b3a731d908690dbc20324a022873be98 pmc_35008522.pdf 39ea8ace14ca18a88eddedcc3116716df1ee539f pmc_35008522.pdf 40e3e3a72670cc0220fe6c6c864b5834455048cb67f0c6da6d501f018d043094 pmc_35008522.pdf Title: Structure-Based Design of an RNase Chimera for Antimicrobial Therapy Subject: Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs’ ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants’ capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics. Keywords: RNase; protein engineering; structure-function relationship; antimicrobial proteins Author: Guillem Prats-Ejarque, Helena Lorente, Clara Villalba, Raúl Anguita, Lu Lu, Sergi Vázquez-Monteagudo, Pablo Fernández-Millán and Ester Boix Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Dec 22 14:03:06 2021 CET ModDate: Wed Dec 22 14:24:16 2021 CET 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: 26835770 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FYYAHQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 HZCUWH+VnURWPalladioL Type 1 Custom yes yes no 15 0 CLSONJ+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 LPHLZC+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 QSQCLT+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 36 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 40 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 44 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 56 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 59 0 RGUFCP+CMSY10 Type 1 Builtin yes yes no 104 0 GVJUWD+EURM10 Type 1 Builtin yes yes no 108 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 123 0 DDWGJY+URWPalladioL-BoldItal Type 1 Custom yes yes no 127 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 156 0 IYAOHD+PazoMath Type 1 Builtin yes yes no 192 0 IUDKMW+VnURWPalladioL-Italic Type 1 Custom yes yes no 226 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-10 02:52:51 CET RepresentationInformation: pmc_35008522.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-17 03:37:25 CET Size: 26835770 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 531 FreeObjects: 19 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: RNases 3/1-v2 and v3 Rational Design Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Catalytic Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Bactericidal Activity and Cytotoxicity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Lipopolysaccharide Binding and Liposome Leakage Activities Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Interaction with the RNase Inhibitor (RI) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: RNase 3/1 Autophagy Induction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Protein Expression and Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Mutagenesis and Construction of RNase 3/1 Chimera Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Molecular Dynamic Simulations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Circular Dichroism (CD) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Activity Staining Gel Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Minimum Bactericidal Concentration (MBC) Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Cytotoxicity Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: LPS Binding Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Liposome Preparation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Liposome Leakage Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Spectrophotometric Kinetic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Ribonuclease Inhibitor Activity Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Macrophage