5e5eec3f375c196d26dcd162e0834f3a pmc_11546810.pdf 15e02acd5c86d28f89898a5cc57c264abe0e9dc9 pmc_11546810.pdf 68a3e0beea884c88bb29d7e1d08da72ca739c0928f58e2298fa0cf17a616ca41 pmc_11546810.pdf Title: Targeting Retinaldehyde Dehydrogenases to Enhance Temozolomide Therapy in Glioblastoma Subject: Glioblastoma (GB) is an aggressive malignant central nervous system tumor that is currently incurable. One of the main pitfalls of GB treatment is resistance to the chemotherapeutic standard of care, temozolomide (TMZ). The role of aldehyde dehydrogenases (ALDHs) in the glioma stem cell (GSC) subpopulation has been related to chemoresistance. ALDHs take part in processes such as cell proliferation, differentiation, invasiveness or metastasis and have been studied as pharmacological targets in cancer treatment. In the present work, three novel ,-acetylenic amino thiolester compounds, with demonstrated efficacy as ALDH inhibitors, were tested in vitro on a panel of six human GB cell lines and one murine GB cell line. Firstly, the expression of the ALDH1A isoforms was assessed, and then inhibitors were tested for their cytotoxicity and their ability to inhibit cellular ALDH activity. Drug combination assays with TMZ were performed, as well as an assessment of the cell death mechanism and generation of ROS. A knockout of several ALDH genes was carried out in one of the human GB cell lines, allowing us to discuss their role in cell proliferation, migration capacity and resistance to treatment. Our results strongly suggest that ALDH inhibitors could be an interesting approach in the treatment of GB, with EC50 values in the order of micromolar, decreasing ALDH activity in GB cell lines to 40–50%. Keywords: aldehyde dehydrogenase; cancer; enzyme inhibition; glioblastoma; retinoic acid Author: Rafael Jiménez, Andrada Constantinescu, Muhube Yazir, Paula Alfonso-Triguero, Raquel Pequerul, Xavier Parés, Mileidys Pérez-Alea, Ana Paula Candiota, Jaume Farrés and Julia Lorenzo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Sat Oct 26 11:18:32 2024 CEST ModDate: Sat Oct 26 11:23:51 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 26 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5778949 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 16 0 AUICZB+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 RMJPQV+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 QLLWVH+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 SXQPWE+VnURWPalladioL Type 1 Custom yes yes yes 37 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 53 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 56 0 RGUFCP+CMSY10 Type 1 Builtin yes yes yes 80 0 JYUFDF+URWPalladioL-BoldItal Type 1 Custom yes yes yes 85 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 94 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 98 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 102 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 106 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 112 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 118 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 133 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 137 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 141 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 145 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 151 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 157 0 BCDKEE+PalatinoLinotype-BoldItalic CID TrueType Identity-H yes yes yes 163 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 180 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 184 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 188 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 192 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 198 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 204 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 