872b352a35f3d890c5d038a103400220  pmc_34572983.pdf
15cb29261bf152a4af200507367433d7a231d349  pmc_34572983.pdf
a8a95fa8ba13ac58c117676fc09d986c57677b85ba698c00e857178c003a6f3f  pmc_34572983.pdf

Title:          Dual-Specificity Phosphatase 1 (DUSP1) Has a Central Role in Redox Homeostasis and Inflammation in the Mouse Cochlea
Subject:        Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1-/- mice. RNA sequencing analysis (GSE176114) indicated that Dusp1-/- cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1-/- mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1-/- mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation.
Keywords:       hearing; N-acetylcysteine; glutathione; antioxidants; reactive oxygen species (ROS); mitochondria; apoptosis; inflammation; RNAseq
Author:         Jose M. Bermúdez-Muñoz, Adelaida M. Celaya, Ángela García-Mato, Daniel Muñoz-Espín, Lourdes Rodríguez-de la Rosa, Manuel Serrano and Isabel Varela-Nieto
Creator:        LaTeX with hyperref
Producer:       pdfTeX-1.40.21
CreationDate:   Thu Sep  2 08:34:46 2021 CEST
ModDate:        Thu Sep  2 11:31:24 2021 CEST
Tagged:         no
UserProperties: no
Suspects:       no
Form:           none
JavaScript:     no
Pages:          23
Encrypted:      no
Page size:      595.276 x 841.89 pts (A4)
Page rot:       0
File size:      9600382 bytes
Optimized:      no
PDF version:    1.7


name                                 type              encoding         emb sub uni object ID
------------------------------------ ----------------- ---------------- --- --- --- ---------
YQOTQA+VnURWPalladioL                Type 1            Custom           yes yes no      10  0
TMTOXG+URWPalladioL-Ital             Type 1            Custom           yes yes no      15  0
ZDIHAI+URWPalladioL-Bold             Type 1            Custom           yes yes no      20  0
ORVXYN+URWPalladioL-Roma             Type 1            Custom           yes yes no      24  0
RGUFCP+CMSY10                        Type 1            Builtin          yes yes no      28  0
PXSALH+VnURWPalladioL-Bold           Type 1            Custom           yes yes no      32  0
KUDXZB+TimesNewRomanPS-BoldItalicMT  Type 1C           WinAnsi          yes yes no      40  0
MCBCCI+SourceSansRoman-Regular       Type 1C           Custom           yes yes no      62  0
MCBCCM+SourceSansRoman.613wght       Type 1C           Custom           yes yes no      66  0
MCBCCO+SourceSansRoman-Bold          Type 1C           Custom           yes yes no      70  0
KESBIZ+EURM10                        Type 1            Builtin          yes yes no      82  0
MPUDFL+PazoMath                      Type 1            Builtin          yes yes no      92  0
DUJUUF+PazoMath-Italic               Type 1            Builtin          yes yes no     136  0


Jhove (Rel. 1.22.1, 2019-04-17)
 Date: 2022-03-22 02:45:48 CET
 RepresentationInformation: pmc_34572983.pdf
  ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17)
  LastModified: 2021-10-04 03:39:00 CEST
  Size: 9600382
  Format: PDF
  Version: 1.7
  Status: Well-Formed and valid
  SignatureMatches:
   PDF-hul
  MIMEtype: application/pdf
  PDFMetadata: 
   Objects: 450
   FreeObjects: 1
   IncrementalUpdates: 0
   DocumentCatalog: 
    PageLayout: SinglePage
    PageMode: UseNone
    Outlines: 
     Item: 
      Title: Introduction 
      Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba
     Item: 
      Title: Materials and Methods 
      Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40
      Children: 
       Item: 
        Title: Mice 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634
       Item: 
        Title: Antioxidant Treatment 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452
       Item: 
        Title: Hearing Evaluation 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86
       Item: 
        Title: RNA-Sequencing 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec
       Item: 
        Title: RT-qPCR 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d
       Item: 
        Title: Cochlear Morphology and Immunohistochemistry 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686
       Item: 
        Title: Hair Cell Number Quantification and TUNEL Assay 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd
       Item: 
        Title: Protein Extraction and Immunoblotting 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811
       Item: 
        Title: Protein Carbonylation 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9
       Item: 
        Title: Measurement of Glucose-6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase and Glutathione Reductase Activity 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4
       Item: 
        Title: Glutathione and Glutathione Disulfide Analysis 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3