Cell Culture, Infection and Intracellular MIC Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Real-Time qPCR Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Acridine Orange Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Intracellular Aggregates Formation Measurement by Thioflavin-S Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Info: Title: Structure-Based Design of an RNase Chimera for Antimicrobial Therapy Author: Guillem Prats-Ejarque, Helena Lorente, Clara Villalba, Raúl Anguita, Lu Lu, Sergi Vázquez-Monteagudo, Pablo Fernández-Millán and Ester Boix Subject: Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs’ ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants’ capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics. Keywords: RNase; protein engineering; structure-function relationship; antimicrobial proteins Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Dec 22 14:03:06 CET 2021 ModDate: Wed Dec 22 14:24:16 CET 2021 ID: 0xd14dba6982f3cd3fbfb37c83844a7763, 0xd14dba6982f3cd3fbfb37c83844a7763 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: IYAOHD+PazoMath FontSubset: true FirstChar: 68 LastChar: 89 FontDescriptor: FontName: IYAOHD+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: IUDKMW+VnURWPalladioL-Italic FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: IUDKMW+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CLSONJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: CLSONJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LPHLZC+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: LPHLZC+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: RGUFCP+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: RGUFCP+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: FYYAHQ+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 243 FontDescriptor: FontName: FYYAHQ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: QSQCLT+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 153 FontDescriptor: FontName: QSQCLT+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: GVJUWD+EURM10 FontSubset: true FirstChar: 11 LastChar: 34 FontDescriptor: FontName: GVJUWD+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: CRJHCU+PazoMath-Italic FontSubset: true FirstChar: 98 LastChar: 98 FontDescriptor: FontName: CRJHCU+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: HZCUWH+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: HZCUWH+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DDWGJY+URWPalladioL-BoldItal FontSubset: true FirstChar: 46 LastChar: 117 FontDescriptor: FontName: DDWGJY+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 88, 335, 152, 344 Annotation: Subtype: Link Rect: 34, 323, 103, 332 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 150, 656, 161, 670 Annotation: Subtype: Link Rect: 448, 656, 459, 670 Annotation: Subtype: Link Rect: 372, 644, 383, 657 Annotation: Subtype: Link Rect: 279, 201, 286, 210 ActionDest: 223 Annotation: Subtype: Link Rect: 360, 189, 367, 198 ActionDest: 223 Annotation: Subtype: Link Rect: 370, 189, 377, 198 ActionDest: 223 Annotation: Subtype: Link Rect: 445, 164, 452, 173 ActionDest: 223 Annotation: Subtype: Link Rect: 455, 164, 467, 173 ActionDest: 223 Annotation: Subtype: Link Rect: 193, 139, 205, 148 ActionDest: 223 Annotation: Subtype: Link Rect: 208, 139, 220, 148 ActionDest: 223 Annotation: Subtype: Link Rect: 444, 126, 456, 135 ActionDest: 223 Annotation: Subtype: Link Rect: 459, 126, 471, 135 ActionDest: 223 Annotation: Subtype: Link Rect: 531, 113, 543, 122 ActionDest: 230 Annotation: Subtype: Link Rect: 544, 113, 556, 