0ef3672a2c10c38d606f36113344726b biology-10-00802.pdf 1ed7e449028fcfcf0af744a93d77da9a0a6d4d2f biology-10-00802.pdf c585f56ce640b3bbadc37a8e6cf28199217608b42cc6fc0afbaf8f6e8b40e3a8 biology-10-00802.pdf Title: Impact of the Epigenetically Regulated Hoxa-5 Gene in Neural Differentiation from Human Adipose-Derived Stem Cells Subject: Human adipose-derived mesenchymal stem cells (hASCs) may be used in some nervous system pathologies, although obtaining an adequate degree of neuronal differentiation is an important barrier to their applicability. This requires a deep understanding of the expression and epigenetic changes of the most important genes involved in their differentiation. We used hASCs from human lipoaspirates to induce neuronal-like cells through three protocols (Neu1, 2, and 3), determined the degree of neuronal differentiation using specific biomarkers in culture cells and neurospheres, and analyzed epigenetic changes of genes involved in this differentiation. Furthermore, we selected the Hoxa-5 gene to determine its potential to improve neuronal differentiation. Our results showed that an excellent hASC neuronal differentiation process using Neu1 which efficiently modulated NES, CHAT, SNAP25, or SCN9A neuronal marker expression. In addition, epigenetic studies showed relevant changes in Hoxa-5, GRM4, FGFR1, RTEL1, METRN, and PAX9 genes. Functional studies of the Hoxa-5 gene using CRISPR/dCas9 and lentiviral systems showed that its overexpression induced hASCs neuronal differentiation that was accelerated with the exposure to Neu1. These results suggest that Hoxa-5 is an essential gene in hASCs neuronal differentiation and therefore, a potential candidate for the development of cell therapy strategies in neurological disorders. Keywords: mesenchymal stem cells; neuronal differentiation; epigenetic changes; Hoxa-5; CRISPR/ dCas9 Author: Rosa Hernández, Cristina Jiménez-Luna, Raúl Ortiz, Fernando Setién, Miguel López, Gloria Perazzoli, Manel Esteller, María Berdasco, Jose Prados and Consolación Melguizo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Aug 25 12:32:06 2021 CEST ModDate: Wed Aug 25 12:38:21 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 20406280 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- UZYDGW+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 PCSWBS+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 VCVWKY+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 DGBDNV+VnURWPalladioL-Bold Type 1 Custom yes yes no 23 0 GLCFZC+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 32 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 54 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 58 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 62 0 RJZZXM+CMSY10 Type 1 Builtin yes yes no 74 0 KNLZHK+VnURWPalladioL Type 1 Custom yes yes no 81 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 88 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-29 02:30:03 CEST RepresentationInformation: biology-10-00802.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-31 17:27:09 CET Size: 20406280 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 370 FreeObjects: 15 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: hASCs from Human Adipose Tissue, Neuronal Differentiation and Neurospheres Formation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Immunofluorescence Analysis of Neuronal Markers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Quantitative RT-PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Genome-Wide CpG Methylation Profiling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Hierarchical Cluster Analysis and Definition of CpG Methylation Differences Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Bisulfite Genomic Sequencing of Multiple Clones Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Generation of a Stable hASCs Line Overexpressing Hoxa-5 Gene by Lentivirus Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Generation of a Hoxa-5 Expressing hASCs Cell Line Using a CRISPR/dCas9 System Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Neuronal Differentiation of hASCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Neuronal Differentiation in hASCs Neurospheres Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: