6670f2cd8c896acbc71e397d15151ed0 intjoumol_a2022m12v23n24p15543.pdf 6ebd8dc9ee100d9de9a2fce4f776a6c2018e8390 intjoumol_a2022m12v23n24p15543.pdf 21aa0fa47bad87db0408ade18d4969f9142e3a1656e041b207787f950b9c9b63 intjoumol_a2022m12v23n24p15543.pdf Title: Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol Subject: Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the “major vault protein” (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of “ready-to-use” loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs. Keywords: prokaryotic cell factories; vault particles; protein nanocages; drug-delivery systems; self-assembling protein nanoparticles Author: Roger Fernández, Aida Carreño, Rosa Mendoza, Antoni Benito, Neus Ferrer-Miralles, María Virtudes Céspedes and José Luis Corchero Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Dec 8 10:46:38 2022 CET ModDate: Thu Oct 19 18:41:17 2023 CEST 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: 3223067 bytes Optimized: yes PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 619 0 NOFKYJ+URWPalladioL-Bold Type 1 Custom yes yes no 623 0 SJDBED+URWPalladioL-Roma Type 1 Custom yes yes no 626 0 THVEIZ+URWPalladioL-Ital Type 1 Custom yes yes no 629 0 SFUDNE+URWPalladioL-BoldItal Type 1 Custom yes yes no 632 0 IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes no 635 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 644 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 646 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 526 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 205 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 208 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 211 0 ArialMT TrueType WinAnsi no no no 214 0 Arial-BoldMT TrueType WinAnsi no no no 217 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 227 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 230 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 233 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 239 0 ArialMT TrueType WinAnsi no no no 242 0 Arial-BoldMT TrueType WinAnsi no no no 245 0 ArialMT CID TrueType Identity-H yes no yes 251 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 257 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 260 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 263 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 269 0 ArialMT TrueType WinAnsi no no no 272 0 Arial-BoldMT TrueType WinAnsi no no no 275 0 ArialMT CID TrueType Identity-H yes no yes 281 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 294 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 297 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 300 0 ArialMT TrueType WinAnsi no no no 303 0 Arial-BoldMT TrueType WinAnsi no no no 306 0 BCDKEE+Calibri-Bold TrueType WinAnsi yes yes no 309 0 BCDLEE+Calibri TrueType WinAnsi yes yes no 312 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 324 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 327 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 330 0 ArialMT TrueType WinAnsi no no no 333 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 342 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 345 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 348 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 354 0 BCDKEE+Calibri-Bold TrueType WinAnsi yes yes no 357 0 Arial-BoldMT TrueType WinAnsi no no no 360 0 BJINPL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 368 0 BJINPN+PalatinoLinotype TrueType WinAnsi yes yes no 370 0 BJINPP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 372 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 383 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 386 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 389 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 395 0 ArialMT TrueType WinAnsi no no no 398 0 Arial-BoldMT TrueType WinAnsi no no no 401 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 411 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 414 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 417 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 423 0 ArialMT TrueType WinAnsi no no no 426 0 Arial-BoldMT TrueType WinAnsi no no no 429 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 531 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-20 02:27:18 CEST RepresentationInformation: intjoumol_a2022m12v23n24p15543.