122 ActionDest: 230 Annotation: Subtype: Link Rect: 348, 88, 360, 97 ActionDest: 230 Annotation: Subtype: Link Rect: 131, 17, 270, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 420, 86, 439 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 362, 635, 374, 644 ActionDest: 230 Annotation: Subtype: Link Rect: 301, 533, 313, 542 ActionDest: 230 Annotation: Subtype: Link Rect: 249, 508, 261, 517 ActionDest: 223 Annotation: Subtype: Link Rect: 262, 508, 273, 517 ActionDest: 230 Annotation: Subtype: Link Rect: 453, 508, 465, 517 ActionDest: 230 Annotation: Subtype: Link Rect: 468, 508, 480, 517 ActionDest: 230 Annotation: Subtype: Link Rect: 204, 483, 216, 492 ActionDest: 230 Annotation: Subtype: Link Rect: 216, 483, 228, 492 ActionDest: 230 Annotation: Subtype: Link Rect: 380, 421, 392, 429 ActionDest: 223 Annotation: Subtype: Link Rect: 403, 408, 415, 417 ActionDest: 230 Annotation: Subtype: Link Rect: 450, 380, 457, 392 ActionDest: 78 Annotation: Subtype: Link Rect: 516, 154, 527, 163 ActionDest: 230 Annotation: Subtype: Link Rect: 365, 77, 376, 86 ActionDest: 230 Annotation: Subtype: Link Rect: 168, 61, 296, 73 Annotation: Subtype: Link Rect: 302, 65, 313, 73 ActionDest: 230 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 549, 695, 556, 707 ActionDest: 89 Annotation: Subtype: Link Rect: 182, 298, 194, 307 ActionDest: 230 Annotation: Subtype: Link Rect: 293, 207, 300, 219 ActionDest: 100 Annotation: Subtype: Link Rect: 190, 120, 197, 132 ActionDest: 100 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 399, 245, 406, 257 ActionDest: 119 Annotation: Subtype: Link Rect: 428, 170, 435, 182 ActionDest: 119 Annotation: Subtype: Link Rect: 483, 120, 490, 132 ActionDest: 119 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 545, 634, 556, 642 ActionDest: 230 Annotation: Subtype: Link Rect: 374, 65, 385, 73 ActionDest: 230 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 189, 695, 201, 704 ActionDest: 230 Annotation: Subtype: Link Rect: 421, 682, 433, 691 ActionDest: 230 Annotation: Subtype: Link Rect: 279, 654, 286, 666 ActionDest: 138 Annotation: Subtype: Link Rect: 358, 496, 365, 508 ActionDest: 138 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 454, 707, 466, 716 ActionDest: 230 Annotation: Subtype: Link Rect: 224, 682, 236, 691 ActionDest: 230 Annotation: Subtype: Link Rect: 210, 657, 222, 666 ActionDest: 230 Annotation: Subtype: Link Rect: 291, 441, 298, 453 ActionDest: 141 Annotation: Subtype: Link Rect: 287, 99, 299, 108 ActionDest: 230 Annotation: Subtype: Link Rect: 300, 99, 312, 108 ActionDest: 230 Annotation: Subtype: Link Rect: 518, 73, 530, 83 ActionDest: 230 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 254, 670, 261, 682 ActionDest: 152 Annotation: Subtype: Link Rect: 336, 323, 348, 332 ActionDest: 230 Annotation: Subtype: Link Rect: 522, 310, 534, 320 ActionDest: 230 Annotation: Subtype: Link Rect: 435, 260, 447, 269 ActionDest: 223 Annotation: Subtype: Link Rect: 447, 260, 459, 269 ActionDest: 223 Annotation: Subtype: Link Rect: 460, 260, 472, 269 ActionDest: 230 Annotation: Subtype: Link Rect: 402, 233, 409, 245 ActionDest: 167 Annotation: Subtype: Link Rect: 549, 207, 556, 219 ActionDest: 167 Annotation: Subtype: Link Rect: 471, 160, 483, 169 ActionDest: 230 Annotation: Subtype: Link Rect: 206, 85, 218, 94 ActionDest: 230 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 213, 341, 224, 350 ActionDest: 230 Annotation: Subtype: Link Rect: 495, 229, 502, 241 ActionDest: 178 Annotation: Subtype: Link Rect: 194, 206, 206, 215 ActionDest: 230 Annotation: Subtype: Link Rect: 207, 206, 219, 215 ActionDest: 230 