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Journal ArticleDOI

Laser microdissection: A promising tool for exploring microorganisms and their interactions with hosts.

TL;DR: The current paper describes the methodological aspects of commercially available laser microdissection instruments and representative examples that demonstrate the advantages of this method for resolving a variety of issues in microbiology.
About: This article is published in Journal of Microbiological Methods.The article was published on 2017-07-01. It has received 17 citations till now. The article focuses on the topics: Laser capture microdissection.
Citations
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TL;DR: In this article, the authors reported the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN, which is a polyploid species with genome copies ranging from approximately 200-900 per cell.
Abstract: While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

201 citations

Journal ArticleDOI
TL;DR: This review focuses on the design of dual RNA-seq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction and a reduction in sequencing cost and single cell transcriptomics coupled with protein and metabolite level dual approaches are set to enhance understanding of plant-pathogen interactions.
Abstract: RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNA-seq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. A reduction in sequencing cost and single cell transcriptomics coupled with protein and metabolite level dual approaches are set to enhance our understanding of plant-pathogen interactions. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions.

40 citations

Journal ArticleDOI
TL;DR: How the latest developments in protocols and methods have made LM a powerful and sometimes essential tool for genomic and proteomic analyzes of tiny amounts of biomolecules extracted from few cells isolated from a complex tissue, in their physiological context, thus offering new opportunities for understanding fundamental physiological and/or patho-physiological processes is seen.

30 citations

Journal ArticleDOI
TL;DR: In this paper, the authors used laser-capture microdissection (LCM) to select precise areas in the gut lumen from frozen whole larval cryo-sections.
Abstract: Bacillus cereus is a Gram-positive opportunistic pathogen closely related to the entomopathogen, Bacillus thuringiensis, both of which are involved in intestinal infections. Iron is an essential micronutrient for full growth and virulence of pathogens during infection. However, little is known about iron homeostasis during gut infection. Therefore, we aimed to assess the expression of B. cereus genes related to bacterial iron homeostasis, virulence and oxidative stress. The hypothesis is that the expression of such genes would vary between early and later stage colonization in correlation to gut cell damage. To perform the study, a germ-free Galleria mellonella model was set up in order to adapt the use of Laser-capture microdissection (LCM), to select precise areas in the gut lumen from frozen whole larval cryo-sections. Analyses were performed from alive larvae and the expression of targeted genes was assessed byspecific pre-amplification of mRNA followed by quantitative PCR. Firstly, the results reinforce the reliability of LCM, despite a low amount of bacterial RNA recovered. Secondly, bacterial genes involved in iron homeostasis are expressed in the lumen at both 3 and 16 hours post force-feeding. Thirdly, iron gene expression is slightly modulated during gut infection, and lastly, the mRNA of G. mellonella encoding for ferritin and transferrin iron storage and transport are recovered too. Therefore, iron homeostasis should play a role in B. cereus gut colonization. Furthermore, we demonstrate for the first time the value of using LCM for specific in situ gene expression analysis of extracellular bacteria in a whole animal.

7 citations

Journal ArticleDOI
TL;DR: In this article , the authors summarize all aspects of microbiome studies from FFPE tissues including the challenges associated with working highly degraded DNA, best practices for reducing environmental contamination, and propose solutions to address these issues.

5 citations

References
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Journal ArticleDOI
TL;DR: Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts.

9,017 citations

Journal ArticleDOI
10 Jun 2005-Science
TL;DR: A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms, and significant intersubject variability and differences between stool and mucosa community composition were discovered.
Abstract: The human endogenous intestinal microflora is an essential “organ” in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.

7,049 citations

Journal ArticleDOI
TL;DR: A single-cell digital gene expression profiling assay with only a single mouse blastomere is described, which detected the expression of 75% more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads.
Abstract: Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.

2,659 citations

Journal ArticleDOI
08 Nov 1996-Science
TL;DR: Laser capture microdissection under direct microscopic visualization permits rapid one-step procurement of selected human cell populations from a section of complex, heterogeneous tissue.
Abstract: Laser capture microdissection (LCM) under direct microscopic visualization permits rapid one-step procurement of selected human cell populations from a section of complex, heterogeneous tissue. In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied to the surface of the tissue section on a standard glass histopathology slide; a carbon dioxide laser pulse then specifically activates the film above the cells of interest. Strong focal adhesion allows selective procurement of the targeted cells. Multiple examples of LCM transfer and tissue analysis, including polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from transferred tissue are demonstrated.

2,371 citations

Journal ArticleDOI
01 Dec 1987-Nature
TL;DR: The use of infrared (IR) light is used to make much improved laser traps with significantly less optical damage to a variety of living cells, and new manipulative techniques using IR light are capable of producing large forces under damage-free conditions and improve the prospects for wider use of optical manipulation techniques in microbiology.
Abstract: Use of optical traps for the manipulation of biological particles was recently proposed, and initial observations of laser trapping of bacteria and viruses with visible argon-laser light were reported. We report here the use of infrared (IR) light to make much improved laser traps with significantly less optical damage to a variety of living cells. Using IR light we have observed the reproduction of Escherichia coli within optical traps at power levels sufficient to give manipulation at velocities up to approximately 500 micron s-1. Reproduction of yeast cells by budding was also achieved in IR traps capable of manipulating individual cells and clumps of cells at velocities of approximately micron s-1. Damage-free trapping and manipulation of suspensions of red blood cells of humans and of organelles located within individual living cells of spirogyra was also achieved, largely as a result of the reduced absorption of haemoglobin and chlorophyll in the IR. Trapping of many types of small protozoa and manipulation of organelles within protozoa is also possible. The manipulative capabilities of optical techniques were exploited in experiments showing separation of individual bacteria from one sample and their introduction into another sample. Optical orientation of individual bacterial cells in space was also achieved using a pair of laser-beam traps. These new manipulative techniques using IR light are capable of producing large forces under damage-free conditions and improve the prospects for wider use of optical manipulation techniques in microbiology.

2,201 citations