Study for superficial microcirculatory hemodynamics of colorectal adenomas using novel high-resolution magnifying endoscopy
Ontology highlight
ABSTRACT:
DISEASE(S): Colorectal Adenoma
PROVIDER: 2691963 | ecrin-mdr-crc |
REPOSITORIES: ECRIN MDR
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2012-06-22 | E-GEOD-17015 | biostudies-arrayexpress
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| S-EPMC7402393 | biostudies-literature
Project description:<p>This postmortem study examines molecular, genetic and epigenetic signatures in the brains of hundreds of subjects with or without mental disorders conducted by the DIRP NIMH Human Brain Collection Core (HBCC). The brain tissues are obtained under protocols approved by the CNS IRB (<a href="https://clinicaltrials.gov/ct2/show/NCT00001260" target="_blank">NCT00001260</a>), with the permission of the next-of-kin (NOK) through the Offices of the Chief Medical Examiners (MEOs) in the District of Columbia, Northern Virginia and Central Virginia. Additional samples were obtained from the University of Maryland Brain and Tissue Bank (contracts NO1-HD-4-3368 and NO1-HD-4-3383) (<a href="http://www.medschool.umaryland.edu/btbank/" target="_blank">http://www.medschool.umaryland.edu/btbank/</a> and the Stanley Medical Research Institute (<a href="http://www.stanleyresearch.org/brain-research/" target="_blank">http://www.stanleyresearch.org/brain-research/</a>).</p> <p>Clinical characterization, neuropathological screening, toxicological analyses, and dissections of various brain regions were performed as previously described (Lipska et al. 2006; PMID: <a href="https://www.ncbi.nlm.nih.gov/pubmed/16997002" target="_blank">16997002</a>). All patients met DSM-IV criteria for a lifetime Axis I diagnosis of psychiatric disorders including schizophrenia or schizoaffective disorder, bipolar disorder and major depression. Controls had no history of psychiatric diagnoses or addictions.</p> <p><b>SNP array:</b></p> <p>Array-based genotyping was performed on most samples published in this collection. The number of SNPs assayed via Illumina chips varied between 650,000 and 5 Million. Cerebellar tissue was generally used for genotyping studies.</p> <table width="481" border="1"> <tbody> <tr> <td width="64" align="center"> <strong>#</strong> </td> <td width="275"> <strong>Diagnosis</strong> </td> <td width="57" align="center"> <strong>SNP Array</strong> </td> </tr> <tr> <td width="64" align="center">1</td> <td width="275">Anxiety Disorder</td> <td width="57" align="center">1</td> </tr> <tr> <td width="64" align="center">2</td> <td width="275">Autism Spectrum Disorder</td> <td width="57" align="center">13</td> </tr> <tr> <td width="64" align="center">3</td> <td width="275">Bipolar Disorder</td> <td width="57" align="center">114</td> </tr> <tr> <td width="64" align="center">4</td> <td width="275">Control</td> <td width="57" align="center">387</td> </tr> <tr> <td width="64" align="center">5</td> <td width="275">Eating Disorder (ED)</td> <td width="57" align="center">2</td> </tr> <tr> <td width="64" align="center">6</td> <td width="275">Major Depressive Disorder (MDD)</td> <td width="57" align="center">186</td> </tr> <tr> <td width="64" align="center">7</td> <td width="275">Obsessive Compulsive Disorder (OCD)</td> <td width="57" align="center">5</td> </tr> <tr> <td width="64" align="center">8</td> <td width="275">Post-Traumatic Stress Disorder (PTSD)</td> <td width="57" align="center">0</td> </tr> <tr> <td width="64" align="center">9</td> <td width="275">Schizophrenia</td> <td width="57" align="center">220</td> </tr> <tr> <td width="64" align="center">10</td> <td width="275">Other</td> <td width="57" align="center">7</td> </tr> <tr> <td width="64" align="center">11</td> <td width="275">Tic Disorder</td> <td width="57" align="center">3</td> </tr> <tr> <td width="64" align="center">12</td> <td width="275">Undetermined</td> <td width="57" align="center">1</td> </tr> <tr> <td width="64" align="center">13</td> <td width="275">Williams Syndrome</td> <td width="57" align="center">2</td> </tr> </tbody> </table> <p>Table: <i>Numbers of samples in each diagnostic category</i>.