5e749579a202011b206093f1d2f08c92 pmc_34834337.pdf 5459f20f2683bc748c7210fbc95eacffa3f665d6 pmc_34834337.pdf aa7ca03298566c170a6ce6ff7876dab03fcef7c6b9affece1e6a1ff5163a9314 pmc_34834337.pdf Title: Antibacterial Activity of T22, a Specific Peptidic Ligand of the Tumoral Marker CXCR4 Subject: CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms. Keywords: antimicrobial peptides; nanoparticles; fusion proteins; inhibition of biofilm formation; multivalent drugs Author: Naroa Serna, José Vicente Carratalá, Oscar Conchillo-Solé, Carlos Martínez-Torró, Ugutz Unzueta, Ramón Mangues, Neus Ferrer-Miralles, Xavier Daura, Esther Vázquez and Antonio Villaverde Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Nov 13 06:50:50 2021 CET ModDate: Sat Nov 13 07:14:24 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5201257 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 IGDFIY+VnURWPalladioL Type 1 Custom yes yes no 15 0 VSTZGU+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 REWVCY+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 YFTYOM+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 37 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 65 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 69 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 73 0 QMDDXF+CMSY10 Type 1 Builtin yes yes no 85 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 92 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 104 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 110 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 116 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 129 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 135 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 141 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 156 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 162 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 168 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 184 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 190 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 196 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 211 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 217 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 223 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 02:54:24 CEST RepresentationInformation: pmc_34834337.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:06:21 CEST Size: 5201257 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 608 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: Material and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Peptides, Proteins and Protein Nanoparticles Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Bacterial Growth and Determination of the Minimum Inhibitory Concentration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Time-Killing Kinetic Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Evaluation of Biofilm Formation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Mammalian Cell Viability Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Hemolysis Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Measurement of the Nanoparticle Size Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Structure-Based Calculations and Molecular Modeling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Results 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: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Info: Title: Antibacterial Activity of T22, a Specific