Browsing by Subject "Laser capture microdissection"

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    Altered microbial biogeography in an innate model of colitis
    (Taylor & Francis, 2022) Boger-May, Antonia; Reed, Theodore; LaTorre, Diana; Ruley-Haase, Katelyn; Hoffman, Hunter; English, Lauren; Roncagli, Connor; Overstreet, Anne-Marie; Boone, David; Microbiology and Immunology, School of Medicine
    Changes in the spatial organization, or biogeography, of colonic microbes have been observed in human inflammatory bowel disease (IBD) and mouse models of IBD. We have developed a mouse model of IBD that occurs spontaneously and consistently in the absence of adaptive immunity. Mice expressing tumor necrosis factor-induced protein 3 (TNFAIP3) in intestinal epithelial cells (villin-TNFAIP3) develop colitis when interbred with Recombination Activating 1-deficient mice (RAG1−/−). The colitis in villin-TNFAIP3 × RAG1−/− (TRAG) mice is prevented by antibiotics, indicating a role for microbes in this innate colitis. We therefore explored the biogeography of microbes and responses to antibiotics in TRAG colitis. Laser capture microdissection and 16S rRNA sequencing revealed altered microbial populations across the transverse axis of the colon as the inner mucus layer of TRAG, but not RAG1−/−, mice was infiltrated by microbes, which included increased abundance of the classes Gammaproteobacteria and Actinobacteria. Along the longitudinal axis differences in the efficacy of antibiotics to prevent colitis were evident. Neomycin was most effective for prevention of inflammation in the cecum, while ampicillin was most effective in the proximal and distal colon. RAG1−/−, but not TRAG, mice exhibited a structured pattern of bacterial abundance with decreased Firmicutes and Proteobacteria but increased Bacteroidetes along the proximal to distal axis of the gut. TRAG mice exhibited increased relative abundance of potential pathobionts including Bifidobacterium animalis along the longitudinal axis of the gut whereas others, like Helicobacter hepaticus were increased only in the cecum. Potential beneficial organisms including Roseburia were decreased in the proximal regions of the TRAG colon, while Bifidobacterium pseudolongulum was decreased in the TRAG distal colon. Thus, the innate immune system maintains a structured, spatially organized, gut microbiome along the transverse and longitudinal axis of the gut, and disruption of this biogeography is a feature of innate immune colitis.
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    Laser Capture Microdissection in the Spatial Analysis of Epigenetic Modifications in Skin: A Comprehensive Review
    (Hindawi, 2022-02-09) Bhamidipati, Theja; Sinha, Mithun; Sen, Chandan K.; Singh, Kanhaiya; Surgery, School of Medicine
    Each cell in the body contains an intricate regulation for the expression of its relevant DNA. While every cell in a multicellular organism contains identical DNA, each tissue-specific cell expresses a different set of active genes. This organizational property exists in a paradigm that is largely controlled by forces external to the DNA sequence via epigenetic regulation. DNA methylation and chromatin modifications represent some of the classical epigenetic modifications that control gene expression. Complex tissues like skin consist of heterogeneous cell types that are spatially distributed and mixed. Furthermore, each individual skin cell has a unique response to physiological and pathological cues. As such, it is difficult to classify skin tissue as homogenous across all cell types and across different environmental exposures. Therefore, it would be prudent to isolate targeted tissue elements prior to any molecular analysis to avoid a possibility of confounding the sample with unwanted cell types. Laser capture microdissection (LCM) is a powerful technique used to isolate a targeted cell group with extreme microscopic precision. LCM presents itself as a solution to tackling the problem of tissue heterogeneity in molecular analysis. This review will cover an overview of LCM technology, the principals surrounding its application, and benefits of its application to the newly defined field of epigenomics, in particular of cutaneous pathology. This presents a comprehensive review about LCM and its use in the spatial analysis of skin epigenetics. Within the realm of skin pathology, this ability to isolate tissues under specific environmental stresses, such as oxidative stress, allows a far more focused investigation.