       Item: 
        Title: Statistical Analysis 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05
     Item: 
      Title: Results 
      Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3
      Children: 
       Item: 
        Title: Dusp1 Deficiency Affects Cellular Programs Involved in Cochlear Homeostasis 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6
       Item: 
        Title: N-Acetylcysteine Treatment of Dusp1-/- Mice Has a Positive Impact on Cochlear Glutathione Metabolism 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297
       Item: 
        Title: N-Acetylcysteine Treatment Delays Hearing Loss Onset and Reduces Hair Cell Death in Dusp1-/- Mice 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4
       Item: 
        Title: N-Acetylcysteine Administration Modulates Redox Systems and Limits Reactive Oxygen Species Production in Dusp1-/- Cochleae 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034
       Item: 
        Title: N-Acetylcysteine Administration Protects Mitochondria and Restrains DNA Damage in Spiral Ganglion Neurons 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6
       Item: 
        Title: N-Acetylcysteine Administration Normalizes the Inflammatory Response and Macrophage Recruitment in Dusp1-/- Cochlea 
        Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f
     Item: 
      Title: Discussion 
      Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003
     Item: 
      Title: Conclusions 
      Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191
     Item: 
      Title: References
      Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb
   Info: 
    Title: Dual-Specificity Phosphatase 1 (DUSP1) Has a Central Role in Redox Homeostasis and Inflammation in the Mouse Cochlea
    Author: Jose M. Bermúdez-Muñoz, Adelaida M. Celaya, Ángela García-Mato, Daniel Muñoz-Espín, Lourdes Rodríguez-de la Rosa, Manuel Serrano and Isabel Varela-Nieto
    Subject: Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1-/- mice. RNA sequencing analysis (GSE176114) indicated that Dusp1-/- cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1-/- mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1-/- mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation.
    Keywords: hearing; N-acetylcysteine; glutathione; antioxidants; reactive oxygen species (ROS); mitochondria; apoptosis; inflammation; RNAseq
    Creator: LaTeX with hyperref
    Producer: pdfTeX-1.40.21
    CreationDate: Thu Sep 02 08:34:46 CEST 2021
    ModDate: Thu Sep 02 11:31:24 CEST 2021
   ID: 0x8dc6ae57aae86a15ef83ac38de1e0917, 0x8dc6ae57aae86a15ef83ac38de1e0917
   Filters: 
    FilterPipeline: FlateDecode
   Fonts: 
    Type1: 
     Font: 
      BaseFont: PXSALH+VnURWPalladioL-Bold
      FontSubset: true
      FirstChar: 129
      LastChar: 243
      FontDescriptor: 
       FontName: PXSALH+VnURWPalladioL-Bold
       Flags: Symbolic
       FontBBox: -152, -301, 1000, 935
       FontFile: true
      EncodingDictionary: 
       Differences: true
     Font: 
      BaseFont: KESBIZ+EURM10
      FontSubset: true
      FirstChar: 11
      LastChar: 22
      FontDescriptor: 
       FontName: KESBIZ+EURM10
       Flags: Symbolic
       FontBBox: -32, -243, 1060, 720
       FontFile: true
     Font: 
      BaseFont: ZDIHAI+URWPalladioL-Bold
      FontSubset: true
      FirstChar: 2
      LastChar: 241
      FontDescriptor: 
       FontName: ZDIHAI+URWPalladioL-Bold
       Flags: Symbolic
       FontBBox: -152, -301, 1000, 935
       FontFile: true
      EncodingDictionary: 
       Differences: true
     Font: 
      BaseFont: ORVXYN+URWPalladioL-Roma
      FontSubset: true
      FirstChar: 2
      LastChar: 250
      FontDescriptor: 
       FontName: ORVXYN+URWPalladioL-Roma
       Flags: Symbolic
       FontBBox: -166, -283, 1021, 943
       FontFile: true
      EncodingDictionary: 
       Differences: true
     Font: 
      BaseFont: DUJUUF+PazoMath-Italic
      FontSubset: true
      FirstChar: 97
      LastChar: 98
      FontDescriptor: 
       FontName: DUJUUF+PazoMath-Italic
       Flags: Symbolic
       FontBBox: -70, -277, 902, 733
       FontFile: true
     Font: 
      BaseFont: YQOTQA+VnURWPalladioL
      FontSubset: true
      FirstChar: 129
      LastChar: 243
      FontDescriptor: 
       FontName: YQOTQA+VnURWPalladioL
       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: MPUDFL+PazoMath
      FontSubset: true
      FirstChar: 68
      LastChar: 68
      FontDescriptor: 
       FontName: MPUDFL+PazoMath
       Flags: Symbolic
       FontBBox: -40, -283, 878, 946
       FontFile: true
     Font: 
      BaseFont: TMTOXG+URWPalladioL-Ital
      FontSubset: true
      FirstChar: 2
      LastChar: 122
      FontDescriptor: 
       FontName: TMTOXG+URWPalladioL-Ital
