857271fd9a10c3d1cd77cace21b10601 pmc_9319574.pdf 034480482307f89876f9e205dd7d53472f1011ce pmc_9319574.pdf 01b3af1225ee0d8cdda970f3cf70213a6d09511200a7ad40a6199436d5457795 pmc_9319574.pdf Title: Characterization of Domiphen Bromide as a New Fast-Acting Antiplasmodial Agent Inhibiting the Apicoplastidic Methyl Erythritol Phosphate Pathway Subject: The evolution of resistance by the malaria parasite to artemisinin, the key component of the combination therapy strategies that are at the core of current antimalarial treatments, calls for the urgent identification of new fast-acting antimalarials. The apicoplast organelle is a preferred target of antimalarial drugs because it contains biochemical processes absent from the human host. Fosmidomycin is the only drug in clinical trials targeting the apicoplast, where it inhibits the methyl erythritol phosphate (MEP) pathway. Here, we characterized the antiplasmodial activity of domiphen bromide (DB), another MEP pathway inhibitor with a rapid mode of action that arrests the in vitro growth of Plasmodium falciparum at the early trophozoite stage. Metabolomic analysis of the MEP pathway and Krebs cycle intermediates in 20 M DB-treated parasites suggested a rapid activation of glycolysis with a concomitant decrease in mitochondrial activity, consistent with a rapid killing of the pathogen. These results present DB as a model compound for the development of new, potentially interesting drugs for future antimalarial combination therapies. Keywords: malaria; Plasmodium falciparum; domiphen bromide; methyl erythritol phosphate pathway; antimalarial drugs; antibiotics Author: Arnau Biosca, Miriam Ramírez, Alex Gomez-Gomez, Aritz Lafuente, Valentín Iglesias, Oscar J. Pozo, Santiago Imperial and Xavier Fernàndez-Busquets Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Jun 22 11:22:15 2022 CEST ModDate: Wed Jun 22 11:27:07 2022 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3189521 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PYPZKK+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 TOZXSU+VnURWPalladioL Type 1 Custom yes yes no 15 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 19 0 RTSUGW+URWPalladioL-Bold Type 1 Custom yes yes no 23 0 TXMIFR+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 PYHJJH+URWPalladioL-Ital Type 1 Custom yes yes no 32 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 84 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 88 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 92 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 96 0 VIWIOF+CMSY10 Type 1 Builtin yes yes no 108 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 116 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 120 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 124 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 128 0 KSAKCU+VnURWPalladioL-Italic Type 1 Custom yes yes no 142 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 154 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 158 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 162 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 166 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 182 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 186 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 190 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 199 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 203 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 207 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 220 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 224 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 228 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 237 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 241 0 BCDGEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 245 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 249 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-06-14 02:41:13 CEST RepresentationInformation: pmc_9319574.