323, 635 Annotation: Subtype: Link Rect: 100, 586, 137, 596 Annotation: Subtype: Link Rect: 343, 559, 380, 570 Annotation: Subtype: Link Rect: 444, 533, 481, 544 Annotation: Subtype: Link Rect: 100, 496, 137, 506 Annotation: Subtype: Link Rect: 141, 457, 178, 467 Annotation: Subtype: Link Rect: 271, 417, 309, 428 Annotation: Subtype: Link Rect: 468, 378, 506, 389 Annotation: Subtype: Link Rect: 160, 341, 198, 351 Annotation: Subtype: Link Rect: 100, 302, 137, 312 Annotation: Subtype: Link Rect: 243, 263, 280, 273 Annotation: Subtype: Link Rect: 292, 224, 329, 234 Annotation: Subtype: Link Rect: 405, 198, 443, 209 Annotation: Subtype: Link Rect: 100, 160, 137, 170 Annotation: Subtype: Link Rect: 340, 120, 378, 131 Annotation: Subtype: Link Rect: 100, 82, 137, 92 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 236, 728, 273, 738 Annotation: Subtype: Link Rect: 245, 701, 283, 712 Annotation: Subtype: Link Rect: 289, 701, 325, 712 Annotation: Subtype: Link Rect: 100, 663, 137, 673 Annotation: Subtype: Link Rect: 352, 624, 390, 635 Annotation: Subtype: Link Rect: 424, 598, 462, 609 Annotation: Subtype: Link Rect: 462, 559, 500, 570 Annotation: Subtype: Link Rect: 195, 521, 232, 531 Annotation: Subtype: Link Rect: 260, 457, 297, 467 Annotation: Subtype: Link Rect: 236, 418, 273, 428 Annotation: Subtype: Link Rect: 159, 379, 196, 389 Annotation: Subtype: Link Rect: 163, 341, 200, 351 Annotation: Subtype: Link Rect: 276, 301, 313, 312 Annotation: Subtype: Link Rect: 320, 301, 356, 312 Annotation: Subtype: Link Rect: 255, 262, 292, 273 Annotation: Subtype: Link Rect: 299, 262, 335, 273 Annotation: Subtype: Link Rect: 150, 237, 187, 247 Annotation: Subtype: Link Rect: 76, 165, 166, 196 Annotation: Subtype: Link Rect: 234, 159, 407, 170 Checksum: 6adf6f51 Type: CRC32 Checksum: be4ad64401113d56e3b0e33111a9950a Type: MD5 Checksum: df8eb5efb90b0d166a747c9415934f94cd7bdaee Type: SHA-1 Checksum: 19ecf67ef00d51979fff82dd53eecb008edbe4156f2edcca5fca9b721af4288e Type: SHA-256