219 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 223 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 227 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 231 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 237 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 243 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 255 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 259 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 263 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 267 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 273 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 279 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 295 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 299 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 303 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 307 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 313 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 319 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 334 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 338 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 342 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 346 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 352 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 358 0 XIRVNA+EURB10 Type 1 Builtin yes yes yes 368 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 381 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 385 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 389 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 393 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 399 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 405 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 421 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 425 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 429 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 433 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 439 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 445 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 460 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 464 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 468 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 477 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 481 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 485 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 489 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 495 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 510 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 514 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 518 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 522 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 528 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 534 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 555 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 559 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 563 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 567 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 573 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 579 0 UZKIAN+CMEX10 Type 1 Builtin yes yes yes 589 0 RFLZJB+CMR10 Type 1 Builtin yes yes yes 594 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-01-30 02:45:04 CET RepresentationInformation: pmc_11546810.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-12-04 02:21:24 CET Size: 5778949 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 955 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: ALDH1A Isoforms Are Expressed Differently in a Panel of GB Cell Lines Destination: subsection.2.1 Item: Title: ALDH Activity in A172 and GL261 Cells Destination: subsection.2.2 Item: Title: ALDH Knockout Impairs Growth Rate and Migration Capacity in A172 Cells and Enhances Sensitivity to the Standard Chemotherapeutic Agent TMZ Destination: subsection.2.3 Item: Title: ALDH Inhibitors Display Potent Cytotoxicity in the Low Micromolar Range in a Panel of GB Cell Lines, Surpassing the Efficacy of the Conventional Standard of Care, TMZ Destination: subsection.2.4 Item: Title: ALDH Inhibitors Reduce the Enzymatic ALDH Activity in A172 and GL261 Cell Extracts Destination: subsection.2.5 Item: Title: Combinations of TMZ with ALDH Inhibitors Act Synergistically to Cause Toxicity in GB Cell Lines A172 and GL261 Destination: subsection.2.6 Item: Title: TMZ and ALDH Inhibitors Trigger Apoptosis and Induce ROS Generation Destination: subsection.2.7 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Lines and Cell Culture Destination: subsection.4.1 Item: Title: Compounds (ALDH Inhibitors) Destination: subsection.4.2 Item: Title: Western Blot Analyses Destination: subsection.4.3 Item: Title: Cell Viability Assays Destination: subsection.4.4 Item: Title: ALDH Activity Assays Destination: subsection.4.5 Item: Title: Cell Death Analyses Destination: subsection.4.6 Item: Title: ROS Production Analyses Destination: subsection.4.7 Item: Title: CRISPR/Cas9 Knockout Destination: subsection.4.8 Item: Title: RT/PCR Analyses Destination: subsection.4.9 Item: Title: Cell Growth Curve Destination: subsection.4.10 Item: Title: Migration Assays Destination: subsection.4.11 Item: Title: Conclusions Destination: section.5 Item: Title: Patents Destination: section.6 Item: Title: References Destination: appendix.A. Info: Title: Targeting Retinaldehyde Dehydrogenases to Enhance Temozolomide Therapy in Glioblastoma Author: Rafael Jiménez, Andrada Constantinescu, Muhube Yazir, Paula Alfonso-Triguero, Raquel Pequerul, Xavier Parés, Mileidys Pérez-Alea, Ana Paula Candiota, Jaume Farrés and Julia Lorenzo Subject: Glioblastoma (GB) is an aggressive malignant central nervous system tumor that is currently incurable. One of the main pitfalls of GB treatment is resistance to the chemotherapeutic standard of care, temozolomide (TMZ). The role of aldehyde dehydrogenases (ALDHs) in the glioma stem cell (GSC) subpopulation has been related to chemoresistance. ALDHs take part in processes such as cell proliferation, differentiation, invasiveness or metastasis and have been studied as pharmacological targets in cancer treatment. In the present work, three novel ,-acetylenic amino thiolester compounds, with demonstrated efficacy as ALDH inhibitors, were tested in vitro on a panel of six human GB cell lines and one murine GB cell line. Firstly, the expression of the ALDH1A isoforms was assessed, and then inhibitors were tested for their cytotoxicity and their ability to inhibit cellular ALDH activity. Drug combination assays with TMZ were performed, as well as an assessment of the cell death mechanism and generation of ROS. A knockout of several ALDH genes was carried out in one of the human GB cell lines, allowing us to discuss their role in cell proliferation, migration capacity and resistance to treatment. Our results strongly suggest that ALDH inhibitors could be an interesting approach in the treatment of GB, with EC50 values in the order of micromolar, decreasing ALDH activity in GB cell lines to 40 50%. Keywords: aldehyde dehydrogenase; cancer; enzyme inhibition; glioblastoma; retinoic acid Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Sat Oct 26 11:18:32 CEST 2024 ModDate: Sat Oct 26 11:23:51 CEST 2024 ID: 0xa26490987cb0c454310f97fabf862829, 0xa26490987cb0c454310f97fabf862829 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: QLLWVH+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: QLLWVH+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: RGUFCP+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: RGUFCP+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true