Biology subjects

Jepsen, K.

Publications and source records attributed to Jepsen, K..

7 recordsLinked to original sources

bioRxiv2026

Single-cell analysis of Plasmodium falciparum transcripts after drug perturbation identifies feedback regulation as well as increased transmission potential

Gene expression analysis in malaria parasites has been used to define transcriptional regulatory networks but has been used less frequently to characterize parasite response to drug treatment or to show how parasites may evade killing. Here, we applied single-cell RNA sequencing (scRNA-seq) to hundreds of thousands of individually infected asynchronous red blood cells to evaluate the parasites response to treatment with three chemotypes that can be used for treatment (artemisinin) or prophylaxis and treatment (atovaquone, ganaplacide). We found that each treatment gave rise to different cell populations with different transcriptional profiles. Comparing single cell transcription patterns in compound-treated cells, to transcript patterns observed previously with synchronized cells showed an enrichment of cells expressing gametocyte-associated genes after artemisinin treatment but fewer lifecycle perturbations after treatment with the two other compounds. In contrast, bulk analysis showed an enrichment of pyrimidine biosynthesis transcripts for atovaquone treatment. Our results show that scRNA-seq may be used to profile diverse drug responses across many lifecycle stages and to potentially classify drug classes. ImportanceDetermining the mechanism of action (MOA) of compounds with antimalarial activity remains a key activity in both drug development and drug resistance studies but remains challenging for some chemotypes. Here we highlight the potential of single cell transcriptional sequencing to augment the process of MOA deconvolution. We develop a new analytical pipeline that involves comparing single cell transcription patterns to existing profiles from synchronized parasites to comprehensively characterize life cycle stage enrichments that may be observed after chemical perturbations. We also show that transcriptional feedback regulation may be present for some drug classes.

microbiology↗

bioRxiv2021

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ

Micro-environmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological and microenvironmental landscape across 85 multiple micro-dissected regions from 39 cases. Most somatic alterations, including whole genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified 3 immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

genomics↗

bioRxiv2020

The unfolded protein response links tumor aneuploidy to local immune dysregulation

Aneuploidy is a chromosomal abnormality associated with poor prognosis in many cancer types. Here we tested the hypothesis that the unfolded protein response (UPR) links mechanistically aneuploidy and local immune dysregulation. Using a single somatic copy-number alteration (SCNA) score inclusive of whole-chromosome, arm and focal chromosome alterations in a pan-cancer analysis of 9,375 samples in The Cancer Genome Atlas (TCGA) database, we found an inverse correlation with a cytotoxicity (CYT) score across disease stages. Co-expression patterns of UPR genes changed substantially between SCNAlow and SCNAhigh groups. Pathway activity scores showed increased activity by multiple branches of the UPR in response to aneuploidy. The PERK branch showed the strongest association with a reduced CYT score. The conditioned medium of aneuploid cells transmitted XBP1 splicing and caused IL-6 and Arginase1 transcription in receiver bone marrow-derived macrophages. We propose the UPR as a mechanistic link between aneuploidy and immune dysregulation in the tumor microenvironment. Statement of SignificanceAneuploidy accumulates over the life of a tumor and is associated with poor prognosis. Tumor progression is also associated with a progressive immune dysregulation. To explain these complex and concurrent disorders we tested the hypothesis that the unfolded protein could represent the link between aneuploidy and a dysregulation of local immunity favoring tumor progression.

cancer biology↗

bioRxiv2020

Mutational profiling of micro-dissected pre-malignant lesions from archived specimens

BackgroundSystematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or potentially insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. MethodsUsing test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). ResultsUsing low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. ConclusionAlternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.

genomics↗

bioRxiv2020

IRE1? regulates macrophage polarization, PD-L1 expression and tumor survival

In the tumor microenvironment local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the upregulation of IL-6, IL-23, Arginase1, as well as surface expression of CD86 and PD-L1. Macrophages in which the IRE1/Xbp1 axis is blocked pharmacologically or deleted genetically have significantly reduced polarization, and CD86 and PD-L1 expression, which was induced independent of IFN{gamma} signaling suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1 gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNASeq analysis showed that bone marrow derived macrophages with IRE1 deletion lose the integrity of the gene connectivity characteristic of regulated IRE1-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.

cancer biology↗

bioRxiv2020

The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication

Grapevine has a major economical and cultural importance since antiquity. A key step in domestication was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. vinifera. While the grapevine sex locus is known to be small, its precise boundaries, gene content and the sex-determining genes are unknown. Here we obtained a high-quality de novo reference genome for V. sylvestris and whole-genome resequencing data of a cross. Studying SNP segregation patterns, gene content and expression in wild and cultivated accessions allowed us to build a model for sex determination in grapevine. In this model, up- and down-regulation of a cytokinin regulator is sufficient to cause female sterility and reversal to hermaphroditism, respectively. This study highlights the importance of neo-functionalization of Y alleles in sex determination and provides a resource for studying genetic diversity in V. sylvestris and the genomic processes of grapevine domestication.

plant biology↗

bioRxiv2017

Cross-Site Comparison of Ribosomal Depletion Kits for Illumina RNAseq Library Construction

Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify [~]14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

genomics↗