d1857160eb0b0b0f3af04c541a33111e 302133.pdf 63755a553b133c2e9b94c820563beca328df348d 302133.pdf 30e858e4cf483182dc861b4c749d393e0869682b65be1d6c27c79de22043c45c 302133.pdf Title: Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins Subject: Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1 branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress. Keywords: sirtuin; Hsp70; STAT3; ER stress Author: Jessica Latorre, Nuria de Vera, Tomàs Santalucía, Rafel Balada, Anna Marazuela-Duque, Alejandro Vaquero, Anna M. Planas and Valérie Petegnief Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Mar 5 10:35:42 2024 CET ModDate: Tue Mar 5 10:47:38 2024 CET Custom Metadata: no Metadata Stream: no 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: 1884000 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GGHKEL+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 KESBIZ+EURM10 Type 1 Builtin yes yes yes 16 0 RSNLEP+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 ELFSWA+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 ZLMWBR+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 YFFKLH+CMSY10 Type 1 Builtin yes yes yes 37 0 DUFXDP+VnURWPalladioL Type 1 Custom yes yes yes 42 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 58 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 61 0 IJOLBE+PalatinoLinotype,Italic TrueType MacRoman yes yes no 84 0 IJOJAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 87 0 IJOJAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 90 0 IJOJAK+PalatinoLinotype TrueType WinAnsi yes yes no 96 0 IJOJAM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 99 0 IJOKAJ+PalatinoLinotype TrueType MacRoman yes yes no 102 0 IJOJAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 112 0 IJOJAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 115 0 IJOJAK+PalatinoLinotype TrueType WinAnsi yes yes no 121 0 IJOJAL+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 124 0 IJOJAM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 130 0 IJOKAJ+PalatinoLinotype TrueType MacRoman yes yes no 133 0 IJOJAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 143 0 IJOJAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 146 0 IJOJAK+PalatinoLinotype TrueType WinAnsi yes yes no 152 0 IJOJAM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 155 0 IJOKAJ+PalatinoLinotype TrueType MacRoman yes yes no 158 0 IJOJAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 168 0 IJOJAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 171 0 IJOJAK+PalatinoLinotype TrueType WinAnsi yes yes no 177 0 IJOJAL+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 180 0 IJOJAM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 186 0 IJOKAJ+PalatinoLinotype TrueType MacRoman yes yes no 189 0 IJOJAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 199 0 IJOJAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 202 0 IJOJAK+PalatinoLinotype TrueType WinAnsi yes yes no 208 0 IJOJAM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 211 0 IJOKAJ+PalatinoLinotype TrueType MacRoman yes yes no 214 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 230 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-11-12 02:44:41 CET RepresentationInformation: 302133.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-11-11 09:49:46 CET Size: 1884000 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 442 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: SIRT1-Deficient Cells Were Less Vulnerable to ER Stress Than wt Cells but Developed Similar UPR, and Resistance Was Independent of mTOR Hyperactivity Destination: subsection.2.1 Item: Title: SIRT1-/- Cells Constitutively Express Heat Shock Proteins Destination: subsection.2.2 Item: Title: Hsp70 Silencing Abolished SIRT1 Resistance to Tunicamycin Toxicity Destination: subsection.2.3 Item: Title: SIRT1 Absence Did Not Impair Proteasome Activity Destination: subsection.2.4 Item: Title: Heat Shock Element (HSE) Was Not Involved in the Constitutive Production of Hsp70 in SIRT1-/- Cells Destination: subsection.2.5 Item: Title: STAT3 and Its Target Gene Socs-3 Were Upregulated in SIRT1-/- Cells Destination: subsection.2.6 