ABSTRACT
Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity, we have developed a single-nucleus RNA-seq2 method tailored for the comprehensive analysis of the nuclear transcriptome from frozen tissues, allowing the dissection of all cell types present in the liver, regardless of cell size or cellular fragility. We use this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that ploidy states are associated with different metabolic potential, and gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobule. Our work reveals a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.
Subject(s)
Liver/metabolism , Ploidies , Sequence Analysis, RNA , Single-Cell Analysis , Animals , Biomarkers/metabolism , Cell Nucleus/metabolism , Chronic Disease , Frozen Sections , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Hepatocytes/metabolism , Liver/pathology , Liver Diseases/pathology , Mice, Inbred C57BL , Regeneration , Stem Cells/metabolism , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
BACKGROUND: Pneumococcal carriage is a reservoir for transmission and a precursor to pneumococcal disease. The experimental human pneumococcal carriage model provides a useful tool to aid vaccine licensure through the measurement of vaccine efficacy against carriage (VEcol). Documentation of the genetic stability of the experimental human pneumococcal carriage model is important to further strengthen confidence in its safety and conclusions, enabling it to further facilitate vaccine licensure through providing evidence of VEcol. METHODS: 229 isolates were sequenced from 10 volunteers in whom experimental human pneumococcal carriage was established, sampled over a period of 35 days. Multiple isolates from within a single volunteer at a single time provided a deep resolution for detecting variation. HiSeq data from the isolates were mapped against a PacBio reference of the inoculum to call variable sites. RESULTS: The observed variation between experimental carriage isolates was minimal with the maximum SNP distance between any isolate and the reference being 3 SNPs. CONCLUSION: The low-level variation described provides evidence for the stability of the experimental human pneumococcal carriage model over 35 days, which can be reliably and confidently used to measure VEcol and aid future progression of pneumococcal vaccination.
Subject(s)
Carrier State/microbiology , Genetic Variation , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/isolation & purification , Adolescent , Adult , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genotype , Healthy Volunteers , Humans , Middle Aged , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Streptococcus pneumoniae/genetics , Young AdultABSTRACT
Cellular introduction of PEBBLEs (photonic explorers for bioanalysis with biologically localized embedding) has been investigated by a wide variety of methods in a range of cell types. These methods include surface functionalization with CPPs (cell-penetrating peptides), pinocytosis, commercial lipid transfection agents, cytochalasin D, picoinjection, and Gene gun bombardment. This paper will overview several of the most popular methods used for the introduction of PEBBLE nanosensors to the cellular environment and discuss the efficacy of the techniques.