</p> <p>DNA extraction: 45-80 mg of cerebellar tissue was pulverized for DNA extractions. The QIAamp DNA mini Kit (Qiagen) method was employed for tissue DNA extraction. The tissue was initially lysed using Tissue Lyser (Qiagen) and extractions were accomplished according to manufacturer's protocol. The DNA was captured in 500uL elution buffer. The concentrations were measured using Thermo Scientific's NanoDrop 1000/NanoDrop ONE. The mean yield was 128.85 uG (+/- 79.48), the mean ratio of 260/280 was 1.87 (+/- 0.105), and the mean ratio of 260/230 was 2.48 (+/-1.75).</p> <p>Genotyping methods: Three types of Illumina Beadarray chips were used: HumanHap650Y, Human1M-Duo, and HumanOmni5M-Quad (San Diego, California). The genotyping was done according to the manufacturer's protocol (Illumina Proprietary, Catalog # WG-901-5003, Part # 15025910 Rev.A, June 2011). Approximately, 400ng DNA was used and each DNA sample was QC tested for 260/280 ratio by nanodrop and DNA band intactness on 2% agarose gel. Briefly, the samples were whole-genome amplified, fragmented, precipitated and resuspended in appropriate hybridization buffer. Denatured samples were hybridized on prepared Bead Array Chips. After hybridization, the Bead Chip oligonucleotides were extended by a single fluorescent labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader, iScan. Normalized bead intensity data obtained for each sample were loaded into the Illumina Genome Studio (Illumina, v.2.0.3) with cluster position files provided by Illumina, and fluorescence intensities were converted into SNP genotypes.</p> <p><b>Microarray:</b></p> <p>We generated RNA expression data using array technology for psychiatric subjects compared to non-psychiatric subjects as controls. We used tissues from three different brain regions i.e. hippocampus, dorsolateral prefrontal cortex (DLPFC), and dura mater for a large cohort of individuals (total number 552 subjects for hippocampus, 800 for DLPFC and 146 for dura). Total RNA was extracted from ~100 mg of tissue using the RNeasy kit (Qiagen) according to the manufacturer's protocol. RNA quality and quantity were examined using the Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc), respectively. Samples with RNA integrity number (RIN) <5 were excluded. Affymetrix 3'IVT express kit protocols (Affymetrix, Inc. catalog#902416) were used to generate the Biotin labeled cRNAs. The Illumina hybridization cocktail containing 2 micrograms of biotin labeled cRNAs was hybridized to the Illumina HumanHT-12_V4 Beadchips. The chips were washed and stained using the standard Illumina protocols and reagents. The Illumina BeadChip Scanner was used to scan the arrays. Gene expression intensities were extracted using the Illumina GenomeStudioV2011.1 software.</p> <table width="481" border="1"> <tbody> <tr> <td width="64" align="center"> <strong>#</strong> </td> <td width="275"> <strong>Diagnosis</strong> </td> <td width="57" align="center"> <strong>DLPFC</strong> </td> <td width="47" align="center"> <strong>Hippo</strong> </td> <td width="39" align="center"> <strong>Dura</strong> </td> </tr> <tr> <td width="64" align="center">1</td> <td width="275">Anxiety Disorder</td> <td width="57" align="center">1</td> <td width="47" align="center">0</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">2</td> <td width="275">Autism Spectrum Disorder</td> <td width="57" align="center">14</td> <td width="47" align="center">6</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">3</td> <td width="275">Bipolar Disorder</td> <td width="57" align="center">90</td> <td width="47" align="center">49</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">4</td> <td width="275">Control</td> <td width="57" align="center">336</td> <td width="47" align="center">270</td> <td