ToUnicode: true Font: BaseFont: RFLZJB+CMR10 FontSubset: true FirstChar: 43 LastChar: 61 FontDescriptor: FontName: RFLZJB+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true ToUnicode: true Font: BaseFont: SXQPWE+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: SXQPWE+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AUICZB+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: AUICZB+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: JYUFDF+URWPalladioL-BoldItal FontSubset: true FirstChar: 84 LastChar: 115 FontDescriptor: FontName: JYUFDF+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: BTFGED+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 178 FontDescriptor: FontName: BTFGED+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: RMJPQV+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: RMJPQV+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: UZKIAN+CMEX10 FontSubset: true FirstChar: 16 LastChar: 17 FontDescriptor: FontName: UZKIAN+CMEX10 Flags: Symbolic FontBBox: -24, -2960, 1454, 772 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 57, 303, 152, 312 Annotation: Subtype: Link Rect: 34, 291, 80, 300 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 149, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 793 Annotation: Subtype: Link Rect: 515, 756, 560, 793 Annotation: Subtype: Link Rect: 97, 649, 108, 662 Annotation: Subtype: Link Rect: 210, 649, 221, 662 Annotation: Subtype: Link Rect: 335, 649, 346, 662 Annotation: Subtype: Link Rect: 422, 649, 433, 662 Annotation: Subtype: Link Rect: 541, 649, 552, 662 Annotation: Subtype: Link Rect: 437, 193, 444, 202 ActionDest: 609 Annotation: Subtype: Link Rect: 272, 155, 279, 164 ActionDest: 609 Annotation: Subtype: Link Rect: 484, 155, 491, 164 ActionDest: 609 Annotation: Subtype: Link Rect: 236, 130, 243, 139 ActionDest: 609 Annotation: Subtype: Link Rect: 521, 130, 528, 139 ActionDest: 609 Annotation: Subtype: Link Rect: 440, 105, 447, 114 ActionDest: 609 Annotation: Subtype: Link Rect: 318, 93, 325, 102 ActionDest: 609 Annotation: Subtype: Link Rect: 328, 92, 335, 102 ActionDest: 612 Annotation: Subtype: Link Rect: 143, 17, 286, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 412, 87, 431 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 282, 731, 294, 740 ActionDest: 612 Annotation: Subtype: Link Rect: 295, 731, 307, 740 ActionDest: 612 Annotation: Subtype: Link Rect: 357, 718, 369, 727 ActionDest: 612 Annotation: Subtype: Link Rect: 494, 693, 506, 702 ActionDest: 612 Annotation: Subtype: Link Rect: 266, 643, 278, 652 ActionDest: 612 Annotation: Subtype: Link Rect: 278, 643, 290, 652 ActionDest: 612 Annotation: Subtype: Link Rect: 488, 618, 500, 627 ActionDest: 612 Annotation: Subtype: Link Rect: 431, 543, 443, 552 ActionDest: 612 Annotation: Subtype: Link Rect: 446, 542, 458, 552 ActionDest: 612 Annotation: Subtype: Link Rect: 248, 492, 260, 501 ActionDest: 612 Annotation: Subtype: Link Rect: 263, 492, 275, 501 ActionDest: 612 Annotation: Subtype: Link Rect: 420, 455, 432, 464 ActionDest: 612 Annotation: Subtype: Link Rect: 318, 405, 330, 414 ActionDest: 612 Annotation: Subtype: Link Rect: 409, 354, 421, 363 ActionDest: 612 Annotation: Subtype: Link Rect: 422, 354, 434, 363 ActionDest: 612 Annotation: Subtype: Link Rect: 399, 329, 411, 338 ActionDest: 612 Annotation: Subtype: Link Rect: 543, 291, 555, 301 ActionDest: 612 Annotation: Subtype: Link Rect: 479, 229, 491, 238 ActionDest: 612 Annotation: Subtype: Link Rect: 492, 229, 504, 238 ActionDest: 612 Annotation: Subtype: Link Rect: 531, 166, 543, 175 ActionDest: 612 Annotation: Subtype: Link Rect: 544, 166, 556, 175 ActionDest: 612 Annotation: Subtype: Link Rect: 332, 128, 344, 137 ActionDest: 612 Annotation: Subtype: Link Rect: 495, 128, 507, 137 ActionDest: 612 Annotation: Subtype: Link Rect: 309, 116, 321, 125 ActionDest: 