Biology subjects

Ritchie, M. E.

Publications and source records attributed to Ritchie, M. E..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Metabolic scaling in small life forms

Metabolic scaling is one of the most important patterns in biology. Theory explaining the 3/4-power size-scaling of biological metabolic rate does not predict the non-linear scaling observed for smaller life forms. Here we present a new model for cells < 10-8 m3 that maximizes power from the reaction-displacement dynamics of enzyme-catalyzed reactions. Maximum metabolic rate is achieved through an allocation of cell volume to optimize a ratio of reaction velocity to molecular movement. Small cells < 10-17 m3 generate power under diffusion by diluting enzyme concentration as cell volume increases. Larger cells require bulk flow of cytoplasm generated by molecular motors. These outcomes predict curves with literature-reported parameters that match the observed scaling of metabolic rates for unicells, and predicts the volume at which Prokaryotes transition to Eukaryotes. We thus reveal multiple size-dependent physical constraints for microbes in a model that extends prior work to provide a parsimonious hypothesis for how metabolism scales across small life.

biophysics↗

bioRxiv2023

Systematic comparison of sequencing-based spatial transcriptomic methods

Recent advancements of sequencing-based spatial transcriptomics (sST) have catalyzed significant advancements by facilitating transcriptome-scale spatial gene expression measurement. Despite this progress, efforts to comprehensively benchmark different platforms are currently lacