3Background: Developmental and reproductive toxicology (DART) testing represents an expensive and time-consuming stage in determining the toxicological profile of new chemical entities. Within DART studies, morphological evaluation of fetal skeletons for developmental abnormalities typically requires 7 to 14 days. Current processing techniques involve digestion of soft tissue using a strong base (KOH), followed by qualitative assessment of the remaining skeletal tissue by a fetal morphologist. Microcomputed tomography (micro-CT) has been proposed as a nondestructive image-based alternative for quantitative assessment of skeletal morphology. Such methods eliminate the need for extensive tissue processing and can be paired with quantitative analysis algorithms. However, due to the significant capital and operational expenses required for micro-CT imaging, this approach has yet to gain widespread traction and regulatory acceptance. Methods: A novel tissue clearing agent was used in 1-week-old rats to render soft tissue optically transparent. Alizarin red was used to stain the skeleton.High dynamic range optical trans-illumination images were then acquired with an optical-CT imaging system and rendered as three-dimensional images of skeletal structure. Results: High dynamic range-based optical-CT imaging of chemically cleared tissues can rapidly generate high resolution (50-250 lm) reconstructions of whole skeletons. Conclusion: In summary, this study demonstrates that the combination of tissue clearing, optical imaging, and novel reconstruction algorithms may present a new paradigm for highthroughput evaluation of tissues in DART testing.
In the last few years several companies have developed 3D in vitro cell culture models and have demonstrated that these 3D models more accurately depict in vivo characteristics compared to traditional 2D monolayer cell culture. Because of this improved in vivo relevancy, these models are being rapidly adopted in the drug discovery space as they can more accurately predict in vivo efficacy and toxicological characteristics. Of the different types of 3D cell culture models being used today, pancreatic islet microtissues have been shown to be a powerful tool for assessing the effectiveness of compounds on diabetes. However, current imaging approaches (e.g. confocal microscopy, wide‐field microscopy) are limited in their ability to completely characterize these 3D models as light attenuation limits imaging depth to approx. 1–3 cell layers. Therefore, current imaging techniques are not able to completely characterize the effect of compounds on beta cell proliferation. Through this work, pancreatic islet microtissues of approx. 250 μm diameter were exposed to several compounds that induce beta cell proliferation and the microtissues were then stained for EdU or Ki67, glucagon, insulin and DAPI. The labeled microtissues were then rendered transparent with a rapid and plate‐compatible tissue clearing technique that allows for the microtissues to be imaged in their entirety using high content confocal imaging. It was shown through this work that the addition of a tissue clearing technique resulted in a 3‐fold increase in the number of cells characterized in the microtissues compared to traditional wide‐field or confocal microscopy.Support or Funding InformationThis work was support by Visikol Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The type I keratin 14 (K14) is mainly expressed in the basal layer of epidermis and related stratified epithelia. Previous work revealed a role for inter-keratin disulfide bonding involving residue cysteine (Cys) 367 of human K14 toward the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in culture. To define the function of K14-dependent disulfide bonding in skin epithelia in vivo, we generated Krt14C373A mice using the Crispr/ Cas9 technology (mouse Cys373 is orthologous to human Cys367). Western blotting revealed a marked decrease in disulfide-bonded K14 species in Krt14C373A ear and tail skin relative to control. Morphological analyses revealed a state of hyperproliferation and hyperkeratosis, dysregulated keratinocyte differentiation, and abnormal cornified envelopes in Krt14C373A skin. Adult Krt14C373A mice show a skin barrier defect at baseline, reflected by increased trans-epidermal water loss. The latter trait is enhanced following topical acetone irritation. Mass spectrometry-based assays identified 14-3-3 as a major K14 interacting protein, and follow-up studies confirmed that 14-3-3 interacts with both WT and C373A mutant forms. 14-3-3 forms aggregates in Krt14C373A keratinocytes in situ, accompanied by abnormal nuclear localization of Yap1, a transcriptional effector of Hippo signaling, in suprabasal differentiated keratinocytes. Mutagenesis shows that the subcellular distribution and regulation of 14-3-3 and Yap1 is specifically regulated by residue Cys373 in mouse K14. The results obtained to date establish that K14-dependent disulfide bonding plays a physiologically important role in epidermal homeostasis and barrier function by impacting the solubility, regulation and function of 14-3-3/Yap1.
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