Peptidic Ligand of the Tumoral Marker CXCR4 Author: Naroa Serna, José Vicente Carratalá, Oscar Conchillo-Solé, Carlos Martínez-Torró, Ugutz Unzueta, Ramón Mangues, Neus Ferrer-Miralles, Xavier Daura, Esther Vázquez and Antonio Villaverde Subject: CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms. Keywords: antimicrobial peptides; nanoparticles; fusion proteins; inhibition of biofilm formation; multivalent drugs Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Nov 13 06:50:50 CET 2021 ModDate: Sat Nov 13 07:14:24 CET 2021 ID: 0xa248cf64cc26798233c7cb9eb19e82bd, 0xa248cf64cc26798233c7cb9eb19e82bd Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: VSTZGU+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: VSTZGU+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QMDDXF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: QMDDXF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: REWVCY+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 135 FontDescriptor: FontName: REWVCY+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JYPLJJ+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: JYPLJJ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YFTYOM+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: YFTYOM+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: IGDFIY+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 230 FontDescriptor: FontName: IGDFIY+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 168, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 371, 662, 382, 675 Annotation: Subtype: Link Rect: 130, 649, 141, 663 Annotation: Subtype: Link Rect: 241, 649, 252, 663 Annotation: Subtype: Link Rect: 370, 649, 381, 663 Annotation: Subtype: Link Rect: 126, 636, 137, 650 Annotation: Subtype: Link Rect: 272, 636, 283, 650 Annotation: Subtype: Link Rect: 125, 383, 154, 391 Annotation: Subtype: Link Rect: 35, 370, 154, 379 Annotation: Subtype: Link Rect: 35, 358, 109, 367 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 218, 99, 225, 108 ActionDest: 236 Annotation: Subtype: Link Rect: 226, 99, 232, 108 ActionDest: 236 Annotation: Subtype: Link Rect: 396, 99, 403, 108 ActionDest: 240 Annotation: Subtype: Link Rect: 478, 74, 485, 83 ActionDest: 236 Annotation: Subtype: Link Rect: 485, 74, 492, 83 ActionDest: 240 Annotation: Subtype: Link Rect: 136, 17, 312, 28 Annotation: Subtype: Link Rect: 383, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 494, 87, 513 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 199, 699, 206, 708 ActionDest: 240 Annotation: Subtype: Link Rect: 209, 699, 221, 708 ActionDest: 240 Annotation: Subtype: Link Rect: 292, 674, 304, 683 ActionDest: 240 Annotation: Subtype: Link Rect: 543, 674, 555, 683 ActionDest: 240 Annotation: Subtype: Link Rect: 544, 661, 556, 670 ActionDest: 240 Annotation: Subtype: Link Rect: 478, 611, 490, 620 ActionDest: 240 Annotation: Subtype: Link Rect: 191, 561, 203, 570 ActionDest: 240 Annotation: Subtype: Link Rect: 206, 561, 218, 570 ActionDest: 240 Annotation: Subtype: Link Rect: 231, 523, 243, 532 ActionDest: 240 Annotation: Subtype: Link Rect: 522, 511, 529, 520 ActionDest: 240 Annotation: Subtype: Link Rect: 530, 511, 541, 520 ActionDest: 240 Annotation: Subtype: Link Rect: 544, 511, 556, 520 ActionDest: 243 Annotation: Subtype: Link Rect: 231, 473, 243, 482 ActionDest: 243 Annotation: Subtype: Link Rect: 244, 473, 255, 482 ActionDest: 243 Annotation: Subtype: Link Rect: 444, 448, 456, 457 ActionDest: 243 Annotation: Subtype: Link Rect: 459, 448, 