Baron, M. — Page 2 | bioRxiv Science

bioRxiv2018

Building a tumor atlas: integrating single-cell RNA-Seq data with spatial transcriptomics in pancreatic ductal adenocarcinoma

To understand tissue architecture it is necessary to understand both which cell types are present and their physical relationships to one another. Single-cell RNA-Seq (scRNA-Seq) has made significant progress towards the unbiased and systematic characterization of cell populations within a tissue by studying hundreds and thousands of cells in a single experiment. However, the characterization of the spatial organization of individual cells within a tissue has been more elusive. The recently introduced spatial transcriptomics method (ST) reveals the spatial pattern of gene expression within a tissue section at a resolution of a thousand 100 m spots across the tissue, each capturing the transcriptomes of [~]10-20 cells. Here, we present an approach for the integration of scRNA-Seq and ST data generated from the same sample of pancreatic cancer tissue. Using markers for cell types identified by scRNA-Seq, we robustly deconvolved the cell type composition of each ST spot to generate a spatial atlas of cell proportions across the tissue. Studying this atlas, we found that distinct spatial localizations accompany each of the cell populations that we identified. Our results provide a framework for creating a tumor atlas by mapping single-cell populations to their spatial region, as well as the inference of cell architecture in any tissue.

cancer biology↗

bioRxiv2017

Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node

Pathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entire Saccharomyces cerevisiae adenine de novo pathway with the cognate human pathway components. A yeast strain was \"humanized\" for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The \"humanized\" yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog of ADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point to PPAT/ADE4 as the central regulatory node. Pathway metabolites are responsible for regulating PPATs protein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation by PPAT and provide further insight into the complex regulation of this critical metabolic pathway.

synthetic biology↗