d44d8dd0898dcc07843929f0991bad6c 265640.pdf 852f4285c174f6ee4c4b7f03597480ce33874bdf 265640.pdf ed2c6d9f0e5356535ede31c8879e825b159e6624eb6301a14fd7dece11e0152e 265640.pdf Title: TMT-Based Proteomic Analysis of Human Spermatozoa from Unexplained Recurrent Miscarriage Patients before and after Oral Antioxidant Treatment Subject: Recently, sperm quality and the presence of double-stranded breaks (DSB) has been pointed out as a possible cause of recurrent miscarriage, and the use of antioxidants has expanded as a treatment for male infertility. The aim of the present study was to analyze the proteomic effects of antioxidants on sperm from RM patients with high incidence of DSB. Proteomic analysis was performed using a tandem mass tag labeling technique, and subsequently compared with the PANTHER database for DEPs, and the STRING database for protein–protein interactions (PPI). Differentially expressed proteins (DEPs) both before and after antioxidant oral treatment were identified. PPI involving DEPs clustered into networks related to cell metabolism, cytoskeleton, and DNA damage. Results show that the sperm proteomic profiles before and after antioxidant treatment do not significantly differ from each other. However, some DEPs found after the antioxidant treatment shifted towards a DEPs profile typical of fertile donors. This indirect measurement suggests an improvement caused by antioxidants on the expression of several proteins. Among them were proteins involved in sperm DNA remodeling (LMO7, MMP28, BNC2, H2B, and PRDM2). The results presented here represent the first approach in the analysis and repair of the proteomic change caused by antioxidants in recurrent miscarriage patients, elucidating biomarkers that may be useful for the diagnosis and further sperm selection in this type of patient. Further studies should be conducted to validate the usefulness of these biomarkers in larger study groups. Keywords: sperm; recurrent miscarriage; antioxidants; sperm DNA fragmentation; proteomics; male infertility Author: Alba Fernandez-Encinas, Jordi Ribas-Maynou, Agustín García-Peiró, Sergio Garcia-Segura, Olga Martinez-Pasarell, Joaquima Navarro, Maria Oliver-Bonet and Jordi Benet Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Aug 19 10:36:31 2022 CEST ModDate: Fri Aug 19 10:44:03 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 948684 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EAGQMG+VnURWPalladioL Type 1 Custom yes yes no 10 0 IPQCZJ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 XULBKR+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 IXSSBX+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 OJYQXC+CMSY10 Type 1 Builtin yes yes no 28 0 YWWVQQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 EBFBTV+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 75 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes no 88 0 ODCCMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 98 0 ODCCNI+PalatinoLinotype TrueType WinAnsi yes yes no 101 0 ODCCNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 104 0 ODCCMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 114 0 ODCCNI+PalatinoLinotype TrueType WinAnsi yes yes no 117 0 ODCCNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 120 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-01-20 02:30:18 CET RepresentationInformation: 265640.