471, 457 ActionDest: 243 Annotation: Subtype: Link Rect: 188, 423, 199, 432 ActionDest: 243 Annotation: Subtype: Link Rect: 200, 423, 212, 432 ActionDest: 243 Annotation: Subtype: Link Rect: 208, 398, 220, 407 ActionDest: 240 Annotation: Subtype: Link Rect: 220, 398, 232, 407 ActionDest: 240 Annotation: Subtype: Link Rect: 233, 398, 245, 407 ActionDest: 243 Annotation: Subtype: Link Rect: 202, 347, 214, 356 ActionDest: 243 Annotation: Subtype: Link Rect: 215, 347, 226, 356 ActionDest: 243 Annotation: Subtype: Link Rect: 399, 322, 411, 331 ActionDest: 243 Annotation: Subtype: Link Rect: 414, 322, 426, 331 ActionDest: 243 Annotation: Subtype: Link Rect: 372, 221, 384, 230 ActionDest: 243 Annotation: Subtype: Link Rect: 452, 221, 464, 230 ActionDest: 243 Annotation: Subtype: Link Rect: 545, 221, 557, 230 ActionDest: 243 Annotation: Subtype: Link Rect: 225, 208, 237, 217 ActionDest: 243 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 483, 257, 495, 266 ActionDest: 243 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 477, 370, 489, 379 ActionDest: 243 Annotation: Subtype: Link Rect: 277, 355, 470, 367 Annotation: Subtype: Link Rect: 358, 345, 370, 354 ActionDest: 243 Annotation: Subtype: Link Rect: 543, 345, 555, 354 ActionDest: 243 Annotation: Subtype: Link Rect: 322, 332, 334, 341 ActionDest: 243 Annotation: Subtype: Link Rect: 493, 332, 505, 341 ActionDest: 243 Annotation: Subtype: Link Rect: 299, 320, 311, 329 ActionDest: 243 Annotation: Subtype: Link Rect: 487, 320, 494, 329 ActionDest: 240 Annotation: Subtype: Link Rect: 169, 279, 416, 291 Annotation: Subtype: Link Rect: 183, 269, 195, 278 ActionDest: 243 Annotation: Subtype: Link Rect: 191, 217, 274, 229 Annotation: Subtype: Link Rect: 218, 182, 230, 191 ActionDest: 243 Annotation: Subtype: Link Rect: 470, 144, 482, 153 ActionDest: 243 Annotation: Subtype: Link Rect: 525, 131, 537, 140 ActionDest: 246 Annotation: Subtype: Link Rect: 169, 104, 424, 116 Annotation: Subtype: Link Rect: 544, 106, 556, 115 ActionDest: 246 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 214, 722, 226, 731 ActionDest: 246 Annotation: Subtype: Link Rect: 363, 720, 559, 732 Annotation: Subtype: Link Rect: 322, 697, 334, 706 ActionDest: 246 Annotation: Subtype: Link Rect: 515, 659, 522, 668 ActionDest: 240 Annotation: Subtype: Link Rect: 451, 634, 463, 643 ActionDest: 243 Annotation: Subtype: Link Rect: 276, 619, 283, 631 ActionDest: 99 Annotation: Subtype: Link Rect: 347, 581, 354, 593 ActionDest: 99 Annotation: Subtype: Link Rect: 397, 569, 404, 581 ActionDest: 99 Annotation: Subtype: Link Rect: 364, 518, 371, 530 ActionDest: 99 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 520, 707, 527, 719 ActionDest: 99 Annotation: Subtype: Link Rect: 465, 697, 477, 706 ActionDest: 246 Annotation: Subtype: Link Rect: 478, 697, 490, 706 ActionDest: 246 Annotation: Subtype: Link Rect: 421, 672, 433, 681 ActionDest: 246 Annotation: Subtype: Link Rect: 239, 644, 246, 656 ActionDest: 124 Annotation: Subtype: Link Rect: 201, 619, 208, 631 ActionDest: 124 Annotation: Subtype: Link Rect: 478, 607, 485, 619 ActionDest: 151 Annotation: Subtype: Link Rect: 471, 594, 478, 606 ActionDest: 151 Annotation: Subtype: Link Rect: 276, 546, 288, 556 ActionDest: 246 Annotation: Subtype: Link Rect: 289, 546, 301, 556 ActionDest: 246 Annotation: Subtype: Link Rect: 254, 531, 261, 543 ActionDest: 179 Annotation: Subtype: Link Rect: 454, 531, 461, 543 ActionDest: 179 Annotation: Subtype: Link Rect: 224, 506, 231, 518 ActionDest: 179 