Batrachochytrium dendrobatidis and B. salamandrivorans are important amphibian pathogens responsible for morbidity and mortality in free-ranging and captive frogs, salamanders, and caecilians. While B. dendrobatidis has a widespread global distribution, B. salamandrivorans has only been detected in amphibians in Asia and Europe. Although molecular detection methods for these fungi are well-characterized, differentiation of the morphologically similar organisms in the tissues of affected amphibians is incredibly difficult. Moreover, an accurate tool to identify and differentiate Batrachochytrium in affected amphibian tissues is essential for a specific diagnosis of the causative agent in chytridiomycosis cases. To address this need, an automated dual-plex chromogenic RNAScope®
in situ hybridization (ISH) assay was developed and characterized for simultaneous detection and differentiation of B. dendrobatidis and B. salamandrivorans. The assay, utilizing double Z target probe pairs designed to hybridize to 28S rRNA sequences, was specific for the identification of both organisms in culture and in formalin-fixed paraffin-embedded amphibian tissues. The assay successfully identified organisms in tissue samples from five salamander and one frog species preserved in formalin for up to 364 days and was sensitive for the detection of Batrachochytrium in animals with qPCR loads as low as 1.1 × 102 zoospores/microliter. ISH staining of B. salamandrivorans also highlighted the infection of dermal cutaneous glands, a feature not observed in amphibian B. dendrobatidis cases and which may play an important role in B. salamandrivorans pathogenesis in salamanders. The developed ISH assay will benefit both amphibian chytridiomycosis surveillance projects and pathogenesis studies by providing a reliable tool for Batrachochytrium differentiation in tissues.
Pheochromocytomas (PCs) are tumors arising from the chromaffin cells of the adrenal glands and are the most common tumors of the adrenal medulla in animals. In people, these are highly correlated to inherited gene mutations in the succinate dehydrogenase (SDH) pathway; however, to date, little work has been done on the genetic basis of these tumors in animals. In humans, immunohistochemistry has proven valuable as a screening technique for SDH mutations. Human PCs that lack succinate dehydrogenase B (SDHB) immunoreactivity have a high rate of mutation in the SDH family of genes, while human PCs lacking succinate dehydrogenase A (SDHA) immunoreactivity have mutations in the SDHA gene. To determine if these results are similar for dogs, we performed SDHA and SDHB immunohistochemistry on 35 canine formalin-fixed, paraffin-embedded (FFPE) PCs. Interestingly, there was a loss of immunoreactivity for both SDHA and SDHB in four samples (11%), suggesting a mutation in SDHx including SDHA. An additional 25 (71%) lacked immunoreactivity for SDHB, while retaining SDHA immunoreactivity. These data suggest that 29 out of the 35 (82%) may have an SDH family mutation other than SDHA. Further work is needed to determine if canine SDH immunohistochemistry on PCs correlates to genetic mutations that are similar to human PCs.
Perspective on This Article from Constitutional Methylation of the <i>BRCA1</i> Promoter Is Specifically Associated with <i>BRCA1</i> Mutation-Associated Pathology in Early-Onset Breast Cancer
Many autoimmune diseases, including multiple sclerosis (MS) exhibit a striking female bias, but the underlying mechanisms remain poorly understood. Using the SJL mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which recapitulates this sex dimorphism, we previously demonstrated that preferential IL-33 expression contributes to male protection. IL-33 expression by mast cells in PLP139–151-immunized males activates type 2 innate lymphoid cells, which in turn drives a non-pathogenic Th2 response to myelin peptide. Testosterone directly activates Il33 in male but not female mast cells, but this male-specific response is also maintained with other modes of activation, including FcɛR1-crosslinking, suggesting there are constraints on the chromatin landscape that limit Il33 expression in females. Here we use ChIP and ATAC-seq to show that the dynamics of chromatin modifications at the Il33 locus is sex-dimorphic. At steady state, ChIP assays reveal activating H3K4me3 and H3K9ac histone modifications at the promoter and conserved non-coding sequences within the Il33 gene are most prevalent in male-derived cells, while H3K27me3 repressive marks dominate in females. This pattern is similar in IgE-DNP-activated mast cells. Increased basal Il33 chromatin accessibility assessed by ATAC-seq is evident in males. Together, these results indicate that while testosterone can directly stimulate IL-33 production, it also exerts effects on Il33 during development conferring a higher potential for expression in males.
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