Experimental evolution of a fungal pathogen into a gut symbiont Tso, Gloria Hoi Wan; Reales-Calderon, Jose Antonio; Tan, Alrina Shin Min ...
Science (American Association for the Advancement of Science),
11/2018, Letnik:
362, Številka:
6414
Journal Article
Recenzirano
Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen
in the mouse ...gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of "trained immunity." Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.
The RNA isoform repertoire is regulated by splicing factor (SF) expression, and alterations in SF levels are associated with disease. SFs contain ultraconserved poison exon (PE) sequences that ...exhibit greater identity across species than nearby coding exons, but their physiological role and molecular regulation is incompletely understood. We show that PEs in serine-arginine-rich (SR) proteins, a family of 14 essential SFs, are differentially spliced during induced pluripotent stem cell (iPSC) differentiation and in tumors versus normal tissues. We uncover an extensive cross-regulatory network of SR proteins controlling their expression via alternative splicing coupled to nonsense-mediated decay. We define sequences that regulate PE inclusion and protein expression of the oncogenic SF TRA2β using an RNA-targeting CRISPR screen. We demonstrate location dependency of RS domain activity on regulation of TRA2β-PE using CRISPR artificial SFs. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2β-PE detected in breast tumors, altering breast cancer cell viability, proliferation, and migration.
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•SR proteins levels are coordinated through splicing of their poison exons•SR protein poison exon splicing is altered during cell differentiation and in tumors•ASOs promotingTRA2β poison exon inclusion have anti-cancer effects in vitro
Leclair et al. demonstrate that expression of 14 SR proteins is coordinated through splicing of their poison exons during cell differentiation and tumorigenesis. Using the TRA2β poison exon as an example, they identify splicing-regulatory sequences and design splice-switching antisense oligonucleotides that decrease TRA2β protein expression and exhibit anti-cancer effects.
Feature selection is a critical step for translating advances afforded by systems-scale molecular profiling into actionable clinical insights. While data-driven methods are commonly utilized for ...selecting candidate genes, knowledge-driven methods must contend with the challenge of efficiently sifting through extensive volumes of biomedical information. This work aimed to assess the utility of large language models (LLMs) for knowledge-driven gene prioritization and selection.
In this proof of concept, we focused on 11 blood transcriptional modules associated with an Erythroid cells signature. We evaluated four leading LLMs across multiple tasks. Next, we established a workflow leveraging LLMs. The steps consisted of: (1) Selecting one of the 11 modules; (2) Identifying functional convergences among constituent genes using the LLMs; (3) Scoring candidate genes across six criteria capturing the gene's biological and clinical relevance; (4) Prioritizing candidate genes and summarizing justifications; (5) Fact-checking justifications and identifying supporting references; (6) Selecting a top candidate gene based on validated scoring justifications; and (7) Factoring in transcriptome profiling data to finalize the selection of the top candidate gene.
Of the four LLMs evaluated, OpenAI's GPT-4 and Anthropic's Claude demonstrated the best performance and were chosen for the implementation of the candidate gene prioritization and selection workflow. This workflow was run in parallel for each of the 11 erythroid cell modules by participants in a data mining workshop. Module M9.2 served as an illustrative use case. The 30 candidate genes forming this module were assessed, and the top five scoring genes were identified as BCL2L1, ALAS2, SLC4A1, CA1, and FECH. Researchers carefully fact-checked the summarized scoring justifications, after which the LLMs were prompted to select a top candidate based on this information. GPT-4 initially chose BCL2L1, while Claude selected ALAS2. When transcriptional profiling data from three reference datasets were provided for additional context, GPT-4 revised its initial choice to ALAS2, whereas Claude reaffirmed its original selection for this module.
Taken together, our findings highlight the ability of LLMs to prioritize candidate genes with minimal human intervention. This suggests the potential of this technology to boost productivity, especially for tasks that require leveraging extensive biomedical knowledge.
Electronic (e)-cigarettes are popular among youth and cigarette smokers attempting to quit. Studies to date have focused on the utility of e-cigarettes as a smoking cessation tool, but the biological ...effects are largely unknown.
To identify transcriptomic differences in the blood and sputum of e-cigarette users compared to conventional cigarettes smokers and healthy controls and describe biological pathways affected by these tobacco products.
Cross-sectional analysis of whole blood and sputum RNA-sequencing data from 8 smokers, 9 e-cigarette users (e-cigs) and 4 controls. Weighted gene co-network analysis (WGCNA) identified gene module associations. Ingenuity Pathway Analysis (IPA) identified canonical pathways associated with tobacco products.
In blood, a three-group comparison showed 16 differentially expressed genes (DEGs); pair-wise comparison showed 7 DEGs between e-cigs and controls, 35 DEGs between smokers and controls, and 13 DEGs between smokers and e-cigs. In sputum, 438 DEGs were in the three-group comparison. In pair-wise comparisons, there were 2 DEGs between e-cigs and controls, 270 DEGs between smokers and controls, and 468 DEGs between smokers and e-cigs. Only 2 genes in the smokers vs. control comparison overlapped between blood and sputum. Most gene modules identified through WGCNA associated with tobacco product exposures also were associated with cotinine and exhaled CO levels. IPA showed more canonical pathways altered by conventional cigarette smoking than by e-cigarette use.
Cigarette smoking and e-cigarette use led to transcriptomic changes in both blood and sputum. However, conventional cigarettes induced much stronger transcriptomic responses in both compartments.
