FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues

The extent of damage surrounding an implanted electrode in the cerebral cortex is a question of significant importance with regard to attaining consistency and validity of physiological recordings. In order to determine the extent of such tissue changes, 150 micron diameter platinum electrodes were implanted in the cortex of four adult baboons, and after eight days the animals were sacrificed by whole body perfusion with a 3% glutaraldehyde in 0.1M phosphate fixative.The calvarium was carefully removed and the electrode tracts were readily discernible in the firm, glutaraldehyde fixed tissue.Careful dissection of the zone of the electrode tract resulted in a small block which was further sectioned into tip, mid-tract and surface areas. Ultrastructurally, damage extended from the electrode sheath to the greatest extent of from 0.2 to 3.5 mm.

Download Full-text

A New Human Breast Tumor Cell Line

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115560 ◽

1975 ◽

Vol 33 ◽

pp. 388-389

Author(s):

R.E. Nordquist ◽

R.M. Wasik ◽

P.J. Riggs ◽

P.L. Munson ◽

F.B. Schafer

Keyword(s):

Electron Microscopy ◽

Cell Line ◽

Calf Serum ◽

Tumor Cell Line ◽

Electron Microscopic Examination ◽

Epithelial Cell Line ◽

Electron Microscopic ◽

Fixed Tissue ◽

Excised Tissue ◽

Caucasian Female

An infiltrating ductal cell carcinoma was removed from the breast of a postmenopausal Caucasian female. The excised tissue was divided into three parts; one part for electron microscopy, one part for tissue culture and the remainder frozen for immunological studies.The tissue for culture was minced finely with sterile razor blades and cultured in Falcon flasks containing Eagel's MEM supplemented with 10% heat denatured fetal calf serum. The tissue for electron microscopy was fixed in 6.25% glutaraldehyde in 0.1 M PO4 buffer plus 5% sucrose and postfixed in 1% OsO4 in the same buffer. The fixed tissue was dehydrated in graded ethanol and embedded in Spurr.The tissue which was cultured began to grow out after approximately six weeks and became a continuous epithelial cell line which was designated BOT-2 (Breast Original Tumor). Electron microscopic examination revealed that these cells had epithelial characteristics, i.e. the presence of tonofilaments and well formed desmosomes.

Download Full-text

Microvasculature and structure of hemal lymph nodes in the wall of the rabbit bladder: a vascular corrosion casting and EM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141743 ◽

1995 ◽

Vol 53 ◽

pp. 1074-1075

Author(s):

F.E. Hossler ◽

M.I. McKamey ◽

F.C. Monson

Keyword(s):

Lymph Nodes ◽

Light Microscopy ◽

Corrosion Casting ◽

Fixed Tissue ◽

Infusion Pressure ◽

India Ink ◽

Cacodylate Buffer ◽

Rabbit Bladder ◽

Lateral Walls ◽

Comprehensive Study

A comprehensive study of the microvasculature of the normal rabbit bladder, revealed unusual "capillary glomeruli" along the lateral walls. Here they are characterized as hemal lymph nodes using light microscopy, SEM, TEM, ink injection, and vascular casting.Bladders were perfused via a cannula placed in the abdominal aorta with either 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for fixation, 10% India ink in 0.9% saline and 0.1M phosphate (pH 7.4) for vessel tracing, or resin (Mercoximethylmethacrylate: catalyst, 4:1:0.3; Ladd Research Industries) for vascular corrosion casting. Infusion pressure was 100mm Hg. Fixed tissue was sectioned from epon-araldyte resin, and stained with toluidine blue for light microscopy, and lead and uranium for TEM. Ink injected tissue was photographed directly from saline-filled bladders illuminated from below. Resin-filled tissue was macerated in 5% KOH and distilled water. Casts were critical point dried, sputter coated with goldpalladium, and examined by routine SEM at 10 KV.