Annotation: Subtype: Link Rect: 252, 494, 259, 506 ActionDest: 179 Page: Sequence: 7 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 306, 410, 318, 419 ActionDest: 246 Annotation: Subtype: Link Rect: 321, 410, 333, 419 ActionDest: 246 Annotation: Subtype: Link Rect: 406, 397, 418, 406 ActionDest: 243 Annotation: Subtype: Link Rect: 419, 397, 431, 406 ActionDest: 243 Annotation: Subtype: Link Rect: 246, 360, 258, 369 ActionDest: 246 Annotation: Subtype: Link Rect: 261, 360, 273, 369 ActionDest: 246 Annotation: Subtype: Link Rect: 478, 347, 490, 356 ActionDest: 246 Annotation: Subtype: Link Rect: 230, 332, 237, 344 ActionDest: 151 Annotation: Subtype: Link Rect: 516, 259, 523, 268 ActionDest: 240 Annotation: Subtype: Link Rect: 523, 259, 530, 268 ActionDest: 240 Annotation: Subtype: Link Rect: 483, 247, 495, 256 ActionDest: 240 Annotation: Subtype: Link Rect: 496, 247, 507, 256 ActionDest: 246 Annotation: Subtype: Link Rect: 473, 219, 480, 231 ActionDest: 206 Annotation: Subtype: Link Rect: 467, 206, 474, 218 ActionDest: 206 Annotation: Subtype: Link Rect: 321, 194, 328, 206 ActionDest: 206 Annotation: Subtype: Link Rect: 226, 169, 233, 181 ActionDest: 124 Annotation: Subtype: Link Rect: 262, 169, 269, 181 ActionDest: 206 Annotation: Subtype: Link Rect: 346, 144, 353, 156 ActionDest: 206 Annotation: Subtype: Link Rect: 422, 131, 429, 143 ActionDest: 206 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 97, 355, 108, 363 ActionDest: 240 Annotation: Subtype: Link Rect: 496, 268, 508, 277 ActionDest: 246 Annotation: Subtype: Link Rect: 508, 268, 520, 277 ActionDest: 246 Annotation: Subtype: Link Rect: 387, 243, 399, 252 ActionDest: 246 Annotation: Subtype: Link Rect: 399, 243, 411, 252 ActionDest: 246 Annotation: Subtype: Link Rect: 349, 218, 361, 227 ActionDest: 246 Annotation: Subtype: Link Rect: 364, 218, 376, 227 ActionDest: 246 Annotation: Subtype: Link Rect: 408, 180, 420, 189 ActionDest: 249 Annotation: Subtype: Link Rect: 423, 180, 435, 189 ActionDest: 249 Annotation: Subtype: Link Rect: 382, 155, 389, 164 ActionDest: 236 Annotation: Subtype: Link Rect: 389, 155, 401, 164 ActionDest: 240 Annotation: Subtype: Link Rect: 402, 155, 414, 164 ActionDest: 240 Annotation: Subtype: Link Rect: 414, 155, 426, 164 ActionDest: 240 Annotation: Subtype: Link Rect: 427, 155, 439, 164 ActionDest: 249 Annotation: Subtype: Link Rect: 439, 155, 451, 164 ActionDest: 249 Annotation: Subtype: Link Rect: 452, 155, 464, 164 ActionDest: 249 Annotation: Subtype: Link Rect: 315, 130, 327, 139 ActionDest: 249 Annotation: Subtype: Link Rect: 330, 130, 342, 139 ActionDest: 249 Annotation: Subtype: Link Rect: 292, 117, 304, 126 ActionDest: 249 Annotation: Subtype: Link Rect: 307, 117, 319, 126 ActionDest: 249 Annotation: Subtype: Link Rect: 384, 117, 396, 126 ActionDest: 249 Annotation: Subtype: Link Rect: 399, 117, 416, 126 ActionDest: 249 Annotation: Subtype: Link Rect: 232, 80, 249, 89 ActionDest: 249 Annotation: Subtype: Link Rect: 252, 80, 269, 89 ActionDest: 252 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 253, 722, 265, 731 ActionDest: 240 Annotation: Subtype: Link Rect: 266, 722, 278, 731 ActionDest: 240 Annotation: Subtype: Link Rect: 544, 710, 556, 719 ActionDest: 240 Annotation: Subtype: Link Rect: 543, 697, 555, 706 ActionDest: 240 Annotation: Subtype: Link Rect: 223, 685, 235, 694 ActionDest: 240 Annotation: Subtype: Link Rect: 236, 685, 248, 694 ActionDest: 243 Annotation: Subtype: Link Rect: 361, 685, 368, 694 ActionDest: 240 Annotation: Subtype: Link Rect: 369, 685, 381, 694 ActionDest: 240 Annotation: Subtype: Link Rect: 299, 657, 306, 669 ActionDest: 124 Annotation: Subtype: Link Rect: 326, 657, 333, 669 ActionDest: 151 Annotation: Subtype: Link Rect: 219, 594, 226, 606 ActionDest: 99 Annotation: Subtype: Link Rect: 282, 572, 294, 581 ActionDest: 243 Annotation: Subtype: Link Rect: 389, 559, 406, 568 ActionDest: 252 Annotation: Subtype: Link Rect: 409, 559, 426, 568 ActionDest: 252 Annotation: Subtype: Link Rect: 364, 534, 376, 543 ActionDest: 243 Annotation: Subtype: Link Rect: 297, 521, 314, 530 ActionDest: 252 Annotation: Subtype: Link Rect: 400, 522, 412, 530 ActionDest: 243 Annotation: Subtype: Link Rect: 412, 521, 429, 530 ActionDest: 252 Annotation: Subtype: Link Rect: 544, 496, 556, 505 ActionDest: 243 Annotation: Subtype: Link Rect: 413, 443, 420, 455 ActionDest: 179 Annotation: Subtype: Link Rect: 294, 431, 301, 443 ActionDest: 124 Annotation: Subtype: Link Rect: 510, 431, 517, 443 ActionDest: 179 Annotation: Subtype: Link Rect: 348, 406, 355, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 498, 358, 515, 367 ActionDest: 252 Annotation: Subtype: Link Rect: 192, 296, 209, 304 ActionDest: 252 Annotation: Subtype: Link Rect: 212, 295, 229, 304 ActionDest: 252 Annotation: Subtype: Link Rect: 488, 233, 500, 242 ActionDest: 243 Annotation: Subtype: Link Rect: 501, 233, 513, 242 ActionDest: 246 Annotation: Subtype: Link Rect: 304, 195, 321, 204 ActionDest: 252 Annotation: Subtype: Link Rect: 439, 145, 451, 154 ActionDest: 246 Annotation: Subtype: Link Rect: 451, 145, 468, 154 ActionDest: 252 Annotation: Subtype: Link Rect: 469, 145, 486, 154 ActionDest: 252 Annotation: Subtype: Link Rect: 505, 132, 522, 141 ActionDest: 252 Annotation: Subtype: Link Rect: 201, 107, 213, 116 ActionDest: 246 Annotation: Subtype: Link Rect: 214, 107, 231, 116 ActionDest: 252 Annotation: Subtype: Link Rect: 231, 107, 248, 116 ActionDest: 252 Annotation: Subtype: Link Rect: 520, 107, 537, 116 ActionDest: 252 Annotation: Subtype: Link Rect: 540, 107, 557, 116 ActionDest: 252 Annotation: Subtype: Link Rect: 186, 82, 203, 91 ActionDest: 252 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 419, 520, 560, 531 Annotation: Subtype: Link Rect: 166, 508, 301, 519 Annotation: Subtype: Link Rect: 288, 226, 560, 237 Annotation: Subtype: Link Rect: 166, 214, 189, 225 Annotation: Subtype: Link Rect: 203, 113, 240, 124 Annotation: Subtype: Link Rect: 459, 79, 497, 90 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 131, 710, 169, 720 Annotation: Subtype: Link Rect: 95, 664, 133, 674 Annotation: Subtype: Link Rect: 298, 629, 336, 640 Annotation: Subtype: Link Rect: 342, 629, 378, 640 Annotation: Subtype: Link Rect: 158, 595, 196, 605 Annotation: Subtype: Link Rect: 445, 571, 483, 582 Annotation: Subtype: Link Rect: 222, 537, 260, 548 Annotation: Subtype: Link Rect: 266, 537, 302, 548 Annotation: Subtype: Link Rect: 225, 502, 263, 513 Annotation: Subtype: Link Rect: 269, 502, 305, 513 Annotation: Subtype: Link Rect: 344, 469, 381, 479 Annotation: Subtype: Link Rect: 179, 434, 216, 444 Annotation: Subtype: Link Rect: 355, 399, 392, 410 Annotation: Subtype: Link Rect: 136, 365, 173, 375 Annotation: Subtype: Link Rect: 131, 331, 169, 341 Annotation: Subtype: Link Rect: 188, 296, 225, 306 Annotation: Subtype: Link Rect: 188, 262, 225, 272 Annotation: Subtype: Link Rect: 167, 227, 205, 237 Annotation: Subtype: Link Rect: 99, 192, 137, 203 Annotation: Subtype: Link Rect: 173, 158, 210, 168 Annotation: Subtype: Link