Evaluation of homogenization techniques for the preparation of mouse tissue samples to support drug discovery

Bioanalysis ◽  
2011 ◽  
Vol 3 (17) ◽  
pp. 1923-1933 ◽  
Author(s):  
Xiaorong Liang ◽  
Savita Ubhayakar ◽  
Bianca M Liederer ◽  
Brian Dean ◽  
Ann Ran-Ran Qin ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mouse Tissue ◽  
Tissue Samples

Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples

2017 ◽  
Vol 38 (12) ◽  
pp. 1602-1608 ◽  
Author(s):  
Boglarka Donczo ◽  
Mate Szarka ◽  
Jozsef Tovari ◽  
Gyorgyi Ostoros ◽  
Eszter Csanky ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Mouse Tissue ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals XenoSarc: A comprehensive platform of patient-derived xenograft (PDX) models of soft tissue sarcoma (STS) for early drug testing.

2019 ◽  
Vol 5 (suppl) ◽  
pp. 37-37 ◽  
Author(s):  
Patrick Schoffski ◽  
Britt Van Renterghem ◽  
Jasmien Cornillie ◽  
Yannick Wang ◽  
Yemarshet Kelemework Gebreyohannes ◽  
...  
Keyword(s):  
Drug Testing ◽  
Treatment Options ◽  
Clinical Information ◽  
Tissue Microarrays ◽  
Mouse Tissue ◽  
Tissue Samples ◽  
Pdx Models ◽  
Early Drug

37 Background: STS is a family of rare, heterogeneous tumors with > 70 subtypes. There is an urgent need for reliable preclinical models, especially for orphan subtypes of STS, given the limited treatment options. Methods: A panel of PDX models was established by s.c. implantation of fresh tumor specimens in athymic NMRI mice. Growing pieces of tumor were re-transplanted to next generations of mice. At each passage fragments were collected for histological/molecular characterization. A model was considered “established” after observing stable features for at least 2 passages. Ex-mouse tissue samples were stored, characterized by immunohistochemistry/flow cytometry and used for in vitro drug testing. Results: Between 2011-2019, 329 samples from 301 consenting patients were transplanted; 56 models are established, 16 additional models are in early passaging. Clinical information about donor and tumor (including sensitivity to standard and experimental agents) is available. The platform includes models of dedifferentiated lipo- (10 models), myxofibro- (8), leiomyo- (7), synovial (2), intimal (2), CIC-positive round cell (1), mesenchymal chondro- (1), extraskeletal osteo- (1), myxoid lipo- (1), myxoinflammatory fibroblastic (1), rhabdomyo- (2) and high-grade undifferentiated pleomorphic sarcoma (7), as well as GIST (8), MPNST (4) and epithelioid hemangioendothelioma (1). Models are well-characterized, with molecular information on copy number changes (low-coverage whole genome sequencing) and gene expression profile (RNA-Seq) available. We also constructed tissue microarrays from the xenografts which are used for target identification and model selection for preclinical studies. Xenografts are available for in vivo testing of novel agents, and results already served as a rationale for a number of prospective clinical trials. Conclusions: XenoSarc offers opportunities for studying the biology of a variety of sarcoma subtypes including ultra-rare entities and is a valuable tool for early drug screening in preparation of clinical STS trials. The platform is well maintained and continuously expanded, and available to collaborators from academia and industry.

scholarly journals Biosynthesis of the Novel Endogenous 15-Lipoxygenase Metabolites N-13-Hydroxy-octodecadienoyl-ethanolamine and 13-Hydroxy-octodecadienoyl-glycerol by Human Neutrophils and Eosinophils

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2322
Author(s):  
Anne-Sophie Archambault ◽  
Francesco Tinto ◽  
Élizabeth Dumais ◽  
Volatiana Rakotoarivelo ◽  
Magdalena Kostrzewa ◽  
...  
Keyword(s):  
Positive Control ◽  
Substrate Preference ◽  
Mouse Tissue ◽  
Human Neutrophils ◽  
Soybean Lipoxygenase ◽  
The Novel ◽  
Lipoxygenase Pathway ◽  
Tissue Samples ◽  
Physiological Processes ◽  
Arachidonoyl Glycerol