width="39" align="center">75</td> </tr> <tr> <td width="64" align="center">5</td> <td width="275">Eating Disorder (ED)</td> <td width="57" align="center">2</td> <td width="47" align="center">1</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">6</td> <td width="275">Major Depressive Disorder (MDD)</td> <td width="57" align="center">144</td> <td width="47" align="center">87</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">7</td> <td width="275">Obsessive Compulsive Disorder (OCD)</td> <td width="57" align="center">5</td> <td width="47" align="center">3</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">8</td> <td width="275">Post-Traumatic Stress Disorder (PTSD)</td> <td width="57" align="center">6</td> <td width="47" align="center">0</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">9</td> <td width="275">Schizophrenia</td> <td width="57" align="center">192</td> <td width="47" align="center">125</td> <td width="39" align="center">71</td> </tr> <tr> <td width="64" align="center">10</td> <td width="275">Other</td> <td width="57" align="center">5</td> <td width="47" align="center">6</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">11</td> <td width="275">Tic Disorder</td> <td width="57" align="center">3</td> <td width="47" align="center">3</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">12</td> <td width="275">Undetermined</td> <td width="57" align="center">1</td> <td width="47" align="center">1</td> <td width="39" align="center">0</td> </tr> <tr> <td width="64" align="center">13</td> <td width="275">Williams Syndrome</td> <td width="57" align="center">2</td> <td width="47" align="center">1</td> <td width="39" align="center">0</td> </tr> </tbody> </table> <p>Table: <i>Numbers of samples in each diagnostic category</i>.</p> <p><b>RNA-Seq of Dorso-lateral prefrontal cortex:</b></p> <p>All brains were collected and the dorsolateral prefrontal cortical (DLPFC) samples dissected at the HBCC, DIRP, NIMH. Dorsolateral prefrontal cortex (DLPFC) specimens were dissected from right or left hemisphere of frozen coronal slabs.</p> <p>The study was funded by the DIRP, NIMH under contract (#HHSN 271201400099C) with Icahn School of Medicine at Mount Sinai,1106402 One Gustave L. Levy Place, Box 3500, New York NY 10029,6574. RNA extraction, library preparation and sequencing were performed under contract at Icahn School of Medicine. The Common Mind Consortium (CMC) provided project management support.</p> <p>RNA isolation: Total RNA from 468 HBCC samples was isolated from approximately 100 mg homogenized tissue from each sample by TRIzol/chloroform extraction and purification with the Qiagen RNeasy kit (Cat#74106) according to manufacturer's protocol. Samples were processed in randomized batches of 12. The order of extraction for schizophrenia, bipolar, and MDD disorders and control samples was assigned randomly with respect to diagnosis and all other sample characteristics. The mean total RNA yield was 24.2 ug (+/- 9.0). The RNA Integrity Number (RIN) was determined by 4200 Agilent TapeStation System. Samples with RIN <5.5 were excluded from the study. Among the remaining samples the mean RIN was 7.5 (+/- 0.9) and the mean ratio of 260/280 was 2.0 (+/- 0.03).</p> <p>DLPFC RNA-Seq quantified expression data are provided for 367 samples. Data were generated, QC'd, processed and quantified as follows: RNA library preparation and sequencing: All samples submitted to the New York Genome Center for RNAseq were prepared for sequencing in randomized batches of 94. The sequencing libraries were prepared using the KAPA Stranded RNAseq Kit with RiboErase (KAPA Biosystems). rRNA was depleted from 1ug of RNA using the KAPA RiboErase protocol that is integrated into the KAPA Stranded RNAseq Kit. The insert size and DNA concentration of the sequencing library was determined on Fragment Analyzer Automated CE System (Advanced Analytical) and Quant-iT PicoGreen (ThermoFisher) respectively.