615 Annotation: Subtype: Link Rect: 395, 91, 407, 100 ActionDest: 615 Annotation: Subtype: Link Rect: 408, 91, 420, 100 ActionDest: 615 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 213, 731, 225, 740 ActionDest: 615 Annotation: Subtype: Link Rect: 228, 731, 240, 740 ActionDest: 615 Annotation: Subtype: Link Rect: 204, 681, 216, 690 ActionDest: 615 Annotation: Subtype: Link Rect: 218, 681, 230, 690 ActionDest: 615 Annotation: Subtype: Link Rect: 476, 618, 487, 627 ActionDest: 615 Annotation: Subtype: Link Rect: 490, 618, 502, 627 ActionDest: 615 Annotation: Subtype: Link Rect: 453, 479, 465, 488 ActionDest: 615 Annotation: Subtype: Link Rect: 228, 451, 235, 463 ActionDest: 76 Annotation: Subtype: Link Rect: 225, 403, 237, 412 ActionDest: 618 Annotation: Subtype: Link Rect: 478, 177, 490, 186 ActionDest: 618 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 416, 463, 423, 475 ActionDest: 76 Annotation: Subtype: Link Rect: 304, 428, 316, 437 ActionDest: 618 Annotation: Subtype: Link Rect: 319, 428, 331, 437 ActionDest: 618 Annotation: Subtype: Link Rect: 244, 378, 256, 387 ActionDest: 618 Annotation: Subtype: Link Rect: 418, 352, 430, 362 ActionDest: 612 Annotation: Subtype: Link Rect: 430, 353, 442, 362 ActionDest: 615 Annotation: Subtype: Link Rect: 443, 352, 455, 362 ActionDest: 618 Annotation: Subtype: Link Rect: 421, 325, 428, 337 ActionDest: 76 Annotation: Subtype: Link Rect: 338, 262, 345, 274 ActionDest: 76 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 294, 602, 301, 614 ActionDest: 125 Annotation: Subtype: Link Rect: 420, 589, 427, 601 ActionDest: 125 Annotation: Subtype: Link Rect: 524, 577, 531, 589 ActionDest: 125 Annotation: Subtype: Link Rect: 198, 170, 205, 182 ActionDest: 172 Annotation: Subtype: Link Rect: 540, 157, 547, 169 ActionDest: 172 Annotation: Subtype: Link Rect: 257, 107, 265, 119 ActionDest: 172 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 516, 401, 523, 413 ActionDest: 211 Annotation: Subtype: Link Rect: 451, 303, 463, 312 ActionDest: 615 Annotation: Subtype: Link Rect: 413, 278, 425, 287 ActionDest: 615 Annotation: Subtype: Link Rect: 426, 278, 438, 287 ActionDest: 615 Annotation: Subtype: Link Rect: 441, 278, 453, 287 ActionDest: 615 Annotation: Subtype: Link Rect: 245, 250, 252, 262 ActionDest: 211 Annotation: Subtype: Link Rect: 321, 240, 333, 249 ActionDest: 615 Annotation: Subtype: Link Rect: 258, 162, 265, 174 ActionDest: 287 Annotation: Subtype: Link Rect: 317, 162, 324, 174 ActionDest: 287 Annotation: Subtype: Link Rect: 529, 127, 541, 136 ActionDest: 618 Annotation: Subtype: Link Rect: 544, 127, 556, 136 ActionDest: 618 Annotation: Subtype: Link Rect: 450, 102, 462, 111 ActionDest: 615 Annotation: Subtype: Link Rect: 506, 89, 518, 99 ActionDest: 615 Annotation: Subtype: Link Rect: 521, 90, 532, 99 ActionDest: 615 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 384, 741, 391, 753 ActionDest: 211 Annotation: Subtype: Link Rect: 446, 706, 458, 715 ActionDest: 615 Annotation: Subtype: Link Rect: 191, 693, 202, 702 ActionDest: 618 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 289, 130, 296, 142 ActionDest: 326 Annotation: Subtype: Link Rect: 422, 130, 429, 142 ActionDest: 326 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 251, 741, 258, 753 ActionDest: 76 Annotation: Subtype: Link Rect: 213, 731, 225, 740 ActionDest: 615 Annotation: Subtype: Link Rect: 416, 706, 428, 715 ActionDest: 615 Annotation: Subtype: Link Rect: 429, 706, 441, 715 ActionDest: 615 Annotation: Subtype: Link Rect: 248, 615, 255, 627 ActionDest: 76 Annotation: Subtype: Link Rect: 407, 514, 414, 526 ActionDest: 365 Annotation: Subtype: Link Rect: 495, 349, 503, 361 ActionDest: 365 Annotation: Subtype: Link Rect: 327, 145, 334, 157 ActionDest: 373 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 418, 328, 425, 340 ActionDest: 413 Annotation: Subtype: Link