Rect: 295, 134, 332, 145 Annotation: Subtype: Link Rect: 161, 100, 199, 111 Annotation: Subtype: Link Rect: 461, 77, 498, 88 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 220, 709, 258, 720 Annotation: Subtype: Link Rect: 389, 686, 427, 697 Annotation: Subtype: Link Rect: 426, 663, 464, 674 Annotation: Subtype: Link Rect: 339, 629, 376, 640 Annotation: Subtype: Link Rect: 520, 606, 557, 617 Annotation: Subtype: Link Rect: 519, 584, 557, 594 Annotation: Subtype: Link Rect: 113, 549, 150, 559 Annotation: Subtype: Link Rect: 254, 526, 292, 536 Annotation: Subtype: Link Rect: 323, 492, 361, 502 Annotation: Subtype: Link Rect: 405, 479, 442, 490 Annotation: Subtype: Link Rect: 374, 456, 412, 467 Annotation: Subtype: Link Rect: 492, 445, 530, 456 Annotation: Subtype: Link Rect: 158, 423, 195, 433 Annotation: Subtype: Link Rect: 445, 410, 483, 421 Annotation: Subtype: Link Rect: 136, 377, 173, 387 Annotation: Subtype: Link Rect: 86, 342, 124, 352 Annotation: Subtype: Link Rect: 95, 308, 133, 318 Annotation: Subtype: Link Rect: 127, 273, 164, 283 Annotation: Subtype: Link Rect: 378, 249, 416, 260 Annotation: Subtype: Link Rect: 95, 215, 132, 226 Annotation: Subtype: Link Rect: 400, 192, 437, 203 Annotation: Subtype: Link Rect: 272, 169, 309, 180 Annotation: Subtype: Link Rect: 197, 135, 234, 145 Annotation: Subtype: Link Rect: 167, 112, 204, 122 Annotation: Subtype: Link Rect: 322, 88, 359, 99 Annotation: Subtype: Link Rect: 470, 77, 508, 88 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 325, 722, 363, 732 Annotation: Subtype: Link Rect: 399, 698, 437, 709 Annotation: Subtype: Link Rect: 455, 686, 492, 697 Annotation: Subtype: Link Rect: 215, 663, 252, 674 Annotation: Subtype: Link Rect: 469, 640, 506, 651 Annotation: Subtype: Link Rect: 512, 640, 548, 651 Annotation: Subtype: Link Rect: 95, 607, 132, 617 Annotation: Subtype: Link Rect: 139, 607, 175, 617 Annotation: Subtype: Link Rect: 186, 571, 224, 582 Annotation: Subtype: Link Rect: 122, 538, 160, 548 Annotation: Subtype: Link Rect: 273, 515, 310, 525 Annotation: Subtype: Link Rect: 317, 515, 353, 525 Annotation: Subtype: Link Rect: 486, 491, 523, 502 Annotation: Subtype: Link Rect: 473, 479, 510, 490 Annotation: Subtype: Link Rect: 517, 479, 553, 490 Annotation: Subtype: Link Rect: 95, 457, 133, 467 Annotation: Subtype: Link Rect: 247, 433, 285, 444 Annotation: Subtype: Link Rect: 291, 433, 327, 444 Annotation: Subtype: Link Rect: 305, 411, 343, 421 Annotation: Subtype: Link Rect: 95, 388, 133, 398 Annotation: Subtype: Link Rect: 139, 388, 175, 398 Annotation: Subtype: Link Rect: 326, 364, 363, 375 Annotation: Subtype: Link Rect: 218, 342, 255, 352 Annotation: Subtype: Link Rect: 262, 342, 298, 352 Annotation: Subtype: Link Rect: 161, 307, 199, 318 Annotation: Subtype: Link Rect: 205, 307, 241, 318 Annotation: Subtype: Link Rect: 270, 284, 308, 295 Annotation: Subtype: Link Rect: 314, 284, 350, 295 Annotation: Subtype: Link Rect: 259, 261, 296, 272 Annotation: Subtype: Link Rect: 303, 261, 339, 272 Annotation: Subtype: Link Rect: 478, 250, 515, 260 Annotation: Subtype: Link Rect: 521, 250, 556, 260 Annotation: Subtype: Link Rect: 291, 227, 328, 237 Annotation: Subtype: Link Rect: 458, 203, 496, 214 Annotation: Subtype: Link Rect: 495, 192, 532, 203 Annotation: Subtype: Link Rect: 329, 169, 367, 180 Annotation: Subtype: Link Rect: 104, 146, 142, 157 Annotation: Subtype: Link Rect: 148, 146, 184, 157 Annotation: Subtype: Link Rect: 122, 123, 160, 134 Annotation: Subtype: Link Rect: 513, 