</p> <table width="317" border="1"> <tbody> <tr> <td width="104" align="center"> <strong>Schizophrenia</strong> </td> <td width="67" align="center"> <strong>Bipolar</strong> </td> <td width="68" align="center"> <strong>Control</strong> </td> </tr> <tr> <td align="center">90</td> <td align="center">65</td> <td align="center">212</td> </tr> </tbody> </table> <p>Table: <i>Numbers of samples in each diagnostic category</i>.</p> <p><b>RNA-Seq of subgenual anterior cingulate cortex (sgACC):</b></p> <p>All the 200 post-mortem brain samples (61 controls; 39 bipolar disorder; 46 schizophrenia; 54 major depressive disorder) were collected by the HBCC, DIRP, NIMH.</p> <p>RNA Extraction and Quality Assessment: Tissue from sgACC was pulverized and stored at -80°C. Total RNA was extracted from 50-80 mg of the tissue using QIAGEN RNeasy Lipid Tissue Mini Kit (QIAGEN, Cat. # 74804) with DNase treatment (QIAGEN, Cat. # 79254). The RNA Integrity Number (RIN) for each sample was assessed with high-resolution capillary electrophoresis on the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, California). The concentration of RNA and their 260/280 ratio (2.1+/- 0.032 SD) were determined with NanoDrop (Thermo Scientific).</p> <p>RNA sequencing: Stranded RNA-Seq libraries were constructed after rRNA depletion using Ribo-Zero GOLD (Illumina). RNA sequencing was performed at National Institute of Health Intramural Sequencing Center (NISC).</p> <table width="317" border="1"> <tbody> <tr> <td width="104" align="center"> <strong>Schizophrenia</strong> </td> <td width="67" align="center"> <strong>Bipolar</strong> </td> <td width="68" align="center"> <strong>Control</strong> </td> <td width="78" align="center"> <strong>MDD</strong> </td> </tr> <tr> <td align="center">46</td> <td align="center">39</td> <td align="center">61</td> <td align="center">54</td> </tr> </tbody> </table> <p>Table: <i>Numbers of samples in each diagnostic category</i>.</p> <p><b>Whole Genome Sequencing:</b></p> <p>All brains were collected and dissected at the HBCC, DIRP, NIMH. This study generates whole genome sequencing data using sequencing of DNA in the dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) or cerebellum of 443 individuals with schizophrenia, bipolar disorder and major depressive disorder and non-psychiatric controls.</p> <p>The study was funded by the DIRP, NIMH under contract (#HHSN 271201400099C) with Icahn School of Medicine at Mount Sinai,1106402 One Gustave L. Levy Place, Box 3500, New York NY 10029,6574. DNA extraction, library preparation and sequencing were performed under contract at Icahn School of Medicine. The Common Mind Consortium (CMC) provided project management support.</p> <p>All specimens were dissected from right or left hemisphere of frozen coronal slabs.</p> <p>DNA Library Preparation and Sequencing: All samples submitted to the New York Genome Center for WGS were prepared for sequencing in randomized batches of 95. The sequencing libraries were prepared using the Illumina PCR-free DNA sample preparation Kit. The insert size and DNA concentration of the sequencing library was determined on Fragment Analyzer Automated CE System (Advanced Analytical) and Quant-iT PicoGreen (ThermoFisher) respectively. A quantitative PCR assay (KAPA), with primers specific to the adapter sequence, was used to determine the yield and efficiency of the adaptor ligation process. Performed on the Illumina HiSeqX with 30X coverage.</p> <table width="233" border="1"> <tbody> <tr> <td width="99" align="center"> <strong>Schizophrenia</strong> </td> <td width="67" align="center"> <strong>Bipolar</strong> </td> <td width="68" align="center"> <strong>Control</strong> </td> </tr> <tr> <td align="center"> <strong>115</strong> </td> <td align="center">78</td> <td align="center">230</td> </tr> </tbody> </table> <p>Table: <i>Numbers of samples in each diagnostic category</i>.