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N-acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N-acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1Z,4Z-pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N-13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G), the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15–30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

Characterization of Tumor-immune Microenvironment by High-throughput Image Analysis of CD8 Immunohistochemistry Combined With Modified Masson’s Trichrome

2021 ◽  
pp. 002215542110349
Author(s):  
Charles Havnar ◽  
Shari Lau ◽  
Jeffrey Hung ◽  
Jeff Eastham-Anderson ◽  
Carmina Espiritu ◽  
...  
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Checkpoint Inhibitors ◽  
Cytotoxic T Cells ◽  
Mouse Tissue ◽  
Flexible Tool ◽  
Tissue Samples ◽  
Automated Method ◽  
Tumor Immune Microenvironment ◽  
Tumor Microenviroment

With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson’s trichrome. Slides are scanned and digital images are analyzed using an adjustable MATLAB algorithm, allowing for high-throughput quantification of cytotoxic T-cells and collagen. This method provides a flexible tool for unbiased quantification of T-cells and their interactions with tumor cells and tumor microenvironment in tissue samples.

scholarly journals Mechanisms of ischemic skeletal muscle regeneration mediated by mechanically constrained human allogeneic mesenchymal stromal cells

2020 ◽  
Vol 3 ◽  
Author(s):  
Alex O'Connor ◽  
Steven Miller ◽  
Michael Loke ◽  
Chang-Hyun Gil ◽  
Katherin Leckie ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Therapy ◽  
Muscle Regeneration ◽  
Significant Risk ◽  
Skeletal Muscle Regeneration ◽  
Mouse Tissue ◽  
Diabetic Patients ◽  
Tissue Samples ◽  
Encapsulated Cells ◽  
Starting Point

Background and Hypothesis: Critical limb threatening ischemia (CLTI) is the end stage of peripheral arterial disease (PAD). CLTI presents a significant risk for lower extremity amputation, especially in diabetic patients with poor options for revascularization. Additionally, using a novel diabetic mouse model of CLTI, we have shown that IM injection of 3D cultured MSCs (spheroids) is more effective in promoting skeletal muscle regeneration of ischemic muscle than monolayer cultured MSC. We hypothesize that this result is due to the mechanical constrained nature of the MSC in spheroid form, which has been shown to alter the cellular phenotype. Alginate encapsulated cells are another form of mechanical confinement, and have additional benefits including resistance to immunological attack, making them a practical therapy for treatment of CLTI patients.    Project Methods: In this work, cells were encapsulated in 2% alginate using a centrifugation method. Media from these cells along with others were analyzed for IL-10 and IL-33 using ELISA. Mouse tissue samples were stained with WGA-555 antibody and analyzed using a scanning microscope. Effects on tissue perfusion were measured with Laser Doppler Perfusion Imaging. At the time of this abstract, the media from encapsulated and naked MSCs is being used to culture myoblasts, looking at cell growth.    Results: ELISA results did not show significant increases in IL-10 or IL-33 for encapsulated vs. non-encapsulated cells. LDPI has shown an increased perfusion rate for hindlimbs treated with encapsulated MSCs vs naked MSCs. Muscle fiber analysis is ongoing, but initial data appears promising.     Conclusion and Potential Impact: This experiment provides a starting point for improving and expanding cell therapy for critical limb ischemia, potentially resulting in better outcomes for diabetic patients and preventing lower limb amputations. The encapsulation process also has value in other types of cell therapy, as it could protect cells from host defenses and increase dwell time. 

scholarly journals The expression of endothelin type A and B receptors in the lateral wall of the mouse cochlea