Sequencing-based microbiome profiling aims at detecting and quantifying individual members of a microbial community in a culture-independent manner. While amplicon-based sequencing (ABS) of bacterial ...or fungal ribosomal DNA is the most widely used technology due to its low cost, it suffers from PCR amplification biases that hinder accurate representation of microbial population structures. Shotgun metagenomics (SMG) conversely allows unbiased microbiome profiling but requires high sequencing depth. Here we report the development of a meta-total RNA sequencing (MeTRS) method based on shotgun sequencing of total RNA and benchmark it on a human stool sample spiked in with known abundances of bacterial and fungal cells. MeTRS displayed the highest overall sensitivity and linearity for both bacteria and fungi, the greatest reproducibility compared to SMG and ABS, while requiring a ~20-fold lower sequencing depth than SMG. We therefore present MeTRS as a valuable alternative to existing technologies for large-scale profiling of complex microbiomes.
The transcription factor NFATc2 regulates dendritic cell (DC) responses to microbial stimulation through the C-type lectin receptor dectin-1. But the genetic targets of NFATc2 and their effects on DC ...function remain largely unknown. Therefore we used ChIP-seq to conduct genome-wide mapping of NFATc2 target sites in dectin-1-activated DCs. By combining binding-site data with a comprehensive gene expression profile, we found that NFATc2 occupancy regulates the expression of a subset of dectin-1-activated genes. Surprisingly, NFATc2 targeted an extensive range of DC-derived cytokines and chemokines, including regulatory cytokines such as IL2, IL23a and IL12b. Furthermore, we demonstrated that NFATc2 binding is required to induce the histone 3 lysine 4 trimethylation (H3K4me3) epigenetic mark, which is associated with enhanced gene expression. Together, these data show that the transcription factor NFATc2 mediates epigenetic modification of DC cytokine and chemokine genes leading to activation of their expression.
The genome is pervasively transcribed to produce a vast array of non-coding RNAs (ncRNAs). Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides and are best known for their ability to ...regulate gene expression. Enhancer RNAs (eRNAs) are subclass of lncRNAs that are synthesized from enhancer regions and have also been shown to coordinate gene expression. The biological function and significance of most lncRNAs and eRNAs remain to be determined. Epithelial to mesenchymal transition (EMT) is a ubiquitous cellular process that occurs during cellular migration, homeostasis, fibrosis, and cancer-cell metastasis. EMT-transcription factors, such as SNAI1 induce a complex transcriptional program that coordinates the morphological and molecular changes associated with EMT. Such complex transcriptional programs are often subject to coordination by networks of ncRNAs and thus can be leveraged to identify novel functional ncRNA loci. Here, using a genome-wide CRISPR activation (CRISPRa) screen targeting ∼10,000 lncRNA loci we identified ncRNA loci that could either promote or attenuate EMT. We discovered a novel locus that we named
(
cis-regulatory eRNAs expressed in monocytes). The
locus contained a cluster of eRNAs that when activated using CRISPRa induced expression of the neighboring gene
, driving concomitant EMT. However, the
eRNA transcripts themselves appeared dispensable for the induction of
expression. Interestingly, the
eRNAs and
were co-expressed in activated monocytes, where the
locus demarcated a monocyte-specific super-enhancer. These findings suggest a potential role for SNAI1 in monocytes. Exploration of the
axis could reveal novel aspects of monocyte biology.
is a resident fungus of the human intestinal microflora. Commonly isolated at low abundance in healthy people,
outcompetes local microbiota during candidiasis episodes. Under normal conditions, ...members of the human gastrointestinal (GI) microbiota were shown to keep
colonization under control. By releasing weak organic acids (WOAs), bacteria are able to moderate yeast growth. This mechanism displays a synergistic effect
with the absence of glucose in medium of culture, which underlines the complex interactions that
faces in its natural environment. Inactivation of the transcriptional regulator
in
results in a lack of sensitivity to this synergistic outcome. To decipher
transcriptional responses to glucose, WOAs, and the role of
, we performed RNA sequencing (RNA-seq) on four biological replicates exposed to combinations of these three parameters. We were able to characterize the (i) glucose response, (ii) response to acetic and butyric acid, (iii)
regulation of
, and (iv) genes responsible for WOA resistance. We identified a group of six genes linked to WOA sensitivity in a glucose-
-dependent manner and inactivated one of these genes, the putative glucose transporter
, in a SC5314 wild-type background. As expected, the mutant displayed a partial complementation to WOA resistance in the absence of glucose. This result points toward a mechanism of WOA sensitivity in
involving membrane transporters, which could be exploited to control yeast colonization in human body niches.
Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for ...establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility.
For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1
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•Human lung tissue dissection and tissue cryopreservation•Lung epithelium organoid generation from cryopreserved human lung tissue•Studying viral infection in organoid-derived air-liquid interface cultures•Viral response assessment via RNA-seq, flow cytometry, viral titer, and imaging
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility.
The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression toward ...severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4–6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an “early” inflammatory/immune signature preceding a “late” type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.
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•SARS-CoV-2 Wuhan-like virus and variants displayed different kinetics of infection•Virus triggers a biphasic & polarized transcriptional response in airway epithelia•Virally induced spatial protein response with CSF3 on epithelial basal side•Virally induced spatial protein response with CCL20 on epithelial apical side
Biological sciences; Molecular biology; Immunology; Microbiology
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