Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a ...substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.
Even when successfully induced, immunological tolerance to solid organs remains vulnerable to inflammatory insults, which can trigger rejection. In a mouse model of cardiac allograft tolerance in ...which infection with Listeria monocytogenes (Lm) precipitates rejection of previously accepted grafts, we showed that recipient CD4+ TCR75 cells reactive to a donor MHC class I-derived peptide become hypofunctional if the allograft is accepted for more than 3 weeks. Paradoxically, infection-induced transplant rejection was not associated with transcriptional or functional reinvigoration of TCR75 cells. We hypothesized that there is heterogeneity in the level of dysfunction of different allospecific T cells, depending on duration of their cognate antigen expression. Unlike CD4+ TCR75 cells, CD4+ TEa cells specific for a peptide derived from donor MHC class II, an alloantigen whose expression declines after transplantation but remains inducible in settings of inflammation, retained function in tolerant mice and expanded during Lm-induced rejection. Repeated injections of alloantigens drove hypofunction in TEa cells and rendered grafts resistant to Lm-dependent rejection. Our results uncover a functional heterogeneity in allospecific T cells of distinct specificities after tolerance induction and reveal a strategy to defunctionalize a greater repertoire of allospecific T cells, thereby mitigating a critical vulnerability of tolerance.
Both gram negative and gram positive bacteria have enzymes for metabolizing alkanes. The alpha proteobacteria Caulobacter crescentus has a medium‐chain alkane hydroxylase that is a member of the ...CYP153 family of cytochrome P450 enzymes. The molecular mechanism controlling transcription of this hydroxylase has not been described. We carried out a reverse genetics screen and RNA‐seq to find the transcription factor regulating this alkane hydroxylase. A deletion of the transcription factor, named CoxR, resulted in upregulation of both the alkane hydroxylase as well as ferredoxin and ferredoxin reductase enzymes. Gel shift assays demonstrated that CoxR bound directly to the promoter of the alkane hydroxylase. In conclusion, we have identified a novel transcription factor involved in metabolism of alkanes.
Abstract only
Some gram negative and gram positive bacteria have evolved enzymes for metabolizing alkanes. A medium‐chain alkane hydroxylase has been found in the model organism Caulobacter ...crescentus, belonging to the CYP153 family of cytochrome P450 enzymes. However, the molecular mechanisms regulating expression of this enzyme are still unknown. We carried out a reverse genetics screen and RNA‐seq to identify CoxR as a transcription factor regulating this alkane hydroxylase. A deletion of coxR resulted in upregulation of both the alkane hydroxylase as well as ferredoxin and ferredoxin reductase enzymes. EMSA assays showed that CoxR bound directly to a short region of the alkane hydroxylase promoter. In summary, we have identified a novel transcription factor involved in metabolism of alkanes.
Alkanes are commonly found in nature and many gram positive and gram negative bacteria have developed enzymes for degrading them. The alphaproteobacteria Caulobacter crescentus has the CYP153 gene ...which codes for a medium‐chain alkane hydroxylase in the cytochrome P450 family. However, the regulatory mechanism controlling this gene has not been well‐characterized. A reverse genetics screen found that a deletion in the TetR family coxR gene results in a strain that is sensitive to oxidative stress. We used RNA‐seq and EMSA with Ni‐NTA purified CoxR to determine which genes CoxR directly regulates. Expression of the CYP153, ferredoxin and ferredoxin reductase genes were shown to be upregulated in the coxR deletion strain. Furthermore, CoxR was shown to directly bind to a 35bp region of the CYP153 promoter. In summary, we have shown that the transcription factor CoxR directly represses CYP153 gene expression.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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