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 280, 412, 292, 421 ActionDest: 230 Annotation: Subtype: Link Rect: 457, 334, 464, 346 ActionDest: 78 Annotation: Subtype: Link Rect: 355, 324, 367, 333 ActionDest: 230 Annotation: Subtype: Link Rect: 487, 274, 499, 283 ActionDest: 230 Annotation: Subtype: Link Rect: 469, 246, 476, 258 ActionDest: 119 Annotation: Subtype: Link Rect: 274, 208, 281, 220 ActionDest: 89 Annotation: Subtype: Link Rect: 392, 108, 399, 120 ActionDest: 119 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 353, 698, 365, 707 ActionDest: 230 Annotation: Subtype: Link Rect: 361, 657, 368, 669 ActionDest: 141 Annotation: Subtype: Link Rect: 293, 547, 304, 556 ActionDest: 230 Annotation: Subtype: Link Rect: 305, 547, 317, 556 ActionDest: 233 Annotation: Subtype: Link Rect: 459, 544, 466, 556 ActionDest: 141 Annotation: Subtype: Link Rect: 304, 484, 316, 493 ActionDest: 233 Annotation: Subtype: Link Rect: 199, 381, 206, 393 ActionDest: 100 Annotation: Subtype: Link Rect: 252, 381, 259, 393 ActionDest: 100 Annotation: Subtype: Link Rect: 550, 369, 557, 381 ActionDest: 100 Annotation: Subtype: Link Rect: 207, 309, 214, 318 ActionDest: 223 Annotation: Subtype: Link Rect: 215, 309, 227, 318 ActionDest: 233 Annotation: Subtype: Link Rect: 227, 309, 239, 318 ActionDest: 233 Annotation: Subtype: Link Rect: 367, 246, 379, 255 ActionDest: 230 Annotation: Subtype: Link Rect: 229, 206, 236, 218 ActionDest: 152 Annotation: Subtype: Link Rect: 352, 193, 359, 205 ActionDest: 78 Annotation: Subtype: Link Rect: 488, 180, 495, 192 ActionDest: 167 Annotation: Subtype: Link Rect: 434, 95, 446, 104 ActionDest: 233 Annotation: Subtype: Link Rect: 449, 95, 461, 104 ActionDest: 233 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 385, 723, 397, 732 ActionDest: 230 Annotation: Subtype: Link Rect: 398, 723, 409, 732 ActionDest: 230 Annotation: Subtype: Link Rect: 430, 670, 437, 682 ActionDest: 178 Annotation: Subtype: Link Rect: 439, 647, 451, 657 ActionDest: 230 Annotation: Subtype: Link Rect: 468, 622, 480, 631 ActionDest: 230 Annotation: Subtype: Link Rect: 480, 622, 492, 631 ActionDest: 233 Annotation: Subtype: Link Rect: 360, 597, 372, 606 ActionDest: 233 Annotation: Subtype: Link Rect: 326, 572, 338, 581 ActionDest: 230 Annotation: Subtype: Link Rect: 339, 572, 351, 581 ActionDest: 233 Annotation: Subtype: Link Rect: 483, 547, 495, 556 ActionDest: 230 Annotation: Subtype: Link Rect: 361, 535, 373, 544 ActionDest: 233 Annotation: Subtype: Link Rect: 196, 457, 202, 469 ActionDest: 178 Annotation: Subtype: Link Rect: 353, 447, 365, 456 ActionDest: 233 Annotation: Subtype: Link Rect: 249, 422, 261, 431 ActionDest: 233 Annotation: Subtype: Link Rect: 261, 422, 273, 431 ActionDest: 233 Annotation: Subtype: Link Rect: 487, 394, 494, 406 ActionDest: 167 Annotation: Subtype: Link Rect: 501, 396, 513, 406 ActionDest: 230 Annotation: Subtype: Link Rect: 415, 321, 426, 330 ActionDest: 223 Annotation: Subtype: Link Rect: 424, 281, 431, 293 ActionDest: 119 Annotation: Subtype: Link Rect: 203, 183, 215, 192 ActionDest: 230 Annotation: Subtype: Link Rect: 247, 155, 254, 167 ActionDest: 200 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 436, 228, 448, 237 ActionDest: 230 Annotation: Subtype: Link Rect: 296, 178, 308, 187 ActionDest: 233 Annotation: Subtype: Link Rect: 335, 115, 347, 124 ActionDest: 233 Annotation: Subtype: Link Rect: 470, 115, 482, 124 ActionDest: 233 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 218, 534, 230, 543 ActionDest: 230 Annotation: Subtype: Link Rect: 516, 371, 528, 380 ActionDest: 233 Annotation: Subtype: Link Rect: 