Biology subjects

Lund, P.

Publications and source records attributed to Lund, P..

9 recordsLinked to original sources

bioRxiv2026

Scalable genotyping in fixed transcriptomes resolves clonal heterogeneity via single-cell sequencing

Despite the promise of single-cell transcriptomics for understanding cell states in heterogeneous populations, widely used platforms have limited ability to link transcriptional states to somatic mutations within the same cells. Here, we introduce Genotyping in Fixed Transcriptomes (GIFT) for the simultaneous detection of large numbers of targeted genetic variants with whole transcriptome profiles in single cells. The core innovation of GIFT is a rationally designed gapfilling reaction between adjacent single-stranded DNA (ssDNA) probes that barcodes native transcript sequence to enable highly-specific targeted mutation detection. GIFT achieves greater than 99% genotyping accuracy and flexible capture of hundreds of mutations per cell, including in formalin-fixed, paraffin-embedded (FFPE) tissue, enabling clonal lineage tracing in heterogeneous settings. We demonstrate the unique scalability of GIFT by profiling more than 700,000 cells from 35 donors with myeloproliferative neoplasms (MPN), revealing mutation-dependent hematopoietic responses to systemic inflammation associated with the characteristic JAK2V617 mutation, including an allelic dose gradient of interferon-associated transcriptional programs and priming of hematopoietic stem cells that develop into divergent disease states. The technical advantages of GIFT enable direct resolution of genotype-to-phenotype relationships via clonal tracing with comprehensive cell-state measurements at single-cell resolution.

genomics↗

bioRxiv2026

Rumen transfaunation between low- and high-methane-yielding dairy cows reveals asymmetric microbiome reconstitution patterns: a pilot study

BackgroundThis study investigated rumen microbiome reconstitution and methane (CH4) emissions following a complete exchange of rumen contents between low- and high-CH4-yielding Norwegian Red dairy cows. Twenty cows were screened for CH4 yield, and two low and two high emitters were selected for rumen cannulation and content swap. Total rumen contents were swapped after complete evacuation and washing of both the rumen and omasum. Rumen samples were collected twice in weeks -1, 1, 3, and 7 for fermentation analysis, metagenomics, and metaproteomics, and at week 8 CH4 production was measured. ResultsPrior to the swap, low and high emitters produced 21.2 {+/-} 0.7 and 26.3 {+/-} 1.4 g CH4/kg dry-matter intake (DMI), respectively. Eight weeks after swap, CH4 yields were 12.7 {+/-} 0.3 and 28.9 {+/-} 0.3 g CH4/kg DMI, respectively, showing that the CH4 phenotype of each cow was maintained. Analysis of metagenome-derived 16S rRNA gene sequences showed that low emitters gradually re-established their original microbial community, whereas high emitters retained donor-like microbiota. Metaproteomic mapping suggested higher expression of Prevotella-associated succinate-propionate pathway enzymes in low emitters at week 7, though these differences were modest. ConclusionThese findings suggest that host factors influence CH4 output and microbial reconstitution, with low emitters restoring their native microbiome while high emitters retained a donor-associated community yet continued to emit high CH4. Results should be interpreted with caution given the small sample size (n = 2 per phenotype) and require confirmation in larger studies. ImportanceReducing enteric methane from cattle requires understanding whether the rumen microbiome or the host animal is the primary driver of methane output. We exchanged the entire rumen contents between low- and high-methane-yielding dairy cows and measured methane production alongside metagenomic and metaproteomic profiling over two months. Despite receiving each others microbiomes, each cows methane phenotype persisted--low emitters stayed low and high emitters stayed high. Microbiome reconstitution was asymmetric: low emitters restored their original microbial community, while high emitters retained the donor microbiota. Methanogen communities did not differ between phenotypes, pointing to host-level rather than microbial-level control of methane yield. These pilot findings suggest that breeding for favorable host traits may be essential for lasting methane reduction, and that microbiome transfer alone is unlikely to shift an animals methane phenotype. Larger studies are needed to confirm these observations.

microbiology↗

bioRxiv2025

Genome-wide screening using TraDIS accelerates identification of key adaptive mutations of longer-term evolution experiment in Escherichia coli.

Long term laboratory-based evolution experiments are a powerful tool that are increasingly being used to study fundamental aspects of evolution and to identify genes that contribute to overall fitness under different conditions. However, even with automation, the time that they take to execute limits the extent to which evolution experiments can be used as part of a high throughput approach to understand the links between genotype and phenotype. Mutations that lead to genetic loss of function are frequently selected for in evolution experiments. Thus in principle these experiments could be done more rapidly by starting not with clonal isolates but with dense transposon libraries that will contain loss of function mutations in all non-essential genes. Here, we test this hypothesis by comparing the results of long term (5 month) evolution experiment, in which E. coli was grown with daily transfers in unbuffered LB starting at pH 4.5, with short term (5 and 10 day) experiments on a high density transposon library in the same strain and under the same conditions. We show that there is very significant overlap in the genes and pathways identified using the two methods. Use of this approach thus has the potential to significantly increase the throughput of laboratory-based evolution and enable rapid testing of a wide range of parameters that may have an impact on evolutionary trajectories. Author summaryUnderstanding how bacterial populations adapt to environmental stress is central to microbiology and evolutionary biology. Laboratory evolution experiments are commonly used to uncover the genetic changes that confer increased fitness, but these experiments are often slow and laborious. In this study, we asked whether the results of long-term adaptive laboratory evolution (ALE) could be predicted using a faster method: short-term selection of a dense transposon mutant library, analysed by transposon-directed insertion site sequencing (TraDIS). We evolved E. coli K-12 MG1655 in unbuffered LB at pH 4.5 for 5 months and compared the mutations that arose to those selected after just 10 days of evolution using a high-density transposon library in the same strain and conditions. We observed significant overlap in the genes identified by both approaches, including independent disruptions in shared regulatory pathways. This suggests that short-term selection on a diverse mutant population can uncover many of the same adaptive changes seen in long-term evolution experiments. We also identified and validated the fitness effects of several mutations uniquely found in the transposon approach. Rather than predicting specific mutations from first principles, this method offers a rapid, empirical means of anticipating which genes are likely to be involved in adaptation under defined conditions, helping to guide further mechanistic studies by offering a powerful shortcut for investigating microbial evolution.