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-10-19 18:41:31 CEST Size: 3223067 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Linearized PDF PDFMetadata: Objects: 0 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Children: Item: Title: MVP-H6 Protein Expression and Solubility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: MVP-H6 Protein Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Item: Title: Physico-Chemical Characterization of Recombinant Nanoparticles Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@69b0fd6f Item: Title: GFP-INT Protein Expression and Solubility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@757942a1 Item: Title: Encapsulation and Purification of GFP-INT Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4a87761d Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@66d1af89 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@8646db9 Children: Item: Title: Synthesis and Cloning of Recombinant Genes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@37374a5e Item: Title: Escherichia coli Strains and Expression Plasmids Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4671e53b Item: Title: Recombinant Protein Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@2db7a79b Item: Title: Bacterial Cell Fractionation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6950e31 Item: Title: SDS-PAGE and Western Blot Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@b7dd107 Item: Title: Protein Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@42eca56e Item: Title: Physico-Chemical Characterization of Recombinant Vault-like Nanoparticles Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@52f759d7 Item: Title: GFP-INT Protein Encapsulation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7cbd213e Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@192d3247 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: SJDBED+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: SJDBED+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: THVEIZ+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: THVEIZ+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SFUDNE+URWPalladioL-BoldItal FontSubset: true FirstChar: 69 LastChar: 115 FontDescriptor: FontName: SFUDNE+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IOJCNK+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 189 FontDescriptor: FontName: IOJCNK+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MWHIBA+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: MWHIBA+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YFFKLH+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: YFFKLH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: NOFKYJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: NOFKYJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true XMP: application/pdf Roger Fernández, Aida Carreño, Rosa Mendoza, Antoni Benito, Neus Ferrer-Miralles, María Virtudes Céspedes and José Luis Corchero Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the “major vault protein” (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of “ready-to-use” loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs. Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol 2022-12-08T17:46:38+08:00 LaTeX with hyperref 2023-10-19T18:41:17+02:00 2023-10-19T18:41:17+02:00 prokaryotic cell factories; vault particles; protein nanocages; drug-delivery systems; self-assembling protein nanoparticles pdfTeX-1.40.21 uuid:d5573b83-f8a5-44b2-960f-06feaffce301 uuid:a5e1c821-07ec-4bb3-968a-059b12412a0d Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 86, 335, 152, 344 Annotation: Subtype: Link Rect: 34, 323, 107, 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: 367, 661, 378, 674 Annotation: Subtype: Link Rect: 495, 661, 507, 674 Annotation: Subtype: Link Rect: 162, 648, 173, 662 Annotation: Subtype: Link Rect: 306, 648, 317, 662 Annotation: Subtype: Link Rect: 243, 93, 250, 102 ActionDest: 137 Annotation: Subtype: Link Rect: 251, 93, 258, 102 ActionDest: 137 Annotation: Subtype: Link Rect: 514, 67, 521, 76 ActionDest: 137 Annotation: Subtype: Link Rect: 143, 17, 286, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 432, 87, 451 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 289, 698, 296, 707 ActionDest: 137 Annotation: Subtype: Link Rect: 296, 698, 303, 707 ActionDest: 137 Annotation: Subtype: Link Rect: 541, 673, 548, 682 ActionDest: 137 Annotation: Subtype: Link Rect: 549, 673, 556, 682 