Rect: 194, 303, 200, 315 ActionDest: 365 Annotation: Subtype: Link Rect: 233, 265, 240, 277 ActionDest: 413 Annotation: Subtype: Link Rect: 200, 203, 207, 215 ActionDest: 76 Annotation: Subtype: Link Rect: 355, 190, 362, 202 ActionDest: 365 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 471, 424, 560, 435 Annotation: Subtype: Link Rect: 165, 411, 202, 422 Annotation: Subtype: Link Rect: 285, 210, 297, 222 ActionDest: 452 Annotation: Subtype: Link Rect: 526, 147, 538, 159 ActionDest: 502 Annotation: Subtype: Link Rect: 417, 84, 424, 96 ActionDest: 413 Page: Sequence: 13 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 235, 351, 247, 360 ActionDest: 618 Annotation: Subtype: Link Rect: 210, 326, 222, 335 ActionDest: 612 Annotation: Subtype: Link Rect: 223, 326, 235, 335 ActionDest: 612 Annotation: Subtype: Link Rect: 235, 326, 247, 335 ActionDest: 618 Annotation: Subtype: Link Rect: 248, 326, 259, 335 ActionDest: 618 Annotation: Subtype: Link Rect: 182, 301, 194, 310 ActionDest: 612 Annotation: Subtype: Link Rect: 195, 301, 207, 310 ActionDest: 615 Annotation: Subtype: Link Rect: 207, 301, 219, 310 ActionDest: 615 Annotation: Subtype: Link Rect: 220, 301, 232, 310 ActionDest: 618 Annotation: Subtype: Link Rect: 232, 301, 244, 310 ActionDest: 618 Annotation: Subtype: Link Rect: 247, 301, 259, 310 ActionDest: 618 Annotation: Subtype: Link Rect: 322, 288, 334, 298 ActionDest: 618 Annotation: Subtype: Link Rect: 268, 238, 280, 247 ActionDest: 618 Annotation: Subtype: Link Rect: 254, 201, 266, 210 ActionDest: 615 Annotation: Subtype: Link Rect: 340, 175, 352, 185 ActionDest: 615 Annotation: Subtype: Link Rect: 355, 175, 367, 185 ActionDest: 615 Annotation: Subtype: Link Rect: 252, 138, 263, 147 ActionDest: 615 Annotation: Subtype: Link Rect: 487, 72, 494, 84 ActionDest: 125 Annotation: Subtype: Link Rect: 514, 72, 521, 84 ActionDest: 172 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 321, 668, 332, 677 ActionDest: 615 Annotation: Subtype: Link Rect: 333, 668, 345, 677 ActionDest: 615 Annotation: Subtype: Link Rect: 346, 668, 357, 677 ActionDest: 618 Annotation: Subtype: Link Rect: 358, 668, 370, 677 ActionDest: 618 Annotation: Subtype: Link Rect: 491, 643, 503, 652 ActionDest: 618 Annotation: Subtype: Link Rect: 504, 643, 516, 652 ActionDest: 618 Annotation: Subtype: Link Rect: 512, 467, 524, 476 ActionDest: 615 Annotation: Subtype: Link Rect: 525, 467, 537, 476 ActionDest: 618 Annotation: Subtype: Link Rect: 205, 455, 217, 464 ActionDest: 621 Annotation: Subtype: Link Rect: 481, 392, 493, 401 ActionDest: 612 Annotation: Subtype: Link Rect: 494, 392, 506, 401 ActionDest: 615 Annotation: Subtype: Link Rect: 506, 392, 518, 401 ActionDest: 615 Annotation: Subtype: Link Rect: 519, 392, 531, 401 ActionDest: 615 Annotation: Subtype: Link Rect: 531, 392, 543, 401 ActionDest: 621 Annotation: Subtype: Link Rect: 544, 392, 556, 401 ActionDest: 621 Annotation: Subtype: Link Rect: 477, 367, 489, 376 ActionDest: 615 Annotation: Subtype: Link Rect: 492, 367, 504, 376 ActionDest: 615 Annotation: Subtype: Link Rect: 505, 367, 517, 376 ActionDest: 615 Annotation: Subtype: Link Rect: 517, 367, 529, 376 ActionDest: 615 Annotation: Subtype: Link Rect: 530, 367, 542, 376 ActionDest: 618 Annotation: Subtype: Link Rect: 416, 292, 428, 301 ActionDest: 612 Annotation: Subtype: Link Rect: 428, 291, 440, 301 ActionDest: 621 Annotation: Subtype: Link Rect: 273, 279, 284, 288 ActionDest: 621 Annotation: Subtype: Link Rect: 287, 279, 299, 288 ActionDest: 621 Annotation: Subtype: Link Rect: 515, 191, 527, 200 ActionDest: 615 Annotation: Subtype: Link Rect: 297, 141, 309, 150 ActionDest: 621 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 196, 728, 203, 740 ActionDest: 76 Annotation: Subtype: Link Rect: 525, 716, 532, 728 ActionDest: 287 Annotation: Subtype: Link Rect: 420, 467, 432, 476 ActionDest: 621 Annotation: Subtype: Link Rect: 396, 404, 408, 414 ActionDest: 621 Annotation: Subtype: Link Rect: 278, 354, 290, 363 ActionDest: 618 Annotation: Subtype: Link Rect: 492, 202, 504, 211 ActionDest: 615 Annotation: Subtype: Link Rect: 207, 99, 219, 111 ActionDest: 547 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 401, 578, 413, 587 ActionDest: 621 Annotation: Subtype: Link Rect: 230, 412, 237, 424 ActionDest: 547 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 431, 499, 559, 511 Annotation: Subtype: Link Rect: 346, 364, 358, 374 ActionDest: 621 Annotation: Subtype: Link Rect: 365, 362, 504, 374 Annotation: Subtype: Link Rect: 365, 339, 377, 348 ActionDest: 621 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 413, 731, 425, 740 ActionDest: 621 Annotation: Subtype: Link Rect: 209, 480, 221, 489 ActionDest: 621 Annotation: Subtype: Link Rect: 272, 442, 284, 451 ActionDest: 618 Annotation: Subtype: Link Rect: 284, 442, 296, 451 ActionDest: 618 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 202, 429, 209, 438 ActionDest: 602 Annotation: Subtype: Link Rect: 342, 125, 560, 137 Annotation: Subtype: Link Rect: 165, 113, 280, 125 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 168, 427, 272, 439 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 479, 654, 560, 665 Annotation: Subtype: Link Rect: 165, 642, 324, 653 Annotation: Subtype: Link Rect: 536, 501, 560, 512 Annotation: Subtype: Link Rect: 165, 489, 264, 500 Annotation: Subtype: Link Rect: 218, 295, 256, 305 Annotation: Subtype: Link Rect: 59, 260, 96, 270 Annotation: Subtype: Link Rect: 135, 237, 173, 247 Annotation: Subtype: Link Rect: 174, 214, 212, 224 Annotation: Subtype: Link Rect: 209, 180, 247, 190 Annotation: Subtype: Link Rect: 392, 156, 430, 167 Annotation: Subtype: Link Rect: 401, 121, 439, 132 Annotation: Subtype: Link Rect: 59, 76, 96, 86 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 95, 719, 133, 729 Annotation: Subtype: Link Rect: 262, 696, 299, 706 Annotation: Subtype: Link Rect: 402, 683, 439, 694 Annotation: Subtype: Link Rect: 59, 650, 96, 660 Annotation: Subtype: Link Rect: 117, 615, 155, 625 Annotation: Subtype: Link Rect: 163, 592, 200, 602 Annotation: Subtype: Link Rect: 217, 568, 254, 579 Annotation: Subtype: Link Rect: 59, 546, 96, 556 Annotation: Subtype: Link Rect: 118, 512, 155, 522 Annotation: Subtype: Link Rect: 250, 476, 288, 487 Annotation: Subtype: Link Rect: 339, 442, 376, 453 Annotation: Subtype: Link Rect: 59, 420, 96, 430 Annotation: Subtype: Link Rect: 215, 396, 252, 407 Annotation: Subtype: Link Rect: 259, 396, 295, 407 Annotation: Subtype: Link Rect: 306, 373, 344, 384 Annotation: Subtype: Link Rect: 350, 373, 386, 384 Annotation: Subtype: Link Rect: 117, 351, 155, 361 Annotation: Subtype: Link Rect: 200, 328, 238, 338 Annotation: Subtype: Link Rect: 292, 304, 330, 315 Annotation: Subtype: Link Rect: 276, 282, 314, 292 Annotation: Subtype: Link Rect: 95, 259, 133, 269 Annotation: Subtype: Link Rect: 296, 235, 334, 246 Annotation: Subtype: Link Rect: 257, 212, 295, 223 Annotation: Subtype: Link Rect: 127, 190, 164, 200 Annotation: Subtype: Link Rect: 446, 166, 484, 177 Annotation: Subtype: Link Rect: 490, 166, 526, 177 Annotation: Subtype: Link Rect: 181, 132, 219, 142 Annotation: Subtype: Link Rect: 225, 132, 261, 142 Annotation: Subtype: Link Rect: 113, 97, 151, 108 Annotation: Subtype: Link Rect: 157, 97, 193, 108 Annotation: Subtype: Link Rect: 190, 74, 228, 85 Annotation: Subtype: Link Rect: 234, 74, 270, 85 Page: Sequence: 24 Annotations: Annotation: Subtype: Link Rect: 332, 718, 369, 729 Annotation: Subtype: Link Rect: 158, 684, 195, 694 Annotation: Subtype: Link Rect: 59, 650, 96, 660 Annotation: Subtype: Link Rect: 59, 615, 96, 625 Annotation: Subtype: Link Rect: 356, 591, 394, 602 Annotation: Subtype: Link Rect: 282, 558, 320, 568 Annotation: Subtype: Link Rect: 256, 535, 293, 545 Annotation: Subtype: Link Rect: 300, 535, 336, 545 Annotation: Subtype: Link Rect: 219, 500, 256, 510 Annotation: Subtype: Link Rect: 263, 500, 299, 510 Annotation: Subtype: Link Rect: 295, 465, 332, 476 Annotation: Subtype: Link Rect: 338, 465, 374, 476 Annotation: Subtype: Link Rect: 519, 442, 557, 453 Annotation: Subtype: Link Rect: 520, 407, 557, 418 Annotation: Subtype: Link Rect: 59, 374, 96, 384 Annotation: Subtype: Link Rect: 166, 339, 203, 349 Annotation: Subtype: Link Rect: 506, 315, 543, 326 Annotation: Subtype: Link Rect: 443, 292, 480, 303 Annotation: Subtype: Link Rect: 157, 247, 194, 257 Annotation: Subtype: Link Rect: 519, 223, 557, 234 Annotation: Subtype: Link Rect: 59, 213, 95, 223 Annotation: Subtype: Link Rect: 178, 177, 215, 188 Annotation: Subtype: Link Rect: 222, 177, 258, 188 Annotation: Subtype: Link Rect: 59, 132, 96, 142 Annotation: Subtype: Link Rect: 102, 132, 138, 142 Annotation: Subtype: Link Rect: 507, 108, 545, 119 Annotation: Subtype: Link Rect: 287, 74, 324, 85 Annotation: Subtype: Link Rect: 330, 74, 366, 85 Page: Sequence: 25 Annotations: Annotation: Subtype: Link Rect: 219, 730, 257, 741 Annotation: Subtype: Link Rect: 454, 706, 491, 718 Annotation: Subtype: Link Rect: 514, 683, 552, 694 Annotation: Subtype: Link Rect: 411, 649, 448, 660 Annotation: Subtype: Link Rect: 354, 626, 391, 637 Annotation: Subtype: Link Rect: 183, 592, 220, 602 Annotation: Subtype: Link Rect: 281, 568, 319, 579 Annotation: Subtype: Link Rect: 255, 534, 293, 545 Annotation: Subtype: Link Rect: 169, 500, 207, 510 Annotation: Subtype: Link Rect: 213, 500, 249, 510 Annotation: Subtype: Link Rect: 364, 476, 401, 487 Annotation: Subtype: Link Rect: 514, 453, 551, 464 Annotation: Subtype: Link Rect: 412, 430, 449, 441 Annotation: Subtype: Link Rect: 495, 407, 533, 418 Annotation: Subtype: Link Rect: 95, 374, 133, 384 Annotation: Subtype: Link Rect: 119, 339, 157, 349 Annotation: Subtype: Link Rect: 333, 315, 370, 326 Annotation: Subtype: Link Rect: 519, 292, 557, 303 Annotation: Subtype: Link Rect: 59, 282, 95, 292 Annotation: Subtype: Link Rect: 427, 259, 465, 269 Annotation: Subtype: Link Rect: 59, 224, 96, 234 Annotation: Subtype: Link Rect: 102, 224, 138, 234 Annotation: Subtype: Link Rect: 313, 200, 350, 211 Annotation: Subtype: Link Rect: 357, 200, 393, 211 Annotation: Subtype: Link Rect: 104, 167, 142, 177 Annotation: Subtype: Link Rect: 235, 131, 273, 142 Annotation: Subtype: Link Rect: 113, 97, 151, 108 Annotation: Subtype: Link Rect: 445, 74, 482, 85 Annotation: Subtype: Link Rect: 489, 74, 525, 85 Page: Sequence: 26 Annotations: Annotation: Subtype: Link Rect: 419, 718, 457, 729 Annotation: Subtype: Link Rect: 172, 684, 209, 694 Annotation: Subtype: Link Rect: 519, 660, 557, 671 Annotation: Subtype: Link Rect: 59, 650, 95, 660 Annotation: Subtype: Link Rect: 364, 626, 402, 637 Annotation: Subtype: Link Rect: 444, 603, 482, 614 Annotation: Subtype: Link Rect: 341, 568, 378, 579 Annotation: Subtype: Link Rect: 385, 568, 421, 579 Annotation: Subtype: Link Rect: 168, 534, 205, 545 Annotation: Subtype: Link Rect: 211, 534, 247, 545 Annotation: Subtype: Link Rect: 519, 488, 557, 499 Annotation: Subtype: Link Rect: 59, 477, 95, 487 Annotation: Subtype: Link Rect: 258, 454, 296, 464 Annotation: Subtype: Link Rect: 179, 420, 217, 430 Annotation: Subtype: Link Rect: 512, 396, 550, 407 Annotation: Subtype: Link Rect: 224, 361, 261, 372 Annotation: Subtype: Link Rect: 267, 361, 303, 372 Annotation: Subtype: Link Rect: 246, 338, 283, 349 Annotation: Subtype: Link Rect: 290, 338, 326, 349 Annotation: Subtype: Link Rect: 401, 327, 438, 338 Annotation: Subtype: Link Rect: 232, 281, 270, 292 Checksum: 47183b4d Type: CRC32 Checksum: 5e5eec3f375c196d26dcd162e0834f3a Type: MD5 Checksum: 15e02acd5c86d28f89898a5cc57c264abe0e9dc9 Type: SHA-1 Checksum: 68a3e0beea884c88bb29d7e1d08da72ca739c0928f58e2298fa0cf17a616ca41 Type: SHA-256