</p> <p><b>ChIP-Seq:</b></p> <p>All brains were collected and the dorsolateral prefrontal cortical (DLPFC) samples dissected at the HBCC, DIRP, NIMH. This study generates epigenetic data using sequencing of DNA after chromatin immunoprecipitation (ChIP-Seq) for marks H3K4me3 and H3K27ac in the dorsolateral prefrontal cortex (DLPFC). Dorsolateral prefrontal cortex (DLPFC) specimens were dissected from right or left hemisphere of frozen coronal slabs.</p> <p>The study was funded by the DIRP, NIMH under contract (#HHSN 271201400099C) with Icahn School of Medicine at Mount Sinai,1106402 One Gustave L. Levy Place, Box 3500, New York NY 10029,6574. Chromatin precipitation, library preparation and sequencing were performed under contract at Icahn School of Medicine. The Common Mind Consortium (CMC) provided project management support.</p> <p>Chromatin immunoprecipitation (ChIP) assays for histone marks H3K4me3 and H3K27ac were carried out using Native ChIP. Micrococcal Nuclease (MNase) (Sigma, N3755) treatment was used to digest chromatin into mononucleosomes. The following antibodies were used for chromatin pull-down: anti-H3K4me3 (Cell Signaling, Cat# 9751BC, lot 7) and anti-H3K27ac (Active Motif, Cat# 39133, Lot # 31814008). Histone modification-enriched genomic DNA fragments were recovered using Protein A/G magnetic beads (Thermo Scientific, 88803-88938 or Millipore 16-663), and then washed, eluted, and treated with RNAse A and proteinase K. Final ChIP DNA products were isolated using phenol-chloroform extraction followed by ethanol precipitation. The efficiency of each ChIP assay was validated using Qubit concentration measurement and qPCR for positive (GRIN2B, DARPP32) and negative (HBB) control genomic regions. Only ChIP assays that passed quality control were further processed for library preparation and sequencing; this included ChIP DNA that was not detectable on Qubit but showed a good signal and expected enrichment patterns in qPCR.</p> <table width="291" border="1"> <tbody> <tr> <td width="64" align="center"> <strong></strong> </td> <td colspan="3" width="153" align="center"> <strong>HISTONE_MARK</strong> </td> <td width="73" align="center"> <strong></strong> </td> </tr> <tr> <td width="64" align="center"> </td> <td colspan="2" width="77" align="center"> <strong>H3K27ac</strong> </td> <td width="77" align="center"> <strong>H3K4me3</strong> </td> <td width="73" align="center"> <strong>Input</strong> </td> </tr> <tr> <td width="64"> <strong>Bipolar</strong> </td> <td width="73" align="center">56</td> <td colspan="2" width="80" align="center">4</td> <td width="73" align="center"> <strong>7</strong> </td> </tr> <tr> <td width="64"> <strong>Control</strong> </td> <td width="73" align="center">158</td> <td colspan="2" width="80" align="center">11</td> <td width="73" align="center">24</td> </tr> <tr> <td width="64"> <strong>Schizophrenia</strong> </td> <td width="73" align="center">79</td> <td colspan="2" width="80" align="center">11</td> <td width="73" align="center">12</td> </tr> </tbody> </table> <p>Table: <i>Numbers of individuals in each assay grouped by histone mark or input</i>.</p>
| phs000979 | dbGaP
Project description:AAA+ ATPases are a diverse protein superfamily which power a vast number of cellular processes, from protein degradation to genome replication and ribosome biogenesis. The latest advances in cryo-EM have resulted in a spectacular increase in the number and quality of AAA+ ATPase structures. This abundance of new information enables closer examination of different types of structural insertions into the conserved core, revealing discrepancies in the current classification of AAA+ modules into clades. Additionally, combined with biochemical data, it has allowed rapid progress in our understanding of structure-functional relationships and provided arguments both in favour and against the existence of a unifying molecular mechanism for the ATPase activity and action on substrates, stimulating further intensive research.
| S-EPMC7973254 | biostudies-literature