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Cultures and Treatments Destination: subsection.4.1 Item: Title: Stable Cell Lines Expressing shRNA-Hsp70 Destination: subsection.4.2 Item: Title: Viability Assays Destination: subsection.4.3 Item: Title: Immunofluorescence Destination: subsection.4.4 Item: Title: Western Blot Destination: subsection.4.5 Item: Title: Real-Time PCR Destination: subsection.4.6 Item: Title: Proteasome Activity Assay Destination: subsection.4.7 Item: Title: HSE-Luciferase Reporter Assay Destination: subsection.4.8 Item: Title: Chromatin Immunoprecipitation (ChIP) Destination: subsection.4.9 Item: Title: Statistical Analysis Destination: subsection.4.10 Item: Title: References Destination: appendix.A. Info: Title: Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins Author: Jessica Latorre, Nuria de Vera, Tomàs Santalucía, Rafel Balada, Anna Marazuela-Duque, Alejandro Vaquero, Anna M. Planas and Valérie Petegnief Subject: Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1 branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress. Keywords: sirtuin; Hsp70; STAT3; ER stress Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Mar 05 10:35:42 CET 2024 ModDate: Tue Mar 05 10:47:38 CET 2024 ID: 0x4b19a8caeeaccc47d1282d145da6cb67, 0x4b19a8caeeaccc47d1282d145da6cb67 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: KESBIZ+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: KESBIZ+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: ZLMWBR+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: ZLMWBR+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: RSNLEP+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: RSNLEP+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: YFFKLH+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: YFFKLH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true ToUnicode: true Font: BaseFont: DUFXDP+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: DUFXDP+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: GGHKEL+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 189 FontDescriptor: FontName: GGHKEL+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ELFSWA+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: ELFSWA+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 279, 152, 288 Annotation: Subtype: Link Rect: 34, 267, 77, 276 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 125, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 149, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 272, 644, 283, 657 Annotation: Subtype: Link Rect: 351, 644, 362, 657 Annotation: Subtype: Link Rect: 127, 631, 138, 645 Annotation: Subtype: Link Rect: 347, 631, 358, 645 Annotation: Subtype: Link Rect: 195, 168, 202, 177 ActionDest: 242 Annotation: Subtype: Link Rect: 195, 143, 202, 152 ActionDest: 242 Annotation: Subtype: Link Rect: 236, 105, 243, 114 ActionDest: 242 Annotation: Subtype: Link Rect: 404, 92, 411, 102 ActionDest: 242 Annotation: Subtype: Link Rect: 411, 93, 418, 102 ActionDest: 242 Annotation: Subtype: Link Rect: 139, 17, 278, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 400, 87, 419 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 464, 744, 471, 752 ActionDest: 242 Annotation: Subtype: Link Rect: 224, 718, 231, 727 ActionDest: 242 Annotation: Subtype: Link Rect: 259, 706, 266, 715 ActionDest: 242 Annotation: Subtype: Link Rect: 266, 706, 273, 715 ActionDest: 242 Annotation: Subtype: Link Rect: 484, 693, 491, 702 ActionDest: 242 Annotation: Subtype: Link Rect: 423, 605, 430, 614 ActionDest: 242 Annotation: Subtype: Link Rect: 219, 517, 231, 526 ActionDest: 242 Annotation: Subtype: Link Rect: 234, 517, 246, 526 ActionDest: 242 Annotation: Subtype: Link Rect: 499, 517, 511, 526 ActionDest: 242 Annotation: Subtype: Link Rect: 383, 480, 395, 489 ActionDest: 242 Annotation: Subtype: Link Rect: 454, 480, 466, 489 ActionDest: 242 Annotation: Subtype: Link Rect: 345, 467, 357, 476 ActionDest: 242 Annotation: Subtype: Link Rect: 510, 467, 522, 476 ActionDest: 242 Annotation: Subtype: Link Rect: 440, 455, 452, 464 ActionDest: 242 Annotation: Subtype: Link Rect: 465, 430, 477, 439 ActionDest: 242 Annotation: Subtype: Link Rect: 222, 417, 234, 