ActionDest: 140 Annotation: Subtype: Link Rect: 446, 635, 453, 644 ActionDest: 140 Annotation: Subtype: Link Rect: 322, 610, 329, 619 ActionDest: 140 Annotation: Subtype: Link Rect: 505, 597, 517, 606 ActionDest: 140 Annotation: Subtype: Link Rect: 329, 572, 341, 581 ActionDest: 140 Annotation: Subtype: Link Rect: 224, 547, 236, 556 ActionDest: 140 Annotation: Subtype: Link Rect: 237, 547, 249, 556 ActionDest: 140 Annotation: Subtype: Link Rect: 355, 535, 367, 544 ActionDest: 140 Annotation: Subtype: Link Rect: 323, 522, 335, 531 ActionDest: 140 Annotation: Subtype: Link Rect: 537, 509, 549, 519 ActionDest: 140 Annotation: Subtype: Link Rect: 458, 472, 470, 481 ActionDest: 140 Annotation: Subtype: Link Rect: 471, 472, 483, 481 ActionDest: 140 Annotation: Subtype: Link Rect: 366, 434, 378, 443 ActionDest: 140 Annotation: Subtype: Link Rect: 206, 396, 218, 406 ActionDest: 140 Annotation: Subtype: Link Rect: 338, 384, 350, 393 ActionDest: 140 Annotation: Subtype: Link Rect: 231, 346, 242, 355 ActionDest: 140 Annotation: Subtype: Link Rect: 227, 321, 239, 330 ActionDest: 140 Annotation: Subtype: Link Rect: 235, 296, 247, 305 ActionDest: 140 Annotation: Subtype: Link Rect: 250, 296, 262, 305 ActionDest: 140 Annotation: Subtype: Link Rect: 343, 296, 355, 305 ActionDest: 140 Annotation: Subtype: Link Rect: 356, 296, 368, 305 ActionDest: 140 Annotation: Subtype: Link Rect: 523, 296, 535, 305 ActionDest: 140 Annotation: Subtype: Link Rect: 344, 283, 356, 293 ActionDest: 140 Annotation: Subtype: Link Rect: 435, 283, 447, 293 ActionDest: 140 Annotation: Subtype: Link Rect: 430, 271, 442, 280 ActionDest: 140 Annotation: Subtype: Link Rect: 472, 258, 484, 267 ActionDest: 140 Annotation: Subtype: Link Rect: 404, 233, 416, 242 ActionDest: 140 Annotation: Subtype: Link Rect: 544, 233, 556, 242 ActionDest: 140 Annotation: Subtype: Link Rect: 311, 183, 323, 192 ActionDest: 140 Annotation: Subtype: Link Rect: 441, 158, 453, 167 ActionDest: 140 Annotation: Subtype: Link Rect: 329, 120, 341, 129 ActionDest: 140 Annotation: Subtype: Link Rect: 442, 83, 454, 92 ActionDest: 143 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 201, 710, 213, 719 ActionDest: 140 Annotation: Subtype: Link Rect: 213, 710, 225, 719 ActionDest: 143 Annotation: Subtype: Link Rect: 464, 531, 471, 543 ActionDest: 4 Annotation: Subtype: Link Rect: 366, 243, 373, 255 ActionDest: 17 Annotation: Subtype: Link Rect: 391, 230, 398, 242 ActionDest: 17 Annotation: Subtype: Link Rect: 542, 218, 549, 230 ActionDest: 17 Annotation: Subtype: Link Rect: 232, 155, 239, 167 ActionDest: 17 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 443, 122, 455, 131 ActionDest: 143 Annotation: Subtype: Link Rect: 550, 94, 557, 106 ActionDest: 54 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 255, 337, 262, 349 ActionDest: 54 Annotation: Subtype: Link Rect: 199, 287, 206, 299 ActionDest: 54 Annotation: Subtype: Link Rect: 351, 249, 358, 261 ActionDest: 54 Annotation: Subtype: Link Rect: 208, 174, 215, 186 ActionDest: 54 Annotation: Subtype: Link Rect: 551, 149, 558, 161 ActionDest: 68 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 474, 357, 482, 369 ActionDest: 78 Annotation: Subtype: Link Rect: 528, 345, 535, 357 ActionDest: 78 Annotation: Subtype: Link Rect: 396, 272, 408, 281 ActionDest: 143 Annotation: Subtype: Link Rect: 383, 184, 395, 193 ActionDest: 143 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 325, 359, 332, 371 ActionDest: 78 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 496, 720, 503, 732 ActionDest: 78 Annotation: Subtype: Link Rect: 314, 544, 321, 556 ActionDest: 103 Annotation: Subtype: Link Rect: 232, 74, 240, 86 ActionDest: 114 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 280, 210, 292, 219 ActionDest: 140 Annotation: Subtype: Link Rect: 190, 197, 202, 207 ActionDest: 140 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 412, 735, 424, 744 ActionDest: 140 Annotation: Subtype: Link Rect: 389, 597, 401, 606 ActionDest: 143 Annotation: Subtype: Link Rect: 402, 597, 414, 606 ActionDest: 143 Annotation: Subtype: Link Rect: 543, 585, 555, 594 ActionDest: 143 Annotation: Subtype: Link Rect: 544, 572, 556, 581 ActionDest: 143 Annotation: Subtype: Link Rect: 298, 560, 310, 569 ActionDest: 143 Annotation: Subtype: Link Rect: 245, 547, 257, 556 ActionDest: 143 Annotation: Subtype: Link Rect: 212, 422, 224, 431 ActionDest: 140 Annotation: Subtype: Link Rect: 455, 271, 467, 280 ActionDest: 143 Annotation: Subtype: Link Rect: 425, 196, 437, 205 ActionDest: 140 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 495, 673, 507, 682 ActionDest: 143 Annotation: Subtype: Link Rect: 507, 673, 519, 682 ActionDest: 143 Annotation: Subtype: Link Rect: 404, 660, 416, 669 ActionDest: 143 Annotation: Subtype: Link Rect: 209, 623, 221, 631 ActionDest: 143 Annotation: Subtype: Link Rect: 406, 622, 418, 631 ActionDest: 140 Annotation: Subtype: Link Rect: 293, 610, 305, 619 ActionDest: 143 Annotation: Subtype: Link Rect: 413, 547, 425, 556 ActionDest: 143 Annotation: Subtype: Link Rect: 425, 547, 437, 556 ActionDest: 143 Annotation: Subtype: Link Rect: 265, 107, 277, 116 ActionDest: 143 Page: Sequence: 12 Page: Sequence: 13 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 336, 262, 560, 273 Annotation: Subtype: Link Rect: 165, 250, 226, 261 Annotation: Subtype: Link Rect: 59, 185, 96, 195 Annotation: Subtype: Link Rect: 102, 185, 138, 195 Annotation: Subtype: Link Rect: 297, 161, 335, 172 Annotation: Subtype: Link Rect: 341, 161, 377, 172 Annotation: Subtype: Link Rect: 413, 138, 451, 149 Annotation: Subtype: Link Rect: 457, 138, 493, 149 Annotation: Subtype: Link Rect: 59, 104, 96, 115 Annotation: Subtype: Link Rect: 95, 81, 132, 92 Annotation: Subtype: Link Rect: 307, 58, 344, 68 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 474, 722, 512, 733 Annotation: Subtype: Link Rect: 506, 710, 543, 721 Annotation: Subtype: Link Rect: 104, 688, 142, 698 Annotation: Subtype: Link Rect: 59, 665, 96, 675 Annotation: Subtype: Link Rect: 108, 642, 146, 652 Annotation: Subtype: Link Rect: 152, 642, 188, 652 Annotation: Subtype: Link Rect: 242, 618, 280, 629 Annotation: Subtype: Link Rect: 286, 618, 322, 629 Annotation: Subtype: Link Rect: 271, 595, 308, 606 Annotation: Subtype: Link Rect: 314, 595, 350, 606 Annotation: Subtype: Link Rect: 59, 573, 96, 583 Annotation: Subtype: Link Rect: 102, 573, 138, 583 Annotation: Subtype: Link Rect: 307, 549, 345, 560 Annotation: Subtype: Link Rect: 351, 549, 387, 560 Annotation: Subtype: Link Rect: 266, 526, 303, 537 Annotation: Subtype: Link Rect: 316, 503, 353, 514 Annotation: Subtype: Link Rect: 95, 481, 133, 491 Annotation: Subtype: Link Rect: 387, 457, 424, 468 Annotation: Subtype: Link Rect: 104, 423, 142, 433 Annotation: Subtype: Link Rect: 113, 389, 150, 399 Annotation: Subtype: Link Rect: 156, 389, 192, 399 Annotation: Subtype: Link Rect: 455, 365, 492, 376 Annotation: Subtype: Link Rect: 498, 365, 534, 376 Annotation: Subtype: Link Rect: 396, 342, 433, 353 Annotation: Subtype: Link Rect: 440, 342, 476, 353 Annotation: Subtype: Link Rect: 189, 308, 227, 318 Annotation: Subtype: Link Rect: 499, 284, 536, 295 Annotation: Subtype: Link Rect: 501, 261, 539, 272 Annotation: Subtype: Link Rect: 338, 238, 376, 249 Annotation: Subtype: Link Rect: 260, 204, 297, 215 Annotation: Subtype: Link Rect: 484, 181, 522, 192 Annotation: Subtype: Link Rect: 185, 158, 222, 169 Annotation: Subtype: Link Rect: 217, 135, 255, 146 Annotation: Subtype: Link Rect: 261, 135, 297, 146 Annotation: Subtype: Link Rect: 59, 113, 96, 123 Annotation: Subtype: Link Rect: 102, 113, 138, 123 Annotation: Subtype: Link Rect: 389, 89, 427, 100 Annotation: Subtype: Link Rect: 433, 89, 469, 100 Annotation: Subtype: Link Rect: 239, 66, 277, 77 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 477, 722, 515, 733 Annotation: Subtype: Link Rect: 521, 722, 557, 733 Annotation: Subtype: Link Rect: 388, 710, 425, 721 Annotation: Subtype: Link Rect: 431, 710, 467, 721 Annotation: Subtype: Link Rect: 59, 676, 96, 687 Annotation: Subtype: Link Rect: 337, 652, 375, 664 Annotation: Subtype: Link Rect: 308, 607, 345, 617 Annotation: Subtype: Link Rect: 174, 584, 211, 594 Annotation: Subtype: Link Rect: 217, 584, 253, 594 Annotation: Subtype: Link Rect: 59, 561, 96, 571 Annotation: Subtype: Link Rect: 520, 549, 557, 560 Annotation: Subtype: Link Rect: 59, 538, 95, 548 Annotation: Subtype: Link Rect: 323, 515, 361, 525 Annotation: Subtype: Link Rect: 367, 515, 403, 525 Annotation: Subtype: Link Rect: 366, 491, 403, 502 Annotation: Subtype: Link Rect: 510, 480, 548, 491 Annotation: Subtype: Link Rect: 394, 457, 431, 468 Annotation: Subtype: Link Rect: 210, 422, 247, 433 Annotation: Subtype: Link Rect: 214, 400, 252, 410 Annotation: Subtype: Link Rect: 173, 377, 210, 387 Checksum: f59dfc41 Type: CRC32 Checksum: 6670f2cd8c896acbc71e397d15151ed0 Type: MD5 Checksum: 6ebd8dc9ee100d9de9a2fce4f776a6c2018e8390 Type: SHA-1