255, 358, 267, 367 ActionDest: 233 Annotation: Subtype: Link Rect: 196, 333, 207, 342 ActionDest: 233 Annotation: Subtype: Link Rect: 442, 321, 454, 330 ActionDest: 233 Annotation: Subtype: Link Rect: 399, 295, 411, 305 ActionDest: 233 Annotation: Subtype: Link Rect: 411, 295, 423, 305 ActionDest: 233 Annotation: Subtype: Link Rect: 256, 270, 268, 279 ActionDest: 233 Annotation: Subtype: Link Rect: 545, 258, 557, 267 ActionDest: 233 Annotation: Subtype: Link Rect: 544, 233, 556, 242 ActionDest: 233 Annotation: Subtype: Link Rect: 246, 208, 258, 217 ActionDest: 233 Annotation: Subtype: Link Rect: 258, 208, 270, 217 ActionDest: 233 Annotation: Subtype: Link Rect: 544, 157, 556, 166 ActionDest: 230 Annotation: Subtype: Link Rect: 382, 94, 394, 103 ActionDest: 233 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 509, 720, 521, 729 ActionDest: 233 Annotation: Subtype: Link Rect: 265, 456, 277, 465 ActionDest: 230 Annotation: Subtype: Link Rect: 273, 280, 285, 289 ActionDest: 230 Annotation: Subtype: Link Rect: 419, 229, 431, 238 ActionDest: 230 Annotation: Subtype: Link Rect: 202, 66, 214, 75 ActionDest: 233 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 389, 560, 401, 568 ActionDest: 233 Annotation: Subtype: Link Rect: 311, 396, 323, 405 ActionDest: 236 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 412, 723, 424, 732 ActionDest: 230 Annotation: Subtype: Link Rect: 544, 547, 556, 556 ActionDest: 230 Annotation: Subtype: Link Rect: 339, 497, 351, 506 ActionDest: 236 Annotation: Subtype: Link Rect: 483, 446, 495, 455 ActionDest: 236 Annotation: Subtype: Link Rect: 176, 308, 188, 317 ActionDest: 236 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 419, 733, 560, 744 Annotation: Subtype: Link Rect: 165, 721, 259, 732 Annotation: Subtype: Link Rect: 189, 430, 227, 440 Annotation: Subtype: Link Rect: 233, 430, 269, 440 Annotation: Subtype: Link Rect: 278, 406, 316, 417 Annotation: Subtype: Link Rect: 322, 406, 358, 417 Annotation: Subtype: Link Rect: 59, 384, 96, 394 Annotation: Subtype: Link Rect: 519, 372, 557, 383 Annotation: Subtype: Link Rect: 59, 361, 95, 371 Annotation: Subtype: Link Rect: 478, 337, 515, 348 Annotation: Subtype: Link Rect: 521, 337, 557, 348 Annotation: Subtype: Link Rect: 227, 314, 265, 325 Annotation: Subtype: Link Rect: 248, 291, 285, 302 Annotation: Subtype: Link Rect: 113, 269, 151, 279 Annotation: Subtype: Link Rect: 283, 245, 320, 256 Annotation: Subtype: Link Rect: 435, 222, 473, 233 Annotation: Subtype: Link Rect: 479, 222, 515, 233 Annotation: Subtype: Link Rect: 473, 199, 510, 210 Annotation: Subtype: Link Rect: 516, 199, 552, 210 Annotation: Subtype: Link Rect: 170, 177, 208, 187 Annotation: Subtype: Link Rect: 214, 177, 250, 187 Annotation: Subtype: Link Rect: 238, 153, 276, 164 Annotation: Subtype: Link Rect: 511, 130, 549, 141 Annotation: Subtype: Link Rect: 496, 107, 533, 118 Annotation: Subtype: Link Rect: 520, 84, 557, 95 Annotation: Subtype: Link Rect: 248, 62, 285, 72 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 59, 711, 96, 721 Annotation: Subtype: Link Rect: 465, 687, 503, 698 Annotation: Subtype: Link Rect: 509, 687, 545, 698 Annotation: Subtype: Link Rect: 59, 653, 96, 664 Annotation: Subtype: Link Rect: 227, 630, 264, 640 Annotation: Subtype: Link Rect: 520, 606, 557, 617 Annotation: Subtype: Link Rect: 59, 596, 95, 606 Annotation: Subtype: Link Rect: 350, 572, 387, 583 Annotation: Subtype: Link Rect: 394, 572, 430, 583 Annotation: Subtype: Link Rect: 431, 549, 468, 560 Annotation: Subtype: Link Rect: 475, 549, 511, 560 Annotation: Subtype: Link Rect: 267, 526, 304, 537 Annotation: Subtype: Link Rect: 310, 526, 346, 537 Annotation: Subtype: Link Rect: 479, 503, 515, 514 Annotation: Subtype: Link Rect: 521, 503, 557, 514 Annotation: Subtype: Link Rect: 201, 480, 238, 491 Annotation: Subtype: Link Rect: 342, 457, 380, 468 Annotation: Subtype: Link Rect: 333, 434, 371, 445 Annotation: Subtype: Link Rect: 59, 412, 96, 422 Annotation: Subtype: Link Rect: 59, 389, 96, 399 Annotation: Subtype: Link Rect: 95, 366, 133, 376 Annotation: Subtype: Link Rect: 350, 342, 387, 353 Annotation: Subtype: Link Rect: 393, 342, 429, 353 Annotation: Subtype: Link Rect: 354, 319, 392, 330 Annotation: Subtype: Link Rect: 398, 319, 434, 330 Annotation: Subtype: Link Rect: 127, 297, 164, 307 Annotation: Subtype: Link Rect: 170, 297, 206, 307 Annotation: Subtype: Link Rect: 321, 273, 358, 284 Annotation: Subtype: Link Rect: 365, 273, 401, 284 Annotation: Subtype: Link Rect: 169, 238, 207, 249 Annotation: Subtype: Link Rect: 337, 215, 375, 226 Annotation: Subtype: Link Rect: 59, 193, 96, 203 Annotation: Subtype: Link Rect: 220, 169, 257, 180 Annotation: Subtype: Link Rect: 104, 136, 142, 146 Annotation: Subtype: Link Rect: 148, 136, 184, 146 Annotation: Subtype: Link Rect: 248, 100, 285, 111 Annotation: Subtype: Link Rect: 291, 100, 327, 111 Annotation: Subtype: Link Rect: 201, 78, 238, 88 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 131, 711, 168, 721 Annotation: Subtype: Link Rect: 174, 711, 211, 721 Annotation: Subtype: Link Rect: 135, 688, 173, 698 Annotation: Subtype: Link Rect: 177, 642, 215, 652 Annotation: Subtype: Link Rect: 221, 642, 257, 652 Annotation: Subtype: Link Rect: 426, 629, 463, 640 Annotation: Subtype: Link Rect: 423, 606, 460, 617 Annotation: Subtype: Link Rect: 484, 583, 521, 594 Annotation: Subtype: Link Rect: 59, 561, 96, 571 Annotation: Subtype: Link Rect: 59, 538, 96, 548 Annotation: Subtype: Link Rect: 102, 538, 138, 548 Annotation: Subtype: Link Rect: 356, 515, 394, 525 Annotation: Subtype: Link Rect: 277, 480, 314, 491 Annotation: Subtype: Link Rect: 321, 480, 357, 491 Annotation: Subtype: Link Rect: 268, 457, 305, 468 Annotation: Subtype: Link Rect: 311, 457, 347, 468 Annotation: Subtype: Link Rect: 410, 434, 447, 445 Annotation: Subtype: Link Rect: 59, 400, 96, 410 Annotation: Subtype: Link Rect: 430, 376, 467, 387 Annotation: Subtype: Link Rect: 285, 353, 323, 364 Annotation: Subtype: Link Rect: 104, 331, 142, 341 Annotation: Subtype: Link Rect: 148, 308, 186, 318 Annotation: Subtype: Link Rect: 167, 284, 205, 295 Annotation: Subtype: Link Rect: 520, 273, 557, 284 Annotation: Subtype: Link Rect: 59, 262, 95, 272 Annotation: Subtype: Link Rect: 221, 239, 258, 249 Annotation: Subtype: Link Rect: 520, 227, 557, 238 Annotation: Subtype: Link Rect: 515, 215, 552, 226 Annotation: Subtype: Link Rect: 104, 193, 142, 203 Annotation: Subtype: Link Rect: 95, 170, 133, 180 Annotation: Subtype: Link Rect: 162, 147, 199, 157 Annotation: Subtype: Link Rect: 206, 147, 242, 157 Annotation: Subtype: Link Rect: 149, 113, 187, 123 Annotation: Subtype: Link Rect: 512, 89, 550, 100 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 520, 722, 557, 733 Annotation: Subtype: Link Rect: 59, 711, 95, 721 Annotation: Subtype: Link Rect: 165, 688, 202, 698 Annotation: Subtype: Link Rect: 208, 688, 244, 698 Annotation: Subtype: Link Rect: 95, 653, 133, 664 Annotation: Subtype: Link Rect: 139, 653, 175, 664 Annotation: Subtype: Link Rect: 241, 629, 278, 640 Annotation: Subtype: Link Rect: 284, 629, 320, 640 Checksum: 4e686da1 Type: CRC32 Checksum: b3a731d908690dbc20324a022873be98 Type: MD5 Checksum: 39ea8ace14ca18a88eddedcc3116716df1ee539f Type: SHA-1