is a bacterial species encompassing both pathogenic and non-pathogenic strains and is frequently found colonizing the same host plants as . This presents the need to develop a detection and genotyping assay able to track these bacteria in microbial consortia with other xanthomonads. Eight -specific DNA markers (XEA1-XEA8) were selected by comparative genomics and validated in silico regarding their specificity and consistency using BLASTn, synteny analysis, CG content, codon usage (CAI/eCAI values) and genomic proximity to plasticity determinants. In silico, the selected eight DNA markers were found to be specific and conserved across the genomes of 11 strains, and in particular, five DNA markers (XEA4, XEA5, XEA6, XEA7 and XEA8) were unfailingly found in these genomes. A multiplex of PCR targeting markers XEA1 (819 bp), XEA8 (648 bp) and XEA5 (295 bp) was shown to successfully detect down to 1 ng of DNA (per PCR reaction). The topology of trees generated with the concatenated sequences of three markers (XEA5, XEA6 and XEA8) and four housekeeping genes (, , and ) underlined the equal discriminatory power of these features and thus the suitability of the DNA markers to discriminate lineages. Overall, this study displays a DNA-marker-based method for the detection and genotyping of strains, contributing to monitoring for its presence in -colonizing habitats. The present study proposes a workflow for the selection of species-specific detection markers. Prospectively, this assay could contribute to unveil alternative host species of ; and improve the control of phytopathogenic strains.
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http://dx.doi.org/10.3390/microorganisms10061078 | DOI Listing |
Methods Mol Biol
January 2025
Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
In the Drosophila brain, neuronal diversity originates from approximately 100 neural stem cells, each dividing asymmetrically. Precise mapping of cell lineages at the single-cell resolution is crucial for understanding the mechanisms that direct neuronal specification. However, existing methods for high-resolution lineage tracing are notably time-consuming and labor-intensive.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
IDG/McGovern Institute of Brain Research, Tsinghua University, Beijing, People's Republic of China.
Mosaic analysis with double markers (MADM) is a powerful in vivo lineage tracing technique. It utilizes Cre recombinase-dependent interchromosomal recombination to restore the stable expression of two fluorescent proteins sparsely in individual dividing stem or progenitor cells and their progenies. Here, we describe the application of this technique for quantitative lineage analysis of radial glial progenitors in the developing mouse neocortex at the single-cell resolution.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Sorbonne Université, Institut du Cerveau (Paris Brain Institute) ICM, Inserm, CNRS, Hôpital de la Pitié Salpêtrière, Paris, France.
Somatic mosaic variants, and especially somatic single nucleotide variants (sSNVs), occur in progenitor cells in the developing human brain frequently enough to provide permanent, unique, and cumulative markers of cell divisions and clones. Here, we describe an experimental workflow to perform lineage studies in the human brain using somatic variants. The workflow consists in two major steps: (1) sSNV calling through whole-genome sequencing (WGS) of bulk (non-single-cell) DNA extracted from human fresh-frozen tissue biopsies, and (2) sSNV validation and cell phylogeny deciphering through single nuclei whole-genome amplification (WGA) followed by targeted sequencing of sSNV loci.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Institute of Science and Technology Austria (ISTA), Klosterneuburg, Austria.
Mosaic Analysis with Double Markers (MADM) represents a mouse genetic approach coupling differential fluorescent labeling to genetic manipulations in dividing cells and their lineages. MADM uniquely enables the generation and visualization of individual control or homozygous mutant cells in a heterozygous genetic environment. Among its diverse applications, MADM has been used to dissect cell-autonomous gene functions important for cortical development and neural development in general.
View Article and Find Full Text PDFCancer Chemother Pharmacol
January 2025
Service de Génomique des Tumeurs et Pharmacologie, Hôpital Saint-Louis, Assistance Publique Hôpitaux de Paris, Paris, France.
The enzyme dihydropyrimidine dehydrogenase (DPD) is the primary catabolic pathway of fluoropyrimidines including 5 fluorouracil (5FU) and capecitabine. Cases of lethal toxicity have been reported in cancer patients with complete DPD deficiency receiving standard dose of 5FU or capecitabine. DPD is encoded by the pharmacogene DPYD in which more than 200 variants have been identified.
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