Advances and challenges in single-cell RNA-seq of microbial communities

Imdahl, Fabian

doi:10.1016/j.mib.2020.10.001

Cited by 31 publications

(15 citation statements)

References 70 publications

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“…While there are examples in the literature, it is not commonplace and consensus methods for chemical fixation and permeabilization have not been established (Emerson et al, 2015;Managadze et al, 2010;Riquelme et al, 2002). A major obstacle for techniques that require the use of affinity probes in intact fixed fungal cells is the presence of a cell wall, which varies in composition throughout the fungal kingdom (Bourett et al, 1998;Imdahl and Saliba, 2020;Patel and Free, 2019). Additionally, we have found that mature, fixed Neurospora hyphae are recalcitrant toward adherence to glass or other surfaces commonly used for light microscopy.…”

Section: Introductionmentioning

confidence: 93%

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Bartholomai

Gladfelter

Loros

et al. 2021

Preprint

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Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

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Section: Introductionmentioning

confidence: 93%

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Bartholomai

Gladfelter

Loros

et al. 2021

Preprint

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“…In recent years, several research groups have developed strategies to sequence and determine the quantitative levels of RNA transcripts in a single bacterial cell [124,125]. An overarching workflow for these different approaches involves isolating a single bacterium, which can be achieved using some of the techniques described in this review (e.g., FACS and the use of microfluidic devices).…”

Section: Next-generation Sequencingmentioning

confidence: 99%

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

et al. 2021

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Antibiotic persistence is a phenomenon in which rare cells of a clonal bacterial population can survive antibiotic doses that kill their kin, even though the entire population is genetically susceptible. With antibiotic treatment failure on the rise, there is growing interest in understanding the molecular mechanisms underlying bacterial phenotypic heterogeneity and antibiotic persistence. However, elucidating these rare cell states can be technically challenging. The advent of single-cell techniques has enabled us to observe and quantitatively investigate individual cells in complex, phenotypically heterogeneous populations. In this review, we will discuss current technologies for studying persister phenotypes, including fluorescent tags and biosensors used to elucidate cellular processes; advances in flow cytometry, mass spectrometry, Raman spectroscopy, and microfluidics that contribute high-throughput and high-content information; and next-generation sequencing for powerful insights into genetic and transcriptomic programs. We will further discuss existing knowledge gaps, cutting-edge technologies that can address them, and how advances in single-cell microbiology can potentially improve infectious disease treatment outcomes.

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“…Single-cell transcriptome sequencing (scRNA-seq) has recently emerged as a powerful method to identify cell types and analyze functional status at single-cell level ( Dal Molin and Di Camillo, 2019 ). scRNA-seq is widely used in various fields including cancer ( Suvà and Tirosh, 2019 ), developmental biology ( Griffiths et al, 2018 ), and microbiology ( Imdahl and Saliba, 2020 ) and has become a promising research tool in the fields of life sciences. scRNA-seq results of COVID-19 find that changes of host factors including EN-RAGE, TNFSF14, oncostatin M, ANXA1, FPR1, and S100A8/9 are correlated with disease severity, revealing the possible pathogenesis of severe COVID-19 and potential host therapeutic targets ( Arunachalam et al, 2020 ; Guo et al, 2021 ; Ren et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Identification of Neutrophil-Related Factor LCN2 for Predicting Severity of Patients With Influenza A Virus and SARS-CoV-2 Infection

Huang

Liu

et al. 2022

Front. Microbiol.

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BackgroundInfluenza and COVID-19 are respiratory infectious diseases that are characterized by high contagiousness and high mutation and pose a serious threat to global health. After Influenza A virus (IAV) and SARS-CoV-2 infection, severe cases may develop into acute lung injury. Immune factors act as an important role during infection and inflammation. However, the molecular immune mechanisms still remain unclear. We aimed to explore immune-related host factors and core biomarker for severe infection, to provide a new therapeutic target of host factor in patients.MethodsGene expression profiles were obtained from Gene Expression Omnibus and the Seurat R package was used for data process of single-cell transcriptome. Differentially expressed gene analysis and cell cluster were used to explore core host genes and source cells of genes. We performed Gene Ontology enrichment, Kyoto Encyclopedia of Genes and Genomes analysis, and gene set enrichment analysis to explore potential biological functions of genes. Gene set variation analysis was used to evaluate the important gene set variation score for different samples. We conduct Enzyme-linked immunosorbent assay (ELISA) to test plasma concentrations of Lipocalin 2 (LCN2).ResultsMultiple virus-related, cytokine-related, and chemokine-related pathways involved in process of IAV infection and inflammatory response mainly derive from macrophages and neutrophils. LCN2 mainly in neutrophils was significantly upregulated after either IAV or SARS-CoV-2 infection and positively correlated with disease severity. The plasma LCN2 of influenza patients were elevated significantly compared with healthy controls by ELISA and positively correlated with disease severity of influenza patients. Further bioinformatics analysis revealed that LCN2 involved in functions of neutrophils, including neutrophil degranulation, neutrophil activation involved in immune response, and neutrophil extracellular trap formation.ConclusionThe neutrophil-related LCN2 could be a promising biomarker for predicting severity of patients with IAV and SARS-CoV-2 infection and may as a new treatment target in severe patients.

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Advances and challenges in single-cell RNA-seq of microbial communities

Cited by 31 publications

References 70 publications

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

Identification of Neutrophil-Related Factor LCN2 for Predicting Severity of Patients With Influenza A Virus and SARS-CoV-2 Infection

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