       Flags: Symbolic
       FontBBox: -170, -305, 1010, 941
       FontFile: true
      EncodingDictionary: 
       Differences: true
   Pages: 
    Page: 
     Sequence: 1
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 35, 759, 175, 795
      Annotation: 
       Subtype: Link
       Rect: 515, 759, 560, 795
      Annotation: 
       Subtype: Link
       Rect: 173, 662, 184, 675
      Annotation: 
       Subtype: Link
       Rect: 401, 662, 412, 675
      Annotation: 
       Subtype: Link
       Rect: 192, 649, 203, 663
      Annotation: 
       Subtype: Link
       Rect: 292, 649, 304, 663
      Annotation: 
       Subtype: Link
       Rect: 438, 649, 449, 663
      Annotation: 
       Subtype: Link
       Rect: 54, 358, 154, 367
      Annotation: 
       Subtype: Link
       Rect: 35, 348, 84, 355
      Annotation: 
       Subtype: Link
       Rect: 35, 162, 97, 184
      Annotation: 
       Subtype: Link
       Rect: 153, 89, 154, 98
      Annotation: 
       Subtype: Link
       Rect: 35, 77, 147, 86
      Annotation: 
       Subtype: Link
       Rect: 516, 119, 523, 128
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 192, 81, 199, 90
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 131, 17, 277, 28
      Annotation: 
       Subtype: Link
       Rect: 390, 17, 558, 28
      Annotation: 
       Subtype: Link
       Rect: 35, 470, 87, 489
    Page: 
     Sequence: 2
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 412, 724, 419, 733
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 541, 674, 548, 683
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 549, 674, 556, 683
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 518, 661, 525, 670
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 469, 636, 476, 645
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 500, 574, 507, 582
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 507, 573, 514, 582
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 549, 511, 556, 520
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 361, 410, 373, 419
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 256, 385, 263, 394
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 263, 385, 275, 394
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 276, 385, 288, 394
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 250, 360, 262, 369
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 315, 360, 327, 369
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 446, 322, 453, 331
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 454, 322, 466, 331
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 370, 310, 382, 319
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 277, 285, 289, 294
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 299, 272, 311, 281
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 385, 259, 397, 269
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 532, 259, 544, 269
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 545, 259, 557, 269
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 266, 247, 278, 256
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 291, 209, 298, 218
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 549, 197, 556, 206
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 359, 172, 366, 181
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 425, 109, 432, 118
       ActionDest: 164
    Page: 
     Sequence: 3
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 544, 558, 556, 567
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 492, 495, 504, 504
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 522, 281, 533, 290
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 415, 168, 427, 177
       ActionDest: 167
    Page: 
     Sequence: 4
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 169, 657, 427, 669
      Annotation: 
       Subtype: Link
       Rect: 388, 644, 560, 656
      Annotation: 
       Subtype: Link
       Rect: 166, 632, 267, 644
      Annotation: 
       Subtype: Link
       Rect: 324, 622, 336, 631
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 424, 622, 436, 631
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 530, 622, 542, 631
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 329, 597, 341, 606
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 222, 581, 468, 594
      Annotation: 
       Subtype: Link
       Rect: 358, 572, 370, 581
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 381, 521, 393, 530
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 487, 521, 499, 530
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 254, 509, 266, 518
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 410, 509, 422, 518
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 439, 494, 446, 506
       ActionDest: 102
      Annotation: 