Download Full-text

Severe Antithrombin III Deficiency in an Infant Associated with Multiple Arterial and Venous Thromboses

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648068 ◽

1976 ◽

Vol 36 (03) ◽

pp. 495-502 ◽

Cited By ~ 14

Author(s):

Geoffrey Mendelsohn ◽

Edward D. Gomperts ◽

Dennis Gurwitz

Keyword(s):

Antithrombin Iii ◽

Adult Life ◽

Rare Condition ◽

Male Infant ◽

Liver Cells ◽

Liver Pathology ◽

Fixed Tissue ◽

Formalin Fixed Tissue ◽

First Case ◽

At Iii

SummaryInherited antithrombin III (AT-II, heparin cofactor) deficiency is a rare condition, presenting with thrombotic disease in adult life. This paper reports an 8 months old South African Black male infant with multiple large vessel venous and arterial thromboses, and E. coli septicaemia. This was associated with an extremely low plasma AT-II level. Micronodular cirrhosis and intracytoplasmic hyaline globules in the liver cells were present. These globules were eosinophilic, and PAS-positive after diastase. They measured approximately 5 μ to 30 μ in diameter, occurred singly in the liver cells and were located mainly in the periportal areas. The histological findings in the liver are similar to those observed in α1-antitrypsin (AAT) deficiency in which the intracytoplasmic globules represent accumulation of altered AAT. Immunochemical studies carried out on formalin fixed tissue failed to detect cross reaction material with anti-α1 antitrypsin or anti-AT III antiserum. This is the first case report of AT-III deficiency presenting in infancy. It is also the first case associated with distinctive liver pathology.The available data presented are insufficient to distinguish between an inborn defect and acquired causes of the severely depressed AT-III plasma level and the distinctive liver pathology.

Download Full-text

Fast Cancer Molecular Diagnosis from FFPE Tissues Based on Metabolic Profiles Using SpiderMass Technology

SSRN Electronic Journal ◽

10.2139/ssrn.3732373 ◽

2020 ◽

Author(s):

Nina Ogrinc ◽

Pierre-Damien Caux ◽

Yves-Marie Robin ◽

Emmanuel Bouchaert ◽

Benoit Fatou ◽

...

Keyword(s):

Molecular Diagnosis ◽

Metabolic Profiles ◽

Ffpe Tissues

Download Full-text

The KLF6 Super Enhancer Modulates Cell Proliferation via MiR-1301 in Human Hepatoma Cells

MicroRNA ◽

10.2174/2211536608666190314122725 ◽

2019 ◽

Vol 9 (1) ◽

pp. 64-69 ◽

Cited By ~ 1

Author(s):

KumChol Ri ◽

Chol Kim ◽

CholJin Pak ◽

PhyongChol Ri ◽

HyonChol Om

Keyword(s):

Cell Proliferation ◽

Cellular Proliferation ◽

Hepatoma Cells ◽

Regulation Mechanism ◽

Dependent Manner ◽

Human Hepatoma ◽

Human Hepatoma Cells ◽

Over Expression ◽

Super Enhancer ◽

Engineering Changes

Background: Recent studies have attempted to elucidate the function of super enhancers by means of microRNAs. Although the functional outcomes of miR-1301 have become clearer, the pathways that regulate the expressions of miR-1301 remain unclear. Objective: The objective of this paper was to consider the pathway regulating expression of miR- 1301 and miR-1301 signaling pathways with the inhibition of cell proliferation. Methods: In this study, we prepared the cell clones that the KLF6 super enhancer was deleted by means of the CRISPR/Cas9 system-mediated genetic engineering. Changes in miR-1301 expression after the deletion of the KLF6 super enhancer were evaluated by RT-PCR analysis, and the signal pathway of miR-1301 with inhibition of the cell proliferation was examined using RNA interference technology. Results: The results showed that miR-1301 expression was significantly increased after the deletion of the KLF6 super enhancer. Over-expression of miR-1301 induced by deletion of the KLF6 super enhancer also regulated the expression of p21 and p53 in human hepatoma cells. functional modeling of findings using siRNA specific to miR-1301 showed that expression level changes had direct biological effects on cellular proliferation in Human hepatoma cells. Furthermore, cellular proliferation assay was shown to be directly associated with miR-1301 levels. Conclusion: As a result, it was demonstrated that the over-expression of miR-1301 induced by the disruption of the KLF6 super enhancer leads to a significant inhibition of proliferation in HepG2 cells. Moreover, it was demonstrated that the KLF6 super enhancer regulates the cell-proliferative effects which are mediated, at least in part, by the induction of p21and p53 in a p53-dependent manner. Our results provide the functional significance of miR-1301 in understanding the transcriptional regulation mechanism of the KLF6 super enhancer.