microbiology↗

bioRxiv2025

The conserved N-terminal SANT1-binding domain (SBD) of EZH2 Regulates PRC2 Activity

Polycomb group proteins maintain gene expression patterns established during early development, with Polycomb Repressive Complex 2 (PRC2) methyltransferase a key regulator of cell differentiation, identity and plasticity. Consequently, extensive somatic mutations in PRC2, including gain- or loss- of function (GOF or LOF), are observed in human cancers. The regulation of chromatin structure by PRC2 is critically dependent on its EZH2 (Enhancer of Zeste Homolog 2) subunit, which catalyzes the methylation of histone H3 lysine 27 (H3K27). Recent structural studies of PRC2 revealed extensive conformational changes in the non-catalytic EZH2 N-terminal SANT-Binding Domain (SBD) during PRC2 activation, though the functional significance remains unclear. Here, we investigate how the SBD regulates PRC2 function. The domain is highly conserved in metazoans, dispensable for PRC2 assembly and chromatin localization, yet required for genome-wide histone H3K27 methylation. Further, we show that an intact SBD is necessary for the proliferation of EZH2- addicted lymphomas, and its deletion in the presence of EZH2 GOF mutations inhibits cancer cell growth. These observations provide new insights to the regulation of PRC2 activity in normal development and malignancy.

genetics↗

bioRxiv2024

A Ketogenic Diet Sensitizes Pancreatic Cancer to Inhibition of Glutamine Metabolism

Pancreatic cancer is the third leading cause of cancer death in the United States, and while conventional chemotherapy remains the standard treatment, responses are poor. Safe and alternative therapeutic strategies are urgently needed1. A ketogenic diet has been shown to have anti-tumor effects across diverse cancer types but will unlikely have a significant effect alone. However, the diet shifts metabolism in tumors to create new vulnerabilities that can be targeted (1). Modulators of glutamine metabolism have shown promise in pre-clinical models but have failed to have a marked impact against cancer in the clinic. We show that a ketogenic diet increases TCA and glutamine-associated metabolites in murine pancreatic cancer models and under metabolic conditions that simulate a ketogenic diet in vitro. The metabolic shift leads to increased reliance on glutamine-mediated anaplerosis to compensate for low glucose abundance associated with a ketogenic diet. As a result, glutamine metabolism inhibitors, such as DON and CB839 in combination with a ketogenic diet had robust anti-cancer effects. These findings provide rationale to study the use of a ketogenic diet with glutamine targeted therapies in a clinical context. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/604377v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ebeefforg.highwire.dtl.DTLVardef@97e2ceorg.highwire.dtl.DTLVardef@1ab45c3org.highwire.dtl.DTLVardef@1713540_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract Description: Mechanistic rationale for combining a ketogenic diet and glutamine metabolism inhibitors. The combination of low glucose from a ketogenic diet and pharmacologic glutamine inhibition impairs nutrient input to mitochondria, reducing cancer growth.

cancer biology↗

bioRxiv2024

An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nanoLC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.

biochemistry↗

bioRxiv2023

snPATHO-seq: unlocking the pathology archives

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low DNA and RNA quality. In this study, leveraging recent single-cell genomic technology advances, we introduce a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples.

molecular biology↗

bioRxiv2018

Inter- and intra-domain functional redundancy in the rumen microbiome during plant biomass degradation

BackgroundRuminant livestock is a major source of the potent greenhouse gas methane (CH4), produced by the complex rumen microbiome. Using an integrated approach, combining quantitative metatranscriptomics with gas- and volatile fatty acid (VFA) profiling, we gained fundamental insights into temporal dynamics of the cow rumen microbiome during feed degradation.\n\nResultsThe microbiome composition was highly individual and remarkably stable within each cow, despite similar gas emission and VFA profiles between cows. Gene expression profiles revealed a fast microbial growth response to feeding, reflected by drastic increases in microbial biomass, CH4 emissions and VFA concentrations. Microbiome individuality was accompanied by high inter- and intra-domain functional redundancy among pro- and eukaryotic microbiome members in the key steps of anaerobic feed degradation. Methyl-reducing but not CO2-reducing methanogens were correlated with increased CH4 emissions during plant biomass degradation.\n\nConclusionsThe major response of the rumen microbiome to feed intake was a general growth of the whole community. The high functional redundancy of the cow-individual microbiomes was possibly linked to the robust performance of the anaerobic degradation process. Furthermore, the strong response of methylotrophic methanogens is suggesting that they might play a more important role in ruminant CH4 emissions than previously assumed, making them potential targets for CH4 mitigation strategies.

microbiology↗

bioRxiv2017

The essential genome of Escherichia coli K-12

Transposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable. To address this, a high density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false positive identification of essential gene candidates, statistical data analysis included corrections for both gene length and genome length. Through this analysis new essential genes and genes previously incorrectly designated as essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects and fine resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis datasets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.\n\nIMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in E. coli, we constructed a very high density transposon mutant library. Initial automated analysis of the resulting data revealed many discrepancies when compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high density TraDIS sequencing data for each putative essential gene for the model laboratory organism, Escherichia coli. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone and a provides new standard for the analysis of TraDIS data.

microbiology↗