       Subtype: Link
       Rect: 406, 484, 417, 493
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 422, 395, 434, 405
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 231, 358, 243, 367
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 524, 295, 536, 304
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 545, 169, 557, 178
       ActionDest: 171
    Page: 
     Sequence: 5
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 421, 682, 433, 691
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 544, 543, 556, 552
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 376, 181, 388, 190
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 211, 155, 223, 165
       ActionDest: 171
    Page: 
     Sequence: 6
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 216, 707, 228, 716
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 457, 376, 464, 388
       ActionDest: 102
      Annotation: 
       Subtype: Link
       Rect: 501, 175, 508, 187
       ActionDest: 108
    Page: 
     Sequence: 7
    Page: 
     Sequence: 8
    Page: 
     Sequence: 9
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 275, 461, 282, 473
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 397, 411, 404, 425
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 460, 386, 467, 400
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 541, 361, 548, 374
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 543, 310, 550, 322
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 247, 285, 254, 297
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 482, 248, 489, 260
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 528, 197, 535, 209
       ActionDest: 108
      Annotation: 
       Subtype: Link
       Rect: 356, 109, 363, 122
       ActionDest: 117
    Page: 
     Sequence: 10
    Page: 
     Sequence: 11
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 542, 539, 549, 553
       ActionDest: 117
      Annotation: 
       Subtype: Link
       Rect: 521, 463, 528, 475
       ActionDest: 117
      Annotation: 
       Subtype: Link
       Rect: 415, 426, 422, 440
       ActionDest: 117
      Annotation: 
       Subtype: Link
       Rect: 279, 401, 286, 413
       ActionDest: 117
      Annotation: 
       Subtype: Link
       Rect: 458, 312, 465, 326
       ActionDest: 127
      Annotation: 
       Subtype: Link
       Rect: 340, 86, 347, 98
       ActionDest: 127
    Page: 
     Sequence: 12
    Page: 
     Sequence: 13
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 225, 682, 232, 694
       ActionDest: 127
      Annotation: 
       Subtype: Link
       Rect: 442, 657, 449, 671
       ActionDest: 127
      Annotation: 
       Subtype: Link
       Rect: 542, 619, 549, 631
       ActionDest: 127
      Annotation: 
       Subtype: Link
       Rect: 476, 556, 483, 570
       ActionDest: 127
      Annotation: 
       Subtype: Link
       Rect: 391, 445, 398, 459
       ActionDest: 140
      Annotation: 
       Subtype: Link
       Rect: 488, 395, 495, 407
       ActionDest: 140
      Annotation: 
       Subtype: Link
       Rect: 519, 320, 526, 332
       ActionDest: 140
      Annotation: 
       Subtype: Link
       Rect: 240, 295, 247, 307
       ActionDest: 102
      Annotation: 
       Subtype: Link
       Rect: 349, 257, 356, 269
       ActionDest: 140
      Annotation: 
       Subtype: Link
       Rect: 230, 244, 237, 256
       ActionDest: 140
      Annotation: 
       Subtype: Link
       Rect: 480, 207, 487, 221
       ActionDest: 140
    Page: 
     Sequence: 14
    Page: 
     Sequence: 15
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 331, 458, 338, 470
       ActionDest: 149
      Annotation: 
       Subtype: Link
       Rect: 200, 308, 207, 320
       ActionDest: 149
      Annotation: 
       Subtype: Link
       Rect: 541, 283, 548, 295
       ActionDest: 149
    Page: 
     Sequence: 16
    Page: 
     Sequence: 17
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 314, 619, 321, 628
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 321, 619, 333, 628
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 435, 606, 447, 615
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 202, 581, 214, 590
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 328, 481, 340, 490
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 418, 443, 430, 452
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 530, 393, 537, 402
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 538, 393, 550, 402
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 334, 343, 346, 352
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 461, 267, 473, 276
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 458, 217, 470, 226
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 202, 167, 214, 176
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 262, 142, 274, 151