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-19 17:00:33 CET Size: 948684 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 347 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4923ab24 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Children: Item: Title: Samples and Study Design Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Semen Collection and Cryopreservation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: DNA Integrity Tests: Alkaline and Neutral Comet Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Proteomic Analysis of Spermatozoa Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Children: Item: Title: Sample Preparation for Proteomic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Trypsin Digestion and TMT Labeling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Liquid Chromatography and Mass Spectrometry Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Protein Identification, Quantification, and Data Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Functional Enrichment Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Sperm DNA Fragmentation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Analysis of Differentially Expressed Proteins (DEPs) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Functional Enrichment and Clustering Analyses of DEPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Info: Title: TMT-Based Proteomic Analysis of Human Spermatozoa from Unexplained Recurrent Miscarriage Patients before and after Oral Antioxidant Treatment Author: Alba Fernandez-Encinas, Jordi Ribas-Maynou, Agustín García-Peiró, Sergio Garcia-Segura, Olga Martinez-Pasarell, Joaquima Navarro, Maria Oliver-Bonet and Jordi Benet Subject: Recently, sperm quality and the presence of double-stranded breaks (DSB) has been pointed out as a possible cause of recurrent miscarriage, and the use of antioxidants has expanded as a treatment for male infertility. The aim of the present study was to analyze the proteomic effects of antioxidants on sperm from RM patients with high incidence of DSB. Proteomic analysis was performed using a tandem mass tag labeling technique, and subsequently compared with the PANTHER database for DEPs, and the STRING database for protein protein interactions (PPI). Differentially expressed proteins (DEPs) both before and after antioxidant oral treatment were identified. PPI involving DEPs clustered into networks related to cell metabolism, cytoskeleton, and DNA damage. Results show that the sperm proteomic profiles before and after antioxidant treatment do not significantly differ from each other. However, some DEPs found after the antioxidant treatment shifted towards a DEPs profile typical of fertile donors. This indirect measurement suggests an improvement caused by antioxidants on the expression of several proteins. Among them were proteins involved in sperm DNA remodeling (LMO7, MMP28, BNC2, H2B, and PRDM2). The results presented here represent the first approach in the analysis and repair of the proteomic change caused by antioxidants in recurrent miscarriage patients, elucidating biomarkers that may be useful for the diagnosis and further sperm selection in this type of patient. Further studies should be conducted to validate the usefulness of these biomarkers in larger study groups. Keywords: sperm; recurrent miscarriage; antioxidants; sperm DNA fragmentation; proteomics; male infertility Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Aug 19 10:36:31 CEST 2022 ModDate: Fri Aug 19 10:44:03 CEST 2022 ID: 0x198270d2318fd3f3c4893edf643e22d4, 0x198270d2318fd3f3c4893edf643e22d4 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: YWWVQQ+VnURWPalladioL-Bold FontSubset: true FirstChar: 189 LastChar: 226 FontDescriptor: FontName: YWWVQQ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XULBKR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: XULBKR+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IXSSBX+URWPalladioL-Ital FontSubset: true FirstChar: 39 LastChar: 122 FontDescriptor: FontName: IXSSBX+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WBVKDJ+URWPalladioL-BoldItal FontSubset: true FirstChar: 112 LastChar: 112 FontDescriptor: FontName: WBVKDJ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: EAGQMG+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: EAGQMG+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: OJYQXC+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: OJYQXC+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: IPQCZJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: IPQCZJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 313, 107, 320 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 197, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 263, 644, 274, 657 Annotation: Subtype: Link Rect: 488, 644, 499, 657 Annotation: Subtype: Link Rect: 349, 631, 360, 645 Annotation: Subtype: Link Rect: 438, 631, 449, 645 Annotation: Subtype: Link Rect: 390, 200, 397, 208 ActionDest: 135 Annotation: Subtype: Link Rect: 398, 200, 405, 208 ActionDest: 135 Annotation: Subtype: Link Rect: 406, 124, 413, 133 ActionDest: 135 Annotation: Subtype: Link Rect: 360, 86, 367, 95 ActionDest: 135 Annotation: Subtype: Link Rect: 368, 87, 375, 95 ActionDest: 135 Annotation: Subtype: Link Rect: 130, 17, 302, 28 Annotation: Subtype: Link Rect: 389, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 456, 87, 475 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 241, 735, 248, 744 ActionDest: 135 Annotation: Subtype: Link Rect: 251, 735, 258, 744 ActionDest: 135 Annotation: Subtype: Link Rect: 346, 710, 353, 719 ActionDest: 135 Annotation: Subtype: Link Rect: 354, 710, 361, 719 ActionDest: 135 Annotation: Subtype: Link Rect: 361, 710, 373, 719 ActionDest: 135 Annotation: Subtype: Link Rect: 291, 673, 303, 682 ActionDest: 135 Annotation: Subtype: Link Rect: 303, 673, 315, 682 ActionDest: 135 Annotation: Subtype: Link Rect: 498, 647, 510, 657 ActionDest: 138 Annotation: Subtype: Link Rect: 513, 647, 525, 657 ActionDest: 138 Annotation: Subtype: Link Rect: 281, 623, 293, 631 ActionDest: 138 Annotation: Subtype: Link Rect: 531, 610, 543, 619 ActionDest: 138 Annotation: Subtype: Link Rect: 543, 610, 555, 619 ActionDest: 138 Annotation: Subtype: Link Rect: 456, 597, 468, 606 ActionDest: 138 Annotation: Subtype: Link Rect: 352, 560, 364, 569 ActionDest: 138 Annotation: Subtype: Link Rect: 366, 535, 378, 544 ActionDest: 138 Annotation: Subtype: Link Rect: 450, 522, 462, 531 ActionDest: 138 Annotation: Subtype: Link Rect: 403, 472, 415, 481 ActionDest: 138 Annotation: Subtype: Link Rect: 544, 434, 556, 443 ActionDest: 138 Annotation: Subtype: Link Rect: 483, 409, 495, 418 ActionDest: 138 Annotation: Subtype: Link Rect: 285, 397, 297, 406 ActionDest: 138 Annotation: Subtype: Link Rect: 292, 384, 303, 393 ActionDest: 138 Annotation: Subtype: Link Rect: 306, 359, 318, 368 ActionDest: 138 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 531, 635, 543, 644 ActionDest: 138 Annotation: Subtype: Link Rect: 544, 635, 556, 644 ActionDest: 138 Annotation: Subtype: Link Rect: 269, 446, 281, 455 ActionDest: 138 Annotation: Subtype: Link Rect: 408, 333, 420, 342 ActionDest: 138 Annotation: Subtype: Link Rect: 483, 318, 490, 330 ActionDest: 72 Page: Sequence: 4 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 459, 692, 558, 704 Annotation: Subtype: Link Rect: 309, 654, 415, 666 Annotation: Subtype: Link Rect: 282, 565, 289, 577 ActionDest: 72 Annotation: Subtype: Link Rect: 511, 440, 518, 452 ActionDest: 85 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 423, 65, 430, 77 ActionDest: 92 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 433, 720, 440, 732 ActionDest: 92 Annotation: Subtype: Link Rect: 344, 683, 