426 ActionDest: 242 Annotation: Subtype: Link Rect: 327, 404, 339, 414 ActionDest: 242 Annotation: Subtype: Link Rect: 544, 405, 556, 414 ActionDest: 242 Annotation: Subtype: Link Rect: 368, 379, 380, 388 ActionDest: 242 Annotation: Subtype: Link Rect: 331, 342, 343, 351 ActionDest: 242 Annotation: Subtype: Link Rect: 241, 304, 253, 313 ActionDest: 242 Annotation: Subtype: Link Rect: 207, 292, 219, 301 ActionDest: 245 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 540, 664, 547, 676 ActionDest: 78 Annotation: Subtype: Link Rect: 268, 641, 275, 650 ActionDest: 242 Annotation: Subtype: Link Rect: 241, 613, 248, 625 ActionDest: 78 Annotation: Subtype: Link Rect: 331, 146, 343, 155 ActionDest: 242 Annotation: Subtype: Link Rect: 194, 133, 206, 142 ActionDest: 245 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 544, 731, 556, 740 ActionDest: 245 Annotation: Subtype: Link Rect: 486, 706, 498, 715 ActionDest: 245 Annotation: Subtype: Link Rect: 462, 565, 469, 577 ActionDest: 78 Annotation: Subtype: Link Rect: 495, 565, 502, 577 ActionDest: 106 Annotation: Subtype: Link Rect: 478, 552, 485, 564 ActionDest: 106 Annotation: Subtype: Link Rect: 424, 540, 431, 552 ActionDest: 106 Annotation: Subtype: Link Rect: 196, 489, 203, 501 ActionDest: 106 Annotation: Subtype: Link Rect: 274, 489, 281, 501 ActionDest: 106 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 196, 653, 203, 665 ActionDest: 137 Annotation: Subtype: Link Rect: 317, 640, 324, 652 ActionDest: 137 Annotation: Subtype: Link Rect: 530, 615, 537, 627 ActionDest: 137 Annotation: Subtype: Link Rect: 330, 590, 337, 602 ActionDest: 137 Annotation: Subtype: Link Rect: 276, 159, 288, 168 ActionDest: 242 Annotation: Subtype: Link Rect: 289, 159, 301, 168 ActionDest: 245 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 502, 702, 509, 714 ActionDest: 162 Annotation: Subtype: Link Rect: 487, 665, 494, 677 ActionDest: 162 Annotation: Subtype: Link Rect: 333, 627, 340, 639 ActionDest: 162 Annotation: Subtype: Link Rect: 379, 118, 391, 127 ActionDest: 242 Annotation: Subtype: Link Rect: 524, 105, 536, 114 ActionDest: 245 Annotation: Subtype: Link Rect: 433, 77, 440, 89 ActionDest: 193 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 248, 741, 255, 753 ActionDest: 193 Annotation: Subtype: Link Rect: 312, 728, 319, 740 ActionDest: 193 Annotation: Subtype: Link Rect: 387, 703, 395, 715 ActionDest: 193 Annotation: Subtype: Link Rect: 196, 681, 208, 690 ActionDest: 245 Annotation: Subtype: Link Rect: 221, 665, 228, 677 ActionDest: 193 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 384, 718, 396, 727 ActionDest: 245 Annotation: Subtype: Link Rect: 549, 706, 556, 715 ActionDest: 224 Annotation: Subtype: Link Rect: 424, 665, 431, 677 ActionDest: 193 Annotation: Subtype: Link Rect: 199, 628, 206, 640 ActionDest: 193 Annotation: Subtype: Link Rect: 198, 603, 205, 615 ActionDest: 193 Annotation: Subtype: Link Rect: 284, 555, 296, 564 ActionDest: 245 Annotation: Subtype: Link Rect: 228, 392, 240, 401 ActionDest: 245 Annotation: Subtype: Link Rect: 241, 392, 253, 401 ActionDest: 245 Annotation: Subtype: Link Rect: 366, 316, 378, 325 ActionDest: 245 Annotation: Subtype: Link Rect: 381, 316, 393, 325 ActionDest: 245 Annotation: Subtype: Link Rect: 227, 291, 239, 300 ActionDest: 245 Annotation: Subtype: Link Rect: 536, 241, 543, 250 ActionDest: 242 Annotation: Subtype: Link Rect: 544, 241, 556, 250 ActionDest: 245 Annotation: Subtype: Link Rect: 355, 203, 367, 212 ActionDest: 242 Annotation: Subtype: Link Rect: 367, 203, 379, 212 ActionDest: 242 Annotation: Subtype: Link Rect: 380, 203, 392, 212 ActionDest: 242 Annotation: Subtype: Link Rect: 392, 203, 404, 212 ActionDest: 245 Annotation: Subtype: Link Rect: 356, 178, 368, 187 ActionDest: 245 Annotation: Subtype: Link Rect: 543, 141, 555, 150 ActionDest: 245 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 544, 744, 556, 752 ActionDest: 245 Annotation: Subtype: Link Rect: 292, 731, 304, 740 ActionDest: 245 Annotation: Subtype: Link Rect: 221, 668, 228, 677 ActionDest: 242 Annotation: Subtype: Link Rect: 228, 565, 235, 577 ActionDest: 162 Annotation: Subtype: Link Rect: 312, 552, 319, 564 ActionDest: 162 Annotation: Subtype: Link Rect: 544, 480, 556, 489 ActionDest: 242 Annotation: Subtype: Link Rect: 197, 417, 209, 426 ActionDest: 242 Annotation: Subtype: Link Rect: 544, 392, 556, 401 ActionDest: 245 Annotation: Subtype: Link Rect: 205, 342, 217, 351 ActionDest: 242 Annotation: Subtype: Link Rect: 461, 241, 473, 250 ActionDest: 245 Annotation: Subtype: Link Rect: 474, 241, 486, 250 ActionDest: 245 Annotation: Subtype: Link Rect: 323, 91, 335, 100 ActionDest: 245 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 301, 731, 313, 740 ActionDest: 242 Annotation: Subtype: Link Rect: 300, 693, 312, 702 ActionDest: 242 Annotation: Subtype: Link Rect: 468, 379, 480, 388 ActionDest: 245 Annotation: Subtype: Link Rect: 373, 77, 385, 86 ActionDest: 245 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 218, 630, 230, 639 ActionDest: 248 Annotation: Subtype: Link Rect: 402, 517, 414, 526 ActionDest: 245 Annotation: Subtype: Link Rect: 292, 190, 304, 199 ActionDest: 248 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 394, 128, 406, 137 ActionDest: 242 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 518, 731, 530, 740 ActionDest: 242 Annotation: Subtype: Link Rect: 460, 693, 471, 702 ActionDest: 245 Annotation: Subtype: Link Rect: 505, 336, 560, 347 Annotation: Subtype: Link Rect: 165, 324, 338, 335 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 59, 716, 96, 726 Annotation: Subtype: Link Rect: 411, 704, 449, 715 Annotation: Subtype: Link Rect: 321, 669, 358, 680 Annotation: Subtype: Link Rect: 520, 657, 557, 668 Annotation: Subtype: Link Rect: 112, 624, 150, 634 Annotation: Subtype: Link Rect: 156, 624, 192, 634 Annotation: Subtype: Link Rect: 185, 589, 222, 599 Annotation: Subtype: Link Rect: 135, 555, 173, 565 Annotation: Subtype: Link Rect: 178, 532, 216, 542 Annotation: Subtype: Link Rect: 131, 497, 168, 507 Annotation: Subtype: Link Rect: 240, 462, 277, 473 Annotation: Subtype: Link Rect: 140, 428, 177, 438 Annotation: Subtype: Link Rect: 113, 405, 150, 415 Annotation: Subtype: Link Rect: 244, 382, 281, 392 Annotation: Subtype: Link Rect: 278, 347, 316, 358 Annotation: Subtype: Link Rect: 322, 347, 358, 358 Annotation: Subtype: Link Rect: 520, 324, 557, 335 Annotation: Subtype: Link Rect: 519, 301, 557, 312 Annotation: Subtype: Link Rect: 59, 267, 96, 277 Annotation: Subtype: Link Rect: 59, 233, 96, 243 Annotation: Subtype: Link Rect: 290, 209, 327, 220 Annotation: Subtype: Link Rect: 289, 186, 326, 197 Annotation: Subtype: Link Rect: 266, 163, 303, 174 Annotation: Subtype: Link Rect: 95, 141, 132, 151 Annotation: Subtype: Link Rect: 167, 118, 204, 128 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 104, 730, 141, 741 Annotation: Subtype: Link Rect: 268, 695, 305, 706 Annotation: Subtype: Link Rect: 190, 673, 228, 683 Annotation: Subtype: Link Rect: 487, 649, 524, 660 Annotation: Subtype: Link Rect: 215, 614, 252, 625 Annotation: Subtype: Link Rect: 104, 581, 142, 591 Annotation: Subtype: Link Rect: 336, 557, 374, 568 Annotation: Subtype: Link Rect: 113, 523, 150, 533 Annotation: Subtype: Link Rect: 228, 500, 266, 510 Annotation: Subtype: Link Rect: 208, 477, 245, 487 Annotation: Subtype: Link Rect: 95, 443, 132, 453 Annotation: Subtype: Link Rect: 361, 419, 399, 430 Annotation: Subtype: Link Rect: 127, 385, 164, 395 Annotation: Subtype: Link Rect: 59, 362, 96, 372 Annotation: Subtype: Link Rect: 438, 338, 476, 349 Annotation: Subtype: Link Rect: 259, 305, 297, 315 Annotation: Subtype: Link Rect: 515, 292, 553, 303 Annotation: Subtype: Link Rect: 440, 269, 478, 280 Annotation: Subtype: Link Rect: 316, 246, 354, 257 Annotation: Subtype: Link Rect: 220, 223, 258, 234 Annotation: Subtype: Link Rect: 126, 201, 164, 211 Annotation: Subtype: Link Rect: 86, 178, 123, 188 Annotation: Subtype: Link Rect: 186, 144, 224, 154 Annotation: Subtype: Link Rect: 95, 109, 133, 119 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 104, 719, 141, 729 Annotation: Subtype: Link Rect: 453, 696, 491, 706 Checksum: 28c02a37 Type: CRC32 Checksum: d1857160eb0b0b0f3af04c541a33111e Type: MD5 Checksum: 63755a553b133c2e9b94c820563beca328df348d Type: SHA-1 Checksum: 30e858e4cf483182dc861b4c749d393e0869682b65be1d6c27c79de22043c45c Type: SHA-256