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 207, 92, 219, 101
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 543, 66, 555, 76
       ActionDest: 171
    Page: 
     Sequence: 18
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 524, 710, 531, 719
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 531, 710, 543, 719
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 544, 710, 556, 719
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 207, 572, 219, 581
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 467, 509, 474, 518
       ActionDest: 164
      Annotation: 
       Subtype: Link
       Rect: 246, 484, 258, 493
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 206, 421, 218, 430
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 472, 383, 484, 392
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 319, 270, 331, 279
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 370, 220, 382, 229
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 544, 208, 556, 217
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 499, 170, 511, 179
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 512, 170, 524, 179
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 194, 107, 206, 116
       ActionDest: 171
    Page: 
     Sequence: 19
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 320, 722, 332, 731
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 348, 672, 360, 681
       ActionDest: 171
      Annotation: 
       Subtype: Link
       Rect: 360, 672, 372, 681
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 384, 622, 396, 631
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 207, 546, 214, 556
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 207, 509, 219, 518
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 411, 433, 423, 443
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 437, 396, 449, 405
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 256, 358, 268, 367
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 242, 320, 253, 330
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 254, 321, 266, 330
       ActionDest: 167
      Annotation: 
       Subtype: Link
       Rect: 266, 320, 278, 330
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 279, 320, 291, 330
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 508, 270, 520, 279
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 419, 106, 560, 117
      Annotation: 
       Subtype: Link
       Rect: 166, 94, 269, 105
    Page: 
     Sequence: 20
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 312, 406, 323, 415
       ActionDest: 174
      Annotation: 
       Subtype: Link
       Rect: 215, 392, 489, 403
      Annotation: 
       Subtype: Link
       Rect: 314, 250, 351, 261
      Annotation: 
       Subtype: Link
       Rect: 287, 238, 560, 249
      Annotation: 
       Subtype: Link
       Rect: 56, 227, 107, 238
      Annotation: 
       Subtype: Link
       Rect: 293, 204, 331, 215
      Annotation: 
       Subtype: Link
       Rect: 337, 204, 373, 215
      Annotation: 
       Subtype: Link
       Rect: 197, 181, 234, 192
      Annotation: 
       Subtype: Link
       Rect: 241, 181, 277, 192
      Annotation: 
       Subtype: Link
       Rect: 284, 146, 321, 157
      Annotation: 
       Subtype: Link
       Rect: 328, 146, 364, 157
      Annotation: 
       Subtype: Link
       Rect: 375, 123, 413, 134
      Annotation: 
       Subtype: Link
       Rect: 419, 123, 455, 134
      Annotation: 
       Subtype: Link
       Rect: 382, 111, 420, 122
      Annotation: 
       Subtype: Link
       Rect: 59, 89, 97, 99
      Annotation: 
       Subtype: Link
       Rect: 103, 89, 139, 99
    Page: 
     Sequence: 21
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 139, 709, 177, 720
      Annotation: 
       Subtype: Link
       Rect: 216, 686, 254, 697
      Annotation: 
       Subtype: Link
       Rect: 140, 653, 178, 663
      Annotation: 
       Subtype: Link
       Rect: 464, 629, 502, 640
      Annotation: 
       Subtype: Link
       Rect: 260, 594, 298, 605
      Annotation: 
       Subtype: Link
       Rect: 304, 594, 340, 605
      Annotation: 
       Subtype: Link
       Rect: 59, 561, 97, 571
      Annotation: 
       Subtype: Link
       Rect: 103, 561, 139, 571
      Annotation: 
       Subtype: Link
       Rect: 197, 537, 234, 548
      Annotation: 
       Subtype: Link
       Rect: 241, 537, 277, 548
      Annotation: 
       Subtype: Link
       Rect: 245, 514, 282, 525
      Annotation: 
       Subtype: Link
       Rect: 59, 480, 97, 490
      Annotation: 
       Subtype: Link
       Rect: 374, 456, 411, 467
      Annotation: 
       Subtype: Link
       Rect: 59, 423, 97, 433
      Annotation: 
       Subtype: Link
       Rect: 282, 399, 319, 410
