Browsing by Subject "RNA-sequencing"

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Bacterial pathogen adaptation during human infections
(2018-06-25) Crofts, Alexander; Davies, Bryan William; Trent, Michael Stephen; Payne, Shelley M; Ochman, Howard; Croyle, Maria A
Like all organisms on earth, bacteria must adapt to changes in their environment to survive. Thus, discovering bacterial adaptations reveals the tools bacteria use to be successful. Identifying how pathogenic bacteria adapt during infections can consequently identify the tools bacteria use to cause disease, and therapy design can then consider inhibiting these tools to treat or prevent infections. Here, the ways in which two worldwide human intestinal pathogens, Campylobacter jejuni and Enterotoxigenic E. coli (ETEC), adapt to the human host during infections are explored. Bacteria were studied directly in infected samples from controlled human infection models. In C. jejuni, genetic adaptations that were selected for during acute and persistent human infections identified the role of a previously uncharacterized flagellar modification gene during persistence. In ETEC, the bacteria’s ability to sense oxygen was linked to global virulence gene expression in human infection samples as well as biofilm formation. As environmental ETEC biofilms are associated with seasonal ETEC epidemics, oxygen sensing likely contributes to human infection inside and outside of the host. Together, these data demonstrate the scope of pathogen adaptation during infections, identified new targetable virulence factors, and can thus aid the design of new therapies
Femtosecond laser microdissection isolates regenerating C. elegans neurons for single cell RNA-sequencing
(2021-05-05) Zhao, Peisen; Ben-Yakar, Adela; Tewfik, Ahmed; Jiang, Ning; Chen, Ray; Arur, Swathi
The complexity of tissue is shaped by profound molecular diversity at a single cell level. Isolating cells of interest based on specific physiology is a crucial step to analyze its molecular underpinnings. Many cell isolation methods have been developed towards such goal. However, none is optimal when highly specific, especially rare cells are desired. For example, cell isolation by fluorescence activated cell sorting (FACS) entails tissue dissociation, which leads to a low yield (only 1 - 10% of labeled cells are collected by FACS), widespread transcriptional artifacts in isolated cells, and loss of the cells’ contextual information. Alternative methods such as laser capture microdissection (LCM) and Patch-seq have been proposed to bypass tissue dissociation, but they also suffer from numerous drawbacks, notably the degradation of cellular content. I developed a new single cell isolation method, femtosecond laser microdissection (fs-LM), to isolate intact single cells directly from living tissue or model organisms. fs-LM resects a single cell from its surrounding tissue by a series of micron-scale ablation spots, which are created by fs-laser ablation in a spherical pattern encasing the cell. I first demonstrated feasibility of fs-LM by isolating neurons of Caenorhabditis elegans and performing single cell RNA-sequencing (scRNA-seq). I achieved a yield of 32% (n = 384) and detected 2,261 ± 132 genes in single C. elegans neuron. In comparison with neurons isolated by the dissociation-FACS method, neurons isolated by fs-LM displayed reduced transcriptional artifacts induced by tissue dissociation, including up-regulation of genes involved in heat shock response and mitochondrial unfolded protein response. Existing clinical interventions to spinal cord injury have been unsatisfactory, and future development requires comprehensive knowledge of the genetic activities driving nerve regeneration. Therefore, I isolated and sequenced regenerating C. elegans neurons following fs-laser axotomy. I revealed transcriptional programs leading to successful regeneration in wild-type animals and regeneration failure in animals lacking DLK-1/p38 kinase. I further investigated the molecular basis of regeneration heterogeneity displayed by neurons of the same type, which has remained understudied despite its clinical importance. In total, 6 distinct gene modules were found to play a potential regulatory role in nerve regeneration.
Molecular mechanisms underlying alcohol use disorder and major depressive disorder comorbidity
(2017-05-03) Wolfe, Sarah Anne; Harris, R. Adron; Raab-Graham, Kimberly F.; Golding, Nace; Morrisett, Richard; Macdonald, Paul
Alcohol Use Disorder (AUD) and Major Depressive Disorder (MDD) are two widespread and debilitating disorders that share a high rate of comorbidity with the presence of either disorder doubling the risk of developing the other. Despite their prevalence, few treatments are available to individuals with comorbid AUD and MDD. Both alcohol and antidepressants promote lasting neuroadaptive changes in synapses and dendrites. With alcohol these changes may provide relief from depressive symptoms, and the initial use of alcohol may be a form of self-medication for individuals with MDD, suggesting ethanol may have antidepressant properties underlying similarities in neurobiological abnormalities. However, the synaptic pathways that are shared by alcohol and antidepressants are unknown. This study aims to identify why acute exposure to ethanol produced lasting antidepressant and anxiolytic behaviors. To understand the functional basis of these behaviors, a molecular pathway activated by rapid antidepressants was investigated. Here ethanol, like rapid antidepressants, altered γ-aminobutyric acid type B receptor (GABA [subscript B] R) expression and signaling, to increase dendritic calcium. New GABA [subscript B] Rs were synthesized in response to ethanol treatment, requiring fragile-X mental retardation protein (FMRP). Ethanol-dependent changes in GABA [subscript B] R expression, dendritic signaling, and antidepressant efficacy were absent in Fmr1-knockout (KO) mice. These findings indicate that FMRP is an important regulator of protein synthesis following acute alcohol exposure, and provided a molecular basis for the antidepressant efficacy of acute ethanol exposure. We identify alterations on a global scale with acute alcohol and antidepressant by sequencing the synaptic transcriptome. We identified parallel alterations in exon usage with acute alcohol and antidepressant treatment. These shared differentially expressed exons may give rise to isoforms and proteins with altered function or localization in the synapse. Some of these differentially expressed exons were identified in genes known to have alternative isoforms with AUD and MDD. These data implicate alternative splicing and isoform expression in the acute antidepressant-like effects of ethanol and the development of comorbid alcohol and depression. Understanding the molecular basis for comorbidity may aid in development of treatment options for afflicted individuals with dual disorders, as well as explore the mechanism for the initiation of addiction with acute exposure to alcohol