351, 695 ActionDest: 92 Annotation: Subtype: Link Rect: 495, 231, 501, 243 ActionDest: 108 Annotation: Subtype: Link Rect: 283, 194, 290, 206 ActionDest: 108 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 544, 657, 556, 666 ActionDest: 138 Annotation: Subtype: Link Rect: 529, 582, 541, 591 ActionDest: 138 Annotation: Subtype: Link Rect: 544, 582, 556, 591 ActionDest: 138 Annotation: Subtype: Link Rect: 495, 569, 507, 578 ActionDest: 138 Annotation: Subtype: Link Rect: 507, 569, 519, 578 ActionDest: 138 Annotation: Subtype: Link Rect: 263, 406, 275, 415 ActionDest: 138 Annotation: Subtype: Link Rect: 276, 406, 288, 415 ActionDest: 138 Annotation: Subtype: Link Rect: 288, 406, 300, 415 ActionDest: 138 Annotation: Subtype: Link Rect: 345, 343, 357, 352 ActionDest: 138 Annotation: Subtype: Link Rect: 202, 130, 214, 139 ActionDest: 138 Annotation: Subtype: Link Rect: 215, 130, 227, 139 ActionDest: 138 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 199, 683, 207, 695 ActionDest: 108 Annotation: Subtype: Link Rect: 338, 610, 349, 619 ActionDest: 138 Annotation: Subtype: Link Rect: 531, 585, 543, 594 ActionDest: 138 Annotation: Subtype: Link Rect: 544, 585, 556, 594 ActionDest: 138 Annotation: Subtype: Link Rect: 191, 560, 203, 569 ActionDest: 138 Annotation: Subtype: Link Rect: 432, 535, 444, 544 ActionDest: 141 Annotation: Subtype: Link Rect: 283, 509, 295, 519 ActionDest: 141 Annotation: Subtype: Link Rect: 296, 509, 308, 519 ActionDest: 141 Annotation: Subtype: Link Rect: 193, 484, 205, 493 ActionDest: 141 Annotation: Subtype: Link Rect: 205, 484, 217, 493 ActionDest: 141 Annotation: Subtype: Link Rect: 396, 346, 407, 355 ActionDest: 141 Annotation: Subtype: Link Rect: 290, 334, 302, 343 ActionDest: 141 Annotation: Subtype: Link Rect: 276, 321, 288, 330 ActionDest: 141 Annotation: Subtype: Link Rect: 466, 196, 478, 205 ActionDest: 141 Annotation: Subtype: Link Rect: 531, 170, 543, 180 ActionDest: 141 Annotation: Subtype: Link Rect: 544, 171, 556, 180 ActionDest: 141 Annotation: Subtype: Link Rect: 285, 120, 297, 129 ActionDest: 141 Annotation: Subtype: Link Rect: 248, 95, 259, 104 ActionDest: 141 Annotation: Subtype: Link Rect: 531, 83, 543, 92 ActionDest: 138 Annotation: Subtype: Link Rect: 544, 83, 556, 92 ActionDest: 141 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 212, 735, 224, 744 ActionDest: 141 Annotation: Subtype: Link Rect: 195, 635, 207, 644 ActionDest: 141 Annotation: Subtype: Link Rect: 210, 635, 222, 644 ActionDest: 141 Annotation: Subtype: Link Rect: 189, 610, 201, 619 ActionDest: 141 Annotation: Subtype: Link Rect: 489, 597, 501, 606 ActionDest: 141 Annotation: Subtype: Link Rect: 268, 560, 280, 569 ActionDest: 141 Annotation: Subtype: Link Rect: 281, 560, 293, 569 ActionDest: 141 Annotation: Subtype: Link Rect: 387, 547, 398, 556 ActionDest: 141 Annotation: Subtype: Link Rect: 203, 522, 215, 531 ActionDest: 141 Annotation: Subtype: Link Rect: 534, 484, 546, 493 ActionDest: 141 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 95, 307, 132, 317 Annotation: Subtype: Link Rect: 325, 295, 362, 306 Annotation: Subtype: Link Rect: 215, 272, 253, 283 Annotation: Subtype: Link Rect: 59, 249, 96, 260 Annotation: Subtype: Link Rect: 324, 214, 362, 225 Annotation: Subtype: Link Rect: 285, 191, 322, 202 Annotation: Subtype: Link Rect: 329, 191, 365, 202 Annotation: Subtype: Link Rect: 478, 179, 515, 191 Annotation: Subtype: Link Rect: 431, 168, 468, 179 Annotation: Subtype: Link Rect: 149, 146, 186, 156 Annotation: Subtype: Link Rect: 462, 122, 499, 133 Annotation: Subtype: Link Rect: 384, 99, 421, 110 Annotation: Subtype: Link Rect: 427, 99, 463, 110 Annotation: Subtype: Link Rect: 285, 76, 322, 87 Annotation: Subtype: Link Rect: 329, 76, 365, 87 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 158, 711, 196, 721 Annotation: Subtype: Link Rect: 202, 711, 238, 721 Annotation: Subtype: Link Rect: 426, 687, 464, 698 Annotation: Subtype: Link Rect: 470, 687, 506, 698 Annotation: Subtype: Link Rect: 450, 664, 487, 675 Annotation: Subtype: Link Rect: 493, 664, 529, 675 Annotation: Subtype: Link Rect: 250, 641, 288, 652 Annotation: Subtype: Link Rect: 294, 641, 330, 652 Annotation: Subtype: Link Rect: 265, 607, 303, 617 Annotation: Subtype: Link Rect: 59, 573, 96, 583 Annotation: Subtype: Link Rect: 322, 549, 359, 560 Annotation: Subtype: Link Rect: 126, 527, 164, 537 Annotation: Subtype: Link Rect: 246, 503, 284, 514 Annotation: Subtype: Link Rect: 290, 503, 326, 514 Annotation: Subtype: Link Rect: 171, 469, 209, 479 Annotation: Subtype: Link Rect: 215, 469, 251, 479 Annotation: Subtype: Link Rect: 271, 445, 309, 456 Annotation: Subtype: Link Rect: 315, 445, 351, 456 Annotation: Subtype: Link Rect: 343, 423, 380, 433 Annotation: Subtype: Link Rect: 386, 423, 422, 433 Annotation: Subtype: Link Rect: 118, 389, 155, 399 Annotation: Subtype: Link Rect: 161, 389, 197, 399 Annotation: Subtype: Link Rect: 152, 353, 190, 364 Annotation: Subtype: Link Rect: 217, 320, 254, 330 Annotation: Subtype: Link Rect: 350, 296, 388, 307 Annotation: Subtype: Link Rect: 336, 273, 373, 284 Annotation: Subtype: Link Rect: 485, 250, 523, 261 Annotation: Subtype: Link Rect: 95, 216, 133, 226 Annotation: Subtype: Link Rect: 139, 216, 175, 226 Annotation: Subtype: Link Rect: 324, 192, 361, 203 Annotation: Subtype: Link Rect: 368, 192, 404, 203 Annotation: Subtype: Link Rect: 415, 169, 453, 180 Annotation: Subtype: Link Rect: 459, 169, 495, 180 Annotation: Subtype: Link Rect: 520, 146, 557, 157 Annotation: Subtype: Link Rect: 59, 136, 95, 146 Annotation: Subtype: Link Rect: 109, 113, 146, 123 Annotation: Subtype: Link Rect: 245, 90, 282, 100 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 59, 711, 96, 721 Annotation: Subtype: Link Rect: 489, 687, 526, 698 Annotation: Subtype: Link Rect: 122, 653, 159, 664 Annotation: Subtype: Link Rect: 59, 630, 96, 640 Annotation: Subtype: Link Rect: 137, 607, 174, 617 Annotation: Subtype: Link Rect: 181, 607, 217, 617 Annotation: Subtype: Link Rect: 191, 583, 229, 594 Annotation: Subtype: Link Rect: 235, 583, 271, 594 Annotation: Subtype: Link Rect: 244, 538, 281, 548 Annotation: Subtype: Link Rect: 471, 526, 508, 537 Annotation: Subtype: Link Rect: 514, 526, 550, 537 Annotation: Subtype: Link Rect: 113, 492, 151, 502 Annotation: Subtype: Link Rect: 184, 469, 221, 479 Annotation: Subtype: Link Rect: 59, 446, 96, 456 Annotation: Subtype: Link Rect: 445, 422, 482, 433 Annotation: Subtype: Link Rect: 95, 389, 133, 399 Annotation: Subtype: Link Rect: 126, 354, 164, 364 Annotation: Subtype: Link Rect: 170, 354, 206, 364 Annotation: Subtype: Link Rect: 266, 330, 303, 341 Annotation: Subtype: Link Rect: 309, 330, 346, 341 Annotation: Subtype: Link Rect: 350, 307, 387, 318 Annotation: Subtype: Link Rect: 447, 284, 484, 295 Annotation: Subtype: Link Rect: 490, 284, 526, 295 Annotation: Subtype: Link Rect: 193, 262, 230, 272 Annotation: Subtype: Link Rect: 236, 262, 272, 272 Annotation: Subtype: Link Rect: 104, 228, 141, 238 Annotation: Subtype: Link Rect: 519, 215, 557, 226 Annotation: Subtype: Link Rect: 59, 205, 95, 215 Annotation: Subtype: Link Rect: 505, 181, 542, 192 Annotation: Subtype: Link Rect: 122, 147, 159, 157 Annotation: Subtype: Link Rect: 354, 77, 392, 88 Annotation: Subtype: Link Rect: 398, 77, 434, 88 Checksum: 0bc3a839 Type: CRC32 Checksum: d44d8dd0898dcc07843929f0991bad6c Type: MD5 Checksum: 852f4285c174f6ee4c4b7f03597480ce33874bdf Type: SHA-1