Download Full-text

EphA2 super-enhancer promotes tumor progression by recruiting FOSL2 and TCF7L2 to activate the target gene EphA2

Cell Death and Disease ◽

10.1038/s41419-021-03538-6 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Shuang Cui ◽

Qiong Wu ◽

Ming Liu ◽

Mu Su ◽

ShiYou Liu ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Target Gene ◽

Super Enhancer ◽

Proliferation And Metastasis ◽

Active Transcription ◽

Hct 116 ◽

Large Clusters

AbstractSuper-enhancers or stretch enhancers (SEs) consist of large clusters of active transcription enhancers which promote the expression of critical genes that define cell identity during development and disease. However, the role of many super-enhancers in tumor cells remains unclear. This study aims to explore the function and mechanism of a new super-enhancer in various tumor cells. A new super-enhancer that exists in a variety of tumors named EphA2-Super-enhancer (EphA2-SE) was found using multiple databases and further identified. CRISPR/Cas9-mediated deletion of EphA2-SE results in the significant downregulation of its target gene EphA2. Mechanistically, we revealed that the core active region of EphA2-SE comprises E1 component enhancer, which recruits TCF7L2 and FOSL2 transcription factors to drive the expression of EphA2, induce cell proliferation and metastasis. Bioinformatics analysis of RNA-seq data and functional experiments in vitro illustrated that EphA2-SE deletion inhibited cell growth and metastasis by blocking PI3K/AKT and Wnt/β-catenin pathway in HeLa, HCT-116 and MCF-7 cells. Overexpression of EphA2 in EphA2-SE−/− clones rescued the effect of EphA2-SE deletion on proliferation and metastasis. Subsequent xenograft animal model revealed that EphA2-SE deletion suppressed tumor proliferation and survival in vivo. Taken together, these findings demonstrate that EphA2-SE plays an oncogenic role and promotes tumor progression in various tumors by recruiting FOSL2 and TCF7L2 to drive the expression of oncogene EphA2.

Download Full-text

GATA2 regulates mast cell identity and responsiveness to antigenic stimulation by promoting chromatin remodeling at super-enhancers

Nature Communications ◽

10.1038/s41467-020-20766-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yapeng Li ◽

Junfeng Gao ◽

Mohammad Kamran ◽

Laura Harmacek ◽

Thomas Danhorn ◽

...

Keyword(s):

Mast Cell ◽

Binding Sites ◽

Cell Lineage ◽

Allergic Inflammation ◽

Chromatin Accessibility ◽

Antigenic Stimulation ◽

Parasitic Infections ◽

Cell Identity ◽

Super Enhancer ◽

Determining Factors

AbstractMast cells are critical effectors of allergic inflammation and protection against parasitic infections. We previously demonstrated that transcription factors GATA2 and MITF are the mast cell lineage-determining factors. However, it is unclear whether these lineage-determining factors regulate chromatin accessibility at mast cell enhancer regions. In this study, we demonstrate that GATA2 promotes chromatin accessibility at the super-enhancers of mast cell identity genes and primes both typical and super-enhancers at genes that respond to antigenic stimulation. We find that the number and densities of GATA2- but not MITF-bound sites at the super-enhancers are several folds higher than that at the typical enhancers. Our studies reveal that GATA2 promotes robust gene transcription to maintain mast cell identity and respond to antigenic stimulation by binding to super-enhancer regions with dense GATA2 binding sites available at key mast cell genes.

Download Full-text