Biology subjects

Fleck, J.

Publications and source records attributed to Fleck, J..

4 recordsLinked to original sources

bioRxiv2024

Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-{beta} expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-{beta} induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-{beta}, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-{beta} mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-{beta} via mitochondrial dsDNA release.

immunology↗

bioRxiv2021

A polyyne toxin produced by an antagonistic bacterium blinds and lyses a green microalga

Microalgae are key contributors to global carbon fixation and the basis of many food webs. In nature, their growth is often supported or suppressed by other microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology. Using a combination of Raman microspectroscopy, genome mining and mutational analysis, we discovered a novel polyyne toxin we name protegencin that is secreted by P. protegens and penetrates algal cells to destroy their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin prevents the phototactic behavior of C. reinhardtii needed to perform optimal photosynthesis. A protegencin-deficient biosynthetic mutant of P. protegens does not affect growth or eyespot carotenoids of C. reinhardtii. Thus, protegencin acts in a direct and destructive way, and reveals at least a two-pronged molecular strategy used by algicidal bacteria.

plant biology↗

bioRxiv2020

Predicting drug sensitivity of cancer cells based on DNA methylation levels

Cancer cell lines, which are cell cultures developed from tumor samples, represent one of the least expensive and most studied preclinical models for drug development. Accurately predicting drug response for a given cell line based on molecular features may help to optimize drug-development pipelines and explain mechanisms behind treatment responses. In this study, we focus on DNA methylation profiles as one type of molecular feature that is known to drive tumorigenesis and modulate treatment responses. Using genome-wide, DNA methylation profiles from 987 cell lines from the Genomics of Drug Sensitivity in Cancer database, we applied machine-learning algorithms to evaluate the potential to predict cytotoxic responses for eight anti-cancer drugs. We compared the performance of five classification algorithms and four regression algorithms that use diverse methodologies, including tree-, probability-, kernel-, ensemble-, and distance-based approaches. For both types of algorithm, we artificially subsampled the data to varying degrees, aiming to understand whether training models based on relatively extreme outcomes would yield improved performance. We also performed an information-gain analysis to examine which genes were most predictive of drug responses. Finally, we used tumor data from The Cancer Genome Atlas to evaluate the feasibility of predicting clinical responses in humans based on models derived from cell lines. When using classification or regression algorithms to predict discrete or continuous responses, respectively, we consistently observed excellent predictive performance when the training and test sets both consisted of cell-line data. However, classification models derived from cell-line data failed to generalize effectively for tumors.

bioinformatics↗

bioRxiv2019

Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

The human brain has changed dramatically since humans diverged from our closest living relatives, chimpanzees and the other great apes1-5. However, the genetic and developmental programs underlying this divergence are not fully understood6-8. Here, we have analyzed stem cell-derived cerebral organoids using single-cell transcriptomics (scRNA-seq) and accessible chromatin profiling (scATAC-seq) to explore gene regulatory changes that are specific to humans. We first analyze cell composition and reconstruct differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We find that brain region composition varies in organoids from different iPSC lines, yet regional gene expression patterns are largely reproducible across individuals. We then analyze chimpanzee and macaque cerebral organoids and find that human neuronal development proceeds at a delayed pace relative to the other two primates. Through pseudotemporal alignment of differentiation paths, we identify human-specific gene expression resolved to distinct cell states along progenitor to neuron lineages in the cortex. We find that chromatin accessibility is dynamic during cortex development, and identify instances of accessibility divergence between human and chimpanzee that correlate with human-specific gene expression and genetic change. Finally, we map human-specific expression in adult prefrontal cortex using single-nucleus RNA-seq and find developmental differences that persist into adulthood, as well as cell state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene regulatory features that are unique to humans.

developmental biology↗