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-05-25 02:35:07 CEST Size: 3189521 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 478 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: P. falciparum Cultures and In Vitro Growth Inhibition Assays Destination: subsection.2.1 Item: Title: Determination of Metabolites from the MEP Pathway and the Tricarboxylic Acid Cycle Destination: subsection.2.2 Item: Title: Statistical Analysis Destination: subsection.2.3 Item: Title: Ethics Statement Destination: subsection.2.4 Item: Title: Results Destination: section.3 Children: Item: Title: Effect of DB on In Vitro P. falciparum Cultures Destination: subsection.3.1 Item: Title: Analysis of MEP Pathway and Citric Acid Cycle Metabolites in DB-Treated Samples Destination: subsection.3.2 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Characterization of Domiphen Bromide as a New Fast-Acting Antiplasmodial Agent Inhibiting the Apicoplastidic Methyl Erythritol Phosphate Pathway Author: Arnau Biosca, Miriam Ramírez, Alex Gomez-Gomez, Aritz Lafuente, Valentín Iglesias, Oscar J. Pozo, Santiago Imperial and Xavier Fernàndez-Busquets Subject: The evolution of resistance by the malaria parasite to artemisinin, the key component of the combination therapy strategies that are at the core of current antimalarial treatments, calls for the urgent identification of new fast-acting antimalarials. The apicoplast organelle is a preferred target of antimalarial drugs because it contains biochemical processes absent from the human host. Fosmidomycin is the only drug in clinical trials targeting the apicoplast, where it inhibits the methyl erythritol phosphate (MEP) pathway. Here, we characterized the antiplasmodial activity of domiphen bromide (DB), another MEP pathway inhibitor with a rapid mode of action that arrests the in vitro growth of Plasmodium falciparum at the early trophozoite stage. Metabolomic analysis of the MEP pathway and Krebs cycle intermediates in 20 M DB-treated parasites suggested a rapid activation of glycolysis with a concomitant decrease in mitochondrial activity, consistent with a rapid killing of the pathogen. These results present DB as a model compound for the development of new, potentially interesting drugs for future antimalarial combination therapies. Keywords: malaria; Plasmodium falciparum; domiphen bromide; methyl erythritol phosphate pathway; antimalarial drugs; antibiotics Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Jun 22 11:22:15 CEST 2022 ModDate: Wed Jun 22 11:27:07 CEST 2022 ID: 0x4696b30b54a0a8490d856460c1e05984, 0x4696b30b54a0a8490d856460c1e05984 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: PYHJJH+URWPalladioL-Ital FontSubset: true FirstChar: 44 LastChar: 151 FontDescriptor: FontName: PYHJJH+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: RTSUGW+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: RTSUGW+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PYPZKK+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 189 FontDescriptor: FontName: PYPZKK+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: TXMIFR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: TXMIFR+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VIWIOF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: VIWIOF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: KSAKCU+VnURWPalladioL-Italic FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: KSAKCU+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: TOZXSU+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: TOZXSU+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 82, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 311, 136, 320 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, 168, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 121, 642, 132, 655 Annotation: Subtype: Link Rect: 335, 642, 346, 655 Annotation: Subtype: Link Rect: 543, 642, 554, 655 Annotation: Subtype: Link Rect: 107, 629, 118, 643 Annotation: Subtype: Link Rect: 216, 629, 227, 643 Annotation: Subtype: Link Rect: 399, 629, 410, 643 Annotation: Subtype: Link Rect: 525, 131, 532, 140 ActionDest: 263 Annotation: Subtype: Link Rect: 135, 17, 311, 28 Annotation: Subtype: Link Rect: 385, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 444, 87, 463 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 382, 736, 389, 744 ActionDest: 263 Annotation: Subtype: Link Rect: 445, 723, 452, 732 ActionDest: 263 Annotation: Subtype: Link Rect: 549, 723, 556, 732 ActionDest: 263 Annotation: Subtype: Link Rect: 389, 673, 396, 682 ActionDest: 263 Annotation: Subtype: Link Rect: 466, 622, 473, 631 ActionDest: 263 Annotation: Subtype: Link Rect: 542, 610, 549, 619 ActionDest: 263 Annotation: Subtype: Link Rect: 550, 610, 557, 619 ActionDest: 263 Annotation: Subtype: Link Rect: 336, 547, 343, 556 ActionDest: 263 Annotation: Subtype: Link Rect: 481, 509, 493, 519 ActionDest: 263 Annotation: Subtype: Link Rect: 496, 509, 508, 519 ActionDest: 263 Annotation: Subtype: Link Rect: 263, 434, 275, 443 ActionDest: 263 Annotation: Subtype: Link Rect: 329, 419, 336, 431 ActionDest: 76 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 235, 672, 246, 681 ActionDest: 263 Annotation: Subtype: Link Rect: 269, 641, 281, 650 ActionDest: 263 Annotation: Subtype: Link Rect: 368, 629, 380, 638 ActionDest: 263 Annotation: Subtype: Link Rect: 380, 629, 392, 638 ActionDest: 263 Annotation: Subtype: Link Rect: 517, 616, 524, 625 ActionDest: 263 Annotation: Subtype: Link Rect: 501, 591, 513, 600 ActionDest: 263 Annotation: Subtype: Link Rect: 482, 553, 493, 562 ActionDest: 263 Annotation: Subtype: Link Rect: 415, 541, 426, 550 ActionDest: 267 Annotation: Subtype: Link Rect: 522, 528, 534, 537 ActionDest: 267 Annotation: Subtype: Link Rect: 368, 516, 380, 525 ActionDest: 267 Annotation: Subtype: Link Rect: 480, 516, 492, 525 ActionDest: 263 Annotation: Subtype: Link Rect: 464, 500, 471, 512 ActionDest: 104 Annotation: Subtype: Link Rect: 399, 491, 411, 500 ActionDest: 267 Annotation: Subtype: Link Rect: 543, 491, 555, 500 ActionDest: 267 Annotation: Subtype: Link Rect: 469, 465, 481, 475 ActionDest: 267 Annotation: Subtype: Link Rect: 295, 453, 307, 462 ActionDest: 267 Annotation: Subtype: Link Rect: 531, 453, 543, 462 ActionDest: 267 Annotation: Subtype: Link Rect: 543, 453, 555, 462 ActionDest: 267 Annotation: Subtype: Link Rect: 417, 440, 429, 449 ActionDest: 267 Annotation: Subtype: Link Rect: 430, 440, 442, 449 ActionDest: 267 Annotation: Subtype: Link Rect: 313, 165, 325, 174 ActionDest: 267 Annotation: Subtype: Link Rect: 510, 165, 522, 174 ActionDest: 267 Annotation: Subtype: Link Rect: 398, 102, 410, 111 ActionDest: 267 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 247, 698, 259, 707 ActionDest: 267 Annotation: Subtype: Link Rect: 259, 698, 271, 707 ActionDest: 267 Annotation: Subtype: Link Rect: 460, 660, 472, 669 ActionDest: 267 Annotation: Subtype: Link Rect: 204, 245, 216, 255 ActionDest: 267 Annotation: Subtype: Link Rect: 217, 246, 229, 255 ActionDest: 267 Annotation: Subtype: Link Rect: 380, 108, 392, 117 ActionDest: 267 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 202, 616, 286, 629 Annotation: Subtype: Link Rect: 338, 414, 345, 426 ActionDest: 146 Annotation: Subtype: Link Rect: 198, 289, 205, 301 ActionDest: 174 Annotation: Subtype: Link Rect: 517, 264, 524, 276 ActionDest: 76 Annotation: Subtype: Link Rect: 217, 213, 224, 225 ActionDest: 76 Annotation: Subtype: Link Rect: 282, 151, 289, 163 ActionDest: 212 Annotation: Subtype: Link Rect: 551, 88, 558, 100 ActionDest: 212 Page: Sequence: 6 Page: Sequence: 7 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 372, 176, 384, 185 ActionDest: 267 Annotation: Subtype: Link Rect: 247, 138, 259, 147 ActionDest: 267 Annotation: Subtype: Link Rect: 259, 138, 271, 147 ActionDest: 267 Annotation: Subtype: Link Rect: 225, 63, 237, 72 ActionDest: 267 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 544, 660, 556, 669 ActionDest: 267 Annotation: Subtype: Link Rect: 513, 648, 525, 657 ActionDest: 267 Annotation: Subtype: Link Rect: 271, 622, 283, 631 ActionDest: 267 Annotation: Subtype: Link Rect: 483, 622, 495, 631 ActionDest: 270 Annotation: Subtype: Link Rect: 521, 597, 533, 606 ActionDest: 270 Annotation: Subtype: Link Rect: 200, 572, 212, 581 ActionDest: 270 Annotation: Subtype: Link Rect: 339, 459, 351, 468 ActionDest: 270 Annotation: Subtype: Link Rect: 352, 459, 364, 468 ActionDest: 270 Annotation: Subtype: Link Rect: 543, 447, 555, 456 ActionDest: 270 Annotation: Subtype: Link Rect: 379, 434, 391, 443 ActionDest: 270 Annotation: Subtype: Link Rect: 233, 409, 245, 418 ActionDest: 270 Annotation: Subtype: Link Rect: 254, 371, 266, 380 ActionDest: 270 Annotation: Subtype: Link Rect: 267, 371, 279, 380 ActionDest: 270 Annotation: Subtype: Link Rect: 279, 371, 291, 380 ActionDest: 270 Annotation: Subtype: Link Rect: 494, 346, 505, 355 ActionDest: 270 Annotation: Subtype: Link Rect: 506, 346, 518, 355 ActionDest: 270 Annotation: Subtype: Link Rect: 273, 334, 285, 343 ActionDest: 270 Annotation: Subtype: Link Rect: 286, 334, 298, 343 ActionDest: 270 Annotation: Subtype: Link Rect: 465, 321, 477, 330 ActionDest: 270 Annotation: Subtype: Link Rect: 416, 283, 428, 293 ActionDest: 270 Annotation: Subtype: Link Rect: 482, 221, 494, 230 ActionDest: 270 Annotation: Subtype: Link Rect: 189, 196, 201, 205 ActionDest: 270 Annotation: Subtype: Link Rect: 470, 196, 482, 205 ActionDest: 270 Annotation: Subtype: Link Rect: 270, 145, 282, 154 ActionDest: 267 Annotation: Subtype: Link Rect: 282, 145, 294, 154 ActionDest: 267 Annotation: Subtype: Link Rect: 400, 143, 503, 155 Annotation: Subtype: Link Rect: 316, 83, 328, 92 ActionDest: 267 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 373, 698, 385, 707 ActionDest: 267 Annotation: Subtype: Link Rect: 440, 673, 452, 682 ActionDest: 267 Annotation: Subtype: Link Rect: 424, 622, 436, 631 ActionDest: 267 Annotation: Subtype: Link Rect: 437, 622, 449, 631 ActionDest: 270 Annotation: Subtype: Link Rect: 449, 622, 461, 631 ActionDest: 270 Annotation: Subtype: Link Rect: 424, 433, 560, 444 Annotation: Subtype: Link Rect: 165, 421, 311, 432 Annotation: Subtype: Link Rect: 168, 315, 285, 326 Annotation: Subtype: Link Rect: 168, 303, 269, 314 Annotation: Subtype: Link Rect: 321, 291, 424, 302 Annotation: Subtype: Link Rect: 168, 268, 276, 279 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 125, 671, 438, 682 Annotation: Subtype: Link Rect: 336, 636, 374, 647 Annotation: Subtype: Link Rect: 380, 636, 416, 647 Annotation: Subtype: Link Rect: 266, 602, 303, 613 Annotation: Subtype: Link Rect: 310, 602, 346, 613 Annotation: Subtype: Link Rect: 122, 568, 159, 578 Annotation: Subtype: Link Rect: 166, 568, 202, 578 Annotation: Subtype: Link Rect: 490, 556, 527, 567 Annotation: Subtype: Link Rect: 95, 534, 133, 544 Annotation: Subtype: Link Rect: 170, 499, 208, 509 Annotation: Subtype: Link Rect: 59, 476, 96, 486 Annotation: Subtype: Link Rect: 216, 452, 253, 463 Annotation: Subtype: Link Rect: 122, 419, 159, 429 Annotation: Subtype: Link Rect: 239, 395, 276, 406 Annotation: Subtype: Link Rect: 280, 372, 318, 383 Annotation: Subtype: Link Rect: 59, 349, 96, 360 Annotation: Subtype: Link Rect: 102, 349, 138, 360 Annotation: Subtype: Link Rect: 298, 314, 335, 325 Annotation: Subtype: Link Rect: 519, 291, 557, 302 Annotation: Subtype: Link Rect: 59, 280, 95, 291 Annotation: Subtype: Link Rect: 520, 257, 557, 268 Annotation: Subtype: Link Rect: 59, 246, 95, 256 Annotation: Subtype: Link Rect: 466, 211, 504, 222 Annotation: Subtype: Link Rect: 86, 177, 124, 187 Annotation: Subtype: Link Rect: 511, 153, 549, 164 Annotation: Subtype: Link Rect: 285, 118, 323, 129 Annotation: Subtype: Link Rect: 230, 84, 268, 95 Annotation: Subtype: Link Rect: 436, 61, 473, 72 Annotation: Subtype: Link Rect: 479, 61, 515, 72 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 104, 699, 141, 710 Annotation: Subtype: Link Rect: 226, 676, 263, 687 Annotation: Subtype: Link Rect: 270, 676, 306, 687 Annotation: Subtype: Link Rect: 127, 619, 164, 629 Annotation: Subtype: Link Rect: 518, 595, 555, 606 Annotation: Subtype: Link Rect: 322, 572, 359, 583 Annotation: Subtype: Link Rect: 192, 537, 455, 548 Annotation: Subtype: Link Rect: 95, 469, 133, 479 Annotation: Subtype: Link Rect: 502, 445, 540, 456 Annotation: Subtype: Link Rect: 282, 422, 320, 433 Annotation: Subtype: Link Rect: 95, 400, 133, 410 Annotation: Subtype: Link Rect: 139, 400, 175, 410 Annotation: Subtype: Link Rect: 358, 365, 395, 376 Annotation: Subtype: Link Rect: 401, 365, 437, 376 Annotation: Subtype: Link Rect: 59, 331, 96, 341 Annotation: Subtype: Link Rect: 140, 296, 178, 307 Annotation: Subtype: Link Rect: 184, 296, 220, 307 Annotation: Subtype: Link Rect: 504, 261, 542, 272 Annotation: Subtype: Link Rect: 395, 228, 433, 238 Annotation: Subtype: Link Rect: 225, 204, 262, 215 Annotation: Subtype: Link Rect: 211, 181, 249, 192 Annotation: Subtype: Link Rect: 168, 159, 206, 169 Annotation: Subtype: Link Rect: 519, 146, 557, 157 Annotation: Subtype: Link Rect: 59, 136, 95, 146 Annotation: Subtype: Link Rect: 164, 113, 202, 123 Annotation: Subtype: Link Rect: 519, 100, 557, 111 Annotation: Subtype: Link Rect: 59, 90, 95, 100 Annotation: Subtype: Link Rect: 375, 77, 560, 88 Annotation: Subtype: Link Rect: 56, 66, 250, 77 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 251, 722, 288, 733 Annotation: Subtype: Link Rect: 295, 722, 331, 733 Annotation: Subtype: Link Rect: 396, 710, 433, 721 Annotation: Subtype: Link Rect: 439, 710, 476, 721 Annotation: Subtype: Link Rect: 167, 688, 204, 698 Annotation: Subtype: Link Rect: 510, 676, 548, 687 Annotation: Subtype: Link Rect: 318, 641, 355, 652 Annotation: Subtype: Link Rect: 199, 618, 236, 629 Annotation: Subtype: Link Rect: 484, 595, 521, 606 Annotation: Subtype: Link Rect: 173, 573, 210, 583 Annotation: Subtype: Link Rect: 216, 573, 252, 583 Annotation: Subtype: Link Rect: 361, 549, 398, 560 Annotation: Subtype: Link Rect: 404, 549, 440, 560 Annotation: Subtype: Link Rect: 229, 526, 267, 537 Annotation: Subtype: Link Rect: 273, 526, 309, 537 Annotation: Subtype: Link Rect: 456, 503, 494, 514 Annotation: Subtype: Link Rect: 500, 503, 536, 514 Annotation: Subtype: Link Rect: 432, 480, 469, 491 Annotation: Subtype: Link Rect: 519, 457, 557, 468 Annotation: Subtype: Link Rect: 360, 434, 397, 445 Annotation: Subtype: Link Rect: 330, 411, 367, 422 Annotation: Subtype: Link Rect: 248, 377, 285, 387 Annotation: Subtype: Link Rect: 483, 353, 521, 364 Checksum: edee164a Type: CRC32 Checksum: 857271fd9a10c3d1cd77cace21b10601 Type: MD5 Checksum: 034480482307f89876f9e205dd7d53472f1011ce Type: SHA-1 Checksum: 01b3af1225ee0d8cdda970f3cf70213a6d09511200a7ad40a6199436d5457795 Type: SHA-256