2007 ◽  
Vol 12 (4) ◽  
Author(s):  
Yan Luo ◽  
Yuedi Tang ◽  
Qingjie Xia ◽  
Jin Liu
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Lateral Wall ◽  
Type A ◽  
Mouse Tissue ◽  
Receptor Subtypes ◽  
Biological Functions ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Polymerase Chain

AbstractEndothelin (ET), originally characterized as a vasoconstrictive peptide, has been found to have many different biological functions, including acting as a local hormonal regulator of pressure, fluid, ions and neurotransmitters in the inner ear. The objective of this study was to examine and quantify the mRNA expression of the endothelin type A and B receptors (ETAR and ETBR) in the strial vascularies (StV) and non-strial tissues (NSt) of the cochlear lateral wall using the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The mouse tissue samples were harvested and RNA was extracted. RT was performed to obtain cDNA, and then the mRNA expression of each gene was measured via real-time PCR. We found that both receptor subtypes were expressed in the cochlear lateral wall, with a predominance of ETAR over ETBR. We showed that the mRNA expression of the two receptor subtypes was higher in the StV with a 1.8 times higher level of ETAR and an 8.1 times higher level of ETBR mRNAs than in the adjacent NSt of the lateral wall tissue. This study shows the existence and the quantity of ET receptor subtypes in the StV and NSt of the mouse cochlea. Our results suggest that an endothelin-mediated response via two different receptors, ETAR and ETBR, may play an important role in the physiological functions of the cochlear lateral wall by maintaining the homeostatic environment of the cochlea.

scholarly journals Rapid and Sensitive Quantification ofBorrelia burgdorferi-Infected Mouse Tissues by Continuous Fluorescent Monitoring of PCR

1999 ◽  
Vol 37 (4) ◽  
pp. 987-992 ◽  
Author(s):  
Tom B. Morrison ◽  
Ying Ma ◽  
John H. Weis ◽  
Janis J. Weis
Keyword(s):  
Absolute Quantification ◽  
Product Formation ◽  
Mouse Tissue ◽  
Pcr Analysis ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Double Stranded Dna ◽  
Mouse Tissues ◽  
External Standard ◽  
Pcr Method

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization ofB. burgdorferi quantities to the mouse nidogengene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.

scholarly journals LM-GlycomeAtlas Ver. 1.0: A Novel Visualization Tool for Lectin Microarray-Based Glycomic Profiles of Mouse Tissue Sections

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2962 ◽  
Author(s):  
Chiaki Nagai-Okatani ◽  
Kiyoko F Aoki-Kinoshita ◽  
Shuichi Kakuda ◽  
Misugi Nagai ◽  
Kozue Hagiwara ◽  
...  
Keyword(s):  
Protein Glycosylation ◽  
Mouse Tissue ◽  
Tissue Samples ◽  
Online Tool ◽  
Lectin Microarray ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Reproducible Method ◽  
Formalin Fixed

For the effective discovery of the biological roles and disease-specific alterations concerning protein glycosylation in tissue samples, it is important to know beforehand the quantitative and qualitative variations of glycan structures expressed in various types of cells, sites, and tissues. To this end, we used laser microdissection-assisted lectin microarray (LMA) to establish a simple and reproducible method for high-throughput and in-depth glycomic profiling of formalin-fixed paraffin-embedded tissue sections. Using this “tissue glycome mapping” approach, we present 234 glycomic profiling data obtained from nine tissue sections (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of two 8-week-old male C57BL/6J mice. We provided this LMA-based dataset in the similar interface as that of GlycomeAtlas, a previously developed tool for mass spectrometry-based tissue glycomic profiling, allowing easy comparison of the two types of data. This online tool, called “LM-GlycomeAtlas”, allows users to visualize the LMA-based tissue glycomic profiling data associated with the sample information as an atlas. Since the present dataset allows the comparison of glycomic profiles, it will facilitate the evaluation of site- and tissue-specific glycosylation patterns. Taking advantage of its extensibility, this tool will continue to be updated with the expansion of deposited data.

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