100, 550, 111 Annotation: Subtype: Link Rect: 328, 77, 365, 88 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 194, 722, 231, 732 Annotation: Subtype: Link Rect: 86, 699, 124, 709 Annotation: Subtype: Link Rect: 130, 699, 166, 709 Annotation: Subtype: Link Rect: 104, 664, 142, 674 Annotation: Subtype: Link Rect: 104, 641, 141, 651 Annotation: Subtype: Link Rect: 219, 606, 257, 617 Annotation: Subtype: Link Rect: 131, 584, 169, 594 Annotation: Subtype: Link Rect: 291, 560, 328, 571 Annotation: Subtype: Link Rect: 335, 560, 371, 571 Annotation: Subtype: Link Rect: 516, 537, 554, 548 Annotation: Subtype: Link Rect: 318, 514, 356, 525 Annotation: Subtype: Link Rect: 362, 514, 398, 525 Annotation: Subtype: Link Rect: 233, 491, 271, 502 Annotation: Subtype: Link Rect: 171, 457, 209, 467 Annotation: Subtype: Link Rect: 353, 388, 391, 398 Annotation: Subtype: Link Rect: 397, 388, 433, 398 Annotation: Subtype: Link Rect: 440, 364, 478, 375 Annotation: Subtype: Link Rect: 484, 364, 520, 375 Annotation: Subtype: Link Rect: 246, 341, 284, 352 Annotation: Subtype: Link Rect: 327, 318, 364, 329 Annotation: Subtype: Link Rect: 180, 296, 218, 306 Annotation: Subtype: Link Rect: 224, 296, 260, 306 Annotation: Subtype: Link Rect: 109, 273, 146, 283 Annotation: Subtype: Link Rect: 381, 249, 418, 260 Annotation: Subtype: Link Rect: 342, 226, 379, 237 Annotation: Subtype: Link Rect: 154, 203, 191, 214 Annotation: Subtype: Link Rect: 172, 169, 210, 180 Annotation: Subtype: Link Rect: 104, 135, 141, 145 Annotation: Subtype: Link Rect: 148, 135, 184, 145 Annotation: Subtype: Link Rect: 145, 100, 182, 111 Annotation: Subtype: Link Rect: 188, 100, 224, 111 Annotation: Subtype: Link Rect: 113, 66, 151, 76 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 95, 699, 133, 709 Annotation: Subtype: Link Rect: 300, 652, 338, 663 Annotation: Subtype: Link Rect: 211, 630, 249, 640 Annotation: Subtype: Link Rect: 137, 594, 174, 605 Annotation: Subtype: Link Rect: 180, 594, 216, 605 Annotation: Subtype: Link Rect: 271, 560, 308, 571 Annotation: Subtype: Link Rect: 456, 537, 493, 548 Annotation: Subtype: Link Rect: 499, 537, 535, 548 Annotation: Subtype: Link Rect: 376, 514, 413, 525 Annotation: Subtype: Link Rect: 419, 514, 455, 525 Annotation: Subtype: Link Rect: 403, 491, 440, 502 Annotation: Subtype: Link Rect: 479, 468, 515, 479 Annotation: Subtype: Link Rect: 521, 468, 557, 479 Annotation: Subtype: Link Rect: 210, 445, 248, 456 Annotation: Subtype: Link Rect: 254, 445, 290, 456 Annotation: Subtype: Link Rect: 104, 411, 142, 421 Annotation: Subtype: Link Rect: 148, 411, 184, 421 Annotation: Subtype: Link Rect: 374, 387, 412, 398 Annotation: Subtype: Link Rect: 196, 364, 234, 375 Annotation: Subtype: Link Rect: 257, 341, 294, 352 Annotation: Subtype: Link Rect: 301, 341, 337, 352 Annotation: Subtype: Link Rect: 164, 319, 201, 329 Annotation: Subtype: Link Rect: 207, 319, 243, 329 Annotation: Subtype: Link Rect: 185, 284, 222, 295 Annotation: Subtype: Link Rect: 91, 215, 128, 226 Annotation: Subtype: Link Rect: 134, 215, 170, 226 Annotation: Subtype: Link Rect: 353, 192, 391, 203 Annotation: Subtype: Link Rect: 397, 192, 433, 203 Annotation: Subtype: Link Rect: 174, 158, 212, 168 Annotation: Subtype: Link Rect: 218, 158, 254, 168 Annotation: Subtype: Link Rect: 241, 134, 279, 145 Annotation: Subtype: Link Rect: 285, 134, 321, 145 Checksum: 178a0513 Type: CRC32 Checksum: 5e749579a202011b206093f1d2f08c92 Type: MD5 Checksum: 5459f20f2683bc748c7210fbc95eacffa3f665d6 Type: SHA-1