DNA Methylation Changes Associated with Neuronal Differentiation of hASCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Modulation of Neuronal Markers in Stable hASC Line That Overexpresses the Hoxa-5 Gene Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Neuronal Differentiation by Hoxa-5 Gene and Neu1 Medium in hASCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Info: Title: Impact of the Epigenetically Regulated Hoxa-5 Gene in Neural Differentiation from Human Adipose-Derived Stem Cells Author: Rosa Hernández, Cristina Jiménez-Luna, Raúl Ortiz, Fernando Setién, Miguel López, Gloria Perazzoli, Manel Esteller, María Berdasco, Jose Prados and Consolación Melguizo Subject: Human adipose-derived mesenchymal stem cells (hASCs) may be used in some nervous system pathologies, although obtaining an adequate degree of neuronal differentiation is an important barrier to their applicability. This requires a deep understanding of the expression and epigenetic changes of the most important genes involved in their differentiation. We used hASCs from human lipoaspirates to induce neuronal-like cells through three protocols (Neu1, 2, and 3), determined the degree of neuronal differentiation using specific biomarkers in culture cells and neurospheres, and analyzed epigenetic changes of genes involved in this differentiation. Furthermore, we selected the Hoxa-5 gene to determine its potential to improve neuronal differentiation. Our results showed that an excellent hASC neuronal differentiation process using Neu1 which efficiently modulated NES, CHAT, SNAP25, or SCN9A neuronal marker expression. In addition, epigenetic studies showed relevant changes in Hoxa-5, GRM4, FGFR1, RTEL1, METRN, and PAX9 genes. Functional studies of the Hoxa-5 gene using CRISPR/dCas9 and lentiviral systems showed that its overexpression induced hASCs neuronal differentiation that was accelerated with the exposure to Neu1. These results suggest that Hoxa-5 is an essential gene in hASCs neuronal differentiation and therefore, a potential candidate for the development of cell therapy strategies in neurological disorders. Keywords: mesenchymal stem cells; neuronal differentiation; epigenetic changes; Hoxa-5; CRISPR/ dCas9 Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Aug 25 12:32:06 CEST 2021 ModDate: Wed Aug 25 12:38:21 CEST 2021 ID: 0x8b13df7675f6d524f611129ec721123d, 0x8b13df7675f6d524f611129ec721123d Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: KNLZHK+VnURWPalladioL FontSubset: true FirstChar: 189 LastChar: 226 FontDescriptor: FontName: KNLZHK+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VCVWKY+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: VCVWKY+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DGBDNV+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 243 FontDescriptor: FontName: DGBDNV+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: UZYDGW+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 151 FontDescriptor: FontName: UZYDGW+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: RJZZXM+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: RJZZXM+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: PCSWBS+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: PCSWBS+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 151, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 380, 647, 391, 661 Annotation: Subtype: Link Rect: 534, 647, 545, 661 Annotation: Subtype: Link Rect: 79, 322, 154, 331 Annotation: Subtype: Link Rect: 35, 310, 114, 319 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: 109, 17, 260, 28 Annotation: Subtype: Link Rect: 408, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 434, 87, 453 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 494, 683, 501, 692 ActionDest: 178 Annotation: Subtype: Link Rect: 437, 658, 444, 667 ActionDest: 178 Annotation: Subtype: Link Rect: 447, 658, 454, 667 ActionDest: 178 Annotation: Subtype: Link Rect: 333, 608, 340, 617 ActionDest: 178 Annotation: Subtype: Link Rect: 205, 595, 212, 605 ActionDest: 178 Annotation: Subtype: Link Rect: 404, 583, 411, 592 ActionDest: 178 Annotation: Subtype: Link Rect: 447, 558, 454, 567 ActionDest: 178 Annotation: Subtype: Link Rect: 550, 545, 557, 554 ActionDest: 178 Annotation: Subtype: Link Rect: 391, 520, 403, 529 ActionDest: 178 Annotation: Subtype: Link Rect: 269, 483, 281, 492 ActionDest: 178 Annotation: Subtype: Link Rect: 505, 445, 517, 454 ActionDest: 178 Annotation: Subtype: Link Rect: 444, 382, 456, 391 ActionDest: 178 Annotation: Subtype: Link Rect: 350, 357, 362, 366 ActionDest: 178 Annotation: Subtype: Link Rect: 241, 332, 253, 341 ActionDest: 178 Annotation: Subtype: Link Rect: 362, 319, 374, 328 ActionDest: 178 Annotation: Subtype: Link Rect: 394, 294, 406, 303 ActionDest: 178 Annotation: Subtype: Link Rect: 544, 269, 556, 278 ActionDest: 178 Annotation: Subtype: Link Rect: 481, 244, 493, 253 ActionDest: 178 Annotation: Subtype: Link Rect: 493, 244, 505, 253 ActionDest: 178 Annotation: Subtype: Link Rect: 336, 219, 348, 228 ActionDest: 178 Annotation: Subtype: Link Rect: 459, 206, 471, 215 ActionDest: 181 Annotation: Subtype: Link Rect: 208, 144, 219, 153 ActionDest: 181 Annotation: Subtype: Link Rect: 232, 106, 244, 115 ActionDest: 181 Annotation: Subtype: Link Rect: 469, 93, 481, 102 ActionDest: 181 Annotation: Subtype: Link Rect: 396, 81, 408, 90 ActionDest: 181 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 350, 735, 362, 744 ActionDest: 181 Annotation: Subtype: Link Rect: 203, 697, 215, 706 ActionDest: 181 Annotation: Subtype: Link Rect: 442, 470, 454, 479 ActionDest: 181 Annotation: Subtype: Link Rect: 375, 395, 387, 404 ActionDest: 181 Annotation: Subtype: Link Rect: 438, 395, 449, 404 ActionDest: 181 Annotation: Subtype: Link Rect: 545, 395, 557, 404 ActionDest: 181 Annotation: Subtype: Link Rect: 524, 369, 536, 379 ActionDest: 181 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 277, 559, 289, 568 ActionDest: 181 Annotation: Subtype: Link Rect: 544, 546, 556, 555 ActionDest: 181 Annotation: Subtype: Link Rect: 215, 496, 227, 505 ActionDest: 181 Annotation: Subtype: Link Rect: 223, 458, 235, 467 ActionDest: 181 Annotation: Subtype: Link Rect: 416, 383, 428, 392 ActionDest: 181 Annotation: Subtype: Link Rect: 406, 270, 418, 279 ActionDest: 181 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 203, 370, 215, 380 ActionDest: 181 Annotation: Subtype: Link Rect: 474, 370, 486, 380 ActionDest: 181 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 446, 640, 453, 652 ActionDest: 95 Annotation: Subtype: Link Rect: 302, 615, 309, 627 ActionDest: 95 Annotation: Subtype: Link Rect: 352, 602, 359, 614 ActionDest: 78 Annotation: Subtype: Link Rect: 543, 602, 550, 614 ActionDest: 95 Annotation: Subtype: Link Rect: 416, 72, 423, 85 ActionDest: 106 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 470, 117, 477, 129 ActionDest: 117 Annotation: Subtype: Link Rect: 315, 67, 322, 79 ActionDest: 117 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 369, 720, 376, 732 ActionDest: 128 Annotation: Subtype: Link Rect: 308, 619, 315, 631 ActionDest: 128 Annotation: Subtype: Link Rect: 542, 594, 549, 606 ActionDest: 128 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 541, 666, 548, 678 ActionDest: 142 Annotation: Subtype: Link Rect: 218, 629, 225, 641 ActionDest: 78 Annotation: Subtype: Link Rect: 369, 616, 376, 628 ActionDest: 142 Annotation: Subtype: Link Rect: 311, 579, 318, 591 ActionDest: 142 Annotation: Subtype: Link Rect: 200, 264, 207, 276 ActionDest: 154 Annotation: Subtype: Link Rect: 242, 227, 249, 239 ActionDest: 154 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 159, 142, 166, 153 ActionDest: 78 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 260, 517, 266, 528 ActionDest: 78 Annotation: Subtype: Link Rect: 427, 125, 434, 136 ActionDest: 78 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 359, 707, 371, 716 ActionDest: 181 Annotation: Subtype: Link Rect: 283, 694, 295, 703 ActionDest: 181 Annotation: Subtype: Link Rect: 537, 617, 544, 628 ActionDest: 78 Annotation: Subtype: Link Rect: 436, 569, 448, 578 ActionDest: 181 Annotation: Subtype: Link Rect: 254, 518, 266, 527 ActionDest: 181 Annotation: Subtype: Link Rect: 210, 493, 222, 502 ActionDest: 181 Annotation: Subtype: Link Rect: 399, 468, 411, 477 ActionDest: 181 Annotation: Subtype: Link Rect: 480, 456, 492, 465 ActionDest: 181 Annotation: Subtype: Link Rect: 397, 430, 409, 440 ActionDest: 181 Annotation: Subtype: Link Rect: 443, 418, 455, 427 ActionDest: 181 Annotation: Subtype: Link Rect: 543, 380, 555, 389 ActionDest: 181 Annotation: Subtype: Link Rect: 480, 330, 492, 339 ActionDest: 184 Annotation: Subtype: Link Rect: 192, 305, 204, 314 ActionDest: 181 Annotation: Subtype: Link Rect: 368, 280, 375, 289 ActionDest: 178 Annotation: Subtype: Link Rect: 400, 255, 412, 264 ActionDest: 184 Annotation: Subtype: Link Rect: 333, 104, 345, 113 ActionDest: 181 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 204, 659, 216, 669 ActionDest: 184 Annotation: Subtype: Link Rect: 486, 647, 498, 656 ActionDest: 181 Annotation: Subtype: Link Rect: 247, 634, 258, 643 ActionDest: 184 Annotation: Subtype: Link Rect: 207, 546, 219, 556 ActionDest: 181 Annotation: Subtype: Link Rect: 505, 319, 560, 330 Annotation: Subtype: Link Rect: 166, 307, 241, 318 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 353, 721, 503, 732 Annotation: Subtype: Link Rect: 108, 632, 146, 642 Annotation: Subtype: Link Rect: 153, 598, 191, 608 Annotation: Subtype: Link Rect: 266, 562, 304, 573 Annotation: Subtype: Link Rect: 306, 540, 343, 550 Annotation: Subtype: Link Rect: 140, 517, 178, 527 Annotation: Subtype: Link Rect: 136, 483, 173, 493 Annotation: Subtype: Link Rect: 220, 447, 258, 458 Annotation: Subtype: Link Rect: 237, 425, 275, 435 Annotation: Subtype: Link Rect: 249, 390, 287, 401 Annotation: Subtype: Link Rect: 77, 356, 115, 366 Annotation: Subtype: Link Rect: 189, 321, 226, 332 Annotation: Subtype: Link Rect: 171, 286, 209, 297 Annotation: Subtype: Link Rect: 495, 252, 533, 263 Annotation: Subtype: Link Rect: 177, 229, 214, 240 Annotation: Subtype: Link Rect: 220, 229, 256, 240 Annotation: Subtype: Link Rect: 159, 206, 197, 217 Annotation: Subtype: Link Rect: 519, 183, 557, 194 Annotation: Subtype: Link Rect: 59, 172, 95, 182 Annotation: Subtype: Link Rect: 519, 148, 557, 159 Annotation: Subtype: Link Rect: 59, 137, 95, 148 Annotation: Subtype: Link Rect: 59, 114, 97, 125 Annotation: Subtype: Link Rect: 103, 114, 139, 125 Annotation: Subtype: Link Rect: 355, 91, 392, 102 Annotation: Subtype: Link Rect: 398, 91, 434, 102 Annotation: Subtype: Link Rect: 95, 68, 132, 79 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 156, 710, 194, 720 Annotation: Subtype: Link Rect: 520, 698, 557, 709 Annotation: Subtype: Link Rect: 520, 686, 557, 697 Annotation: Subtype: Link Rect: 59, 676, 95, 686 Annotation: Subtype: Link Rect: 483, 652, 521, 663 Annotation: Subtype: Link Rect: 337, 629, 374, 640 Annotation: Subtype: Link Rect: 145, 607, 182, 617 Annotation: Subtype: Link Rect: 393, 583, 430, 594 Annotation: Subtype: Link Rect: 59, 549, 97, 559 Annotation: Subtype: Link Rect: 103, 549, 139, 559 Annotation: Subtype: Link Rect: 415, 525, 453, 536 Annotation: Subtype: Link Rect: 229, 503, 267, 513 Annotation: Subtype: Link Rect: 59, 469, 97, 479 Annotation: Subtype: Link Rect: 311, 445, 348, 456 Annotation: Subtype: Link Rect: 519, 422, 557, 433 Annotation: Subtype: Link Rect: 104, 388, 141, 398 Annotation: Subtype: Link Rect: 466, 364, 503, 375 Annotation: Subtype: Link Rect: 176, 342, 214, 352 Annotation: Subtype: Link Rect: 485, 318, 523, 329 Annotation: Subtype: Link Rect: 184, 296, 221, 306 Annotation: Subtype: Link Rect: 228, 296, 264, 306 Annotation: Subtype: Link Rect: 318, 272, 356, 283 Annotation: Subtype: Link Rect: 362, 272, 398, 283 Annotation: Subtype: Link Rect: 219, 249, 257, 260 Annotation: Subtype: Link Rect: 474, 226, 511, 237 Annotation: Subtype: Link Rect: 405, 203, 443, 214 Annotation: Subtype: Link Rect: 290, 181, 328, 191 Annotation: Subtype: Link Rect: 520, 157, 557, 168 Annotation: Subtype: Link Rect: 420, 134, 458, 145 Annotation: Subtype: Link Rect: 346, 111, 384, 122 Annotation: Subtype: Link Rect: 104, 77, 142, 88 Annotation: Subtype: Link Rect: 148, 77, 184, 88 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 310, 721, 348, 732 Annotation: Subtype: Link Rect: 354, 721, 390, 732 Annotation: Subtype: Link Rect: 452, 698, 489, 709 Annotation: Subtype: Link Rect: 335, 675, 372, 686 Annotation: Subtype: Link Rect: 378, 675, 414, 686 Annotation: Subtype: Link Rect: 285, 652, 322, 663 Annotation: Subtype: Link Rect: 328, 652, 364, 663 Checksum: 19c7172b Type: CRC32 Checksum: 0ef3672a2c10c38d606f36113344726b Type: MD5 Checksum: 1ed7e449028fcfcf0af744a93d77da9a0a6d4d2f Type: SHA-1