      Annotation: 
       Subtype: Link
       Rect: 436, 376, 473, 387
      Annotation: 
       Subtype: Link
       Rect: 489, 353, 525, 364
      Annotation: 
       Subtype: Link
       Rect: 349, 318, 387, 329
      Annotation: 
       Subtype: Link
       Rect: 393, 318, 429, 329
      Annotation: 
       Subtype: Link
       Rect: 354, 296, 392, 306
      Annotation: 
       Subtype: Link
       Rect: 398, 296, 434, 306
      Annotation: 
       Subtype: Link
       Rect: 370, 272, 407, 283
      Annotation: 
       Subtype: Link
       Rect: 59, 238, 97, 249
      Annotation: 
       Subtype: Link
       Rect: 233, 215, 271, 226
      Annotation: 
       Subtype: Link
       Rect: 355, 192, 392, 203
      Annotation: 
       Subtype: Link
       Rect: 257, 158, 294, 168
      Annotation: 
       Subtype: Link
       Rect: 297, 134, 335, 145
      Annotation: 
       Subtype: Link
       Rect: 260, 112, 297, 122
      Annotation: 
       Subtype: Link
       Rect: 56, 88, 241, 99
    Page: 
     Sequence: 22
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 131, 710, 169, 720
      Annotation: 
       Subtype: Link
       Rect: 175, 710, 211, 720
      Annotation: 
       Subtype: Link
       Rect: 248, 687, 286, 697
      Annotation: 
       Subtype: Link
       Rect: 118, 653, 155, 663
      Annotation: 
       Subtype: Link
       Rect: 452, 629, 489, 640
      Annotation: 
       Subtype: Link
       Rect: 59, 595, 97, 605
      Annotation: 
       Subtype: Link
       Rect: 103, 595, 139, 605
      Annotation: 
       Subtype: Link
       Rect: 300, 571, 338, 582
      Annotation: 
       Subtype: Link
       Rect: 82, 549, 119, 559
      Annotation: 
       Subtype: Link
       Rect: 125, 549, 161, 559
      Annotation: 
       Subtype: Link
       Rect: 145, 526, 182, 536
      Annotation: 
       Subtype: Link
       Rect: 86, 503, 124, 513
      Annotation: 
       Subtype: Link
       Rect: 509, 479, 546, 490
      Annotation: 
       Subtype: Link
       Rect: 429, 456, 466, 467
      Annotation: 
       Subtype: Link
       Rect: 238, 433, 275, 444
      Annotation: 
       Subtype: Link
       Rect: 327, 410, 365, 421
      Annotation: 
       Subtype: Link
       Rect: 271, 387, 308, 398
      Annotation: 
       Subtype: Link
       Rect: 315, 387, 351, 398
      Annotation: 
       Subtype: Link
       Rect: 305, 377, 342, 387
      Annotation: 
       Subtype: Link
       Rect: 199, 353, 236, 364
      Annotation: 
       Subtype: Link
       Rect: 193, 330, 231, 341
      Annotation: 
       Subtype: Link
       Rect: 59, 308, 97, 318
      Annotation: 
       Subtype: Link
       Rect: 103, 308, 139, 318
      Annotation: 
       Subtype: Link
       Rect: 152, 272, 189, 283
      Annotation: 
       Subtype: Link
       Rect: 474, 261, 511, 272
      Annotation: 
       Subtype: Link
       Rect: 517, 261, 553, 272
      Annotation: 
       Subtype: Link
       Rect: 184, 238, 222, 249
      Annotation: 
       Subtype: Link
       Rect: 104, 215, 141, 226
      Annotation: 
       Subtype: Link
       Rect: 59, 181, 97, 191
      Annotation: 
       Subtype: Link
       Rect: 144, 158, 181, 168
      Annotation: 
       Subtype: Link
       Rect: 187, 158, 223, 168
      Annotation: 
       Subtype: Link
       Rect: 462, 134, 499, 145
      Annotation: 
       Subtype: Link
       Rect: 95, 112, 132, 122
      Annotation: 
       Subtype: Link
       Rect: 450, 88, 487, 99
      Annotation: 
       Subtype: Link
       Rect: 493, 88, 529, 99
      Annotation: 
       Subtype: Link
       Rect: 454, 65, 491, 76
    Page: 
     Sequence: 23
     Annotations: 
      Annotation: 
       Subtype: Link
       Rect: 255, 721, 293, 732
      Annotation: 
       Subtype: Link
       Rect: 299, 721, 335, 732
      Annotation: 
       Subtype: Link
       Rect: 422, 698, 459, 709
      Annotation: 
       Subtype: Link
       Rect: 354, 675, 392, 686
      Annotation: 
       Subtype: Link
       Rect: 284, 640, 322, 651
      Annotation: 
       Subtype: Link
       Rect: 328, 640, 364, 651
      Annotation: 
       Subtype: Link
       Rect: 520, 629, 557, 640
      Annotation: 
       Subtype: Link
       Rect: 141, 595, 179, 605
      Annotation: 
       Subtype: Link
       Rect: 384, 571, 422, 582
      Annotation: 
       Subtype: Link
       Rect: 428, 571, 464, 582
      Annotation: 
       Subtype: Link
       Rect: 520, 548, 557, 559
      Annotation: 
       Subtype: Link
       Rect: 59, 538, 95, 548
      Annotation: 
       Subtype: Link
       Rect: 156, 515, 194, 525
      Annotation: 
       Subtype: Link
       Rect: 202, 492, 240, 502
  Checksum: a269cf8c
   Type: CRC32
  Checksum: 872b352a35f3d890c5d038a103400220
   Type: MD5
  Checksum: 15cb29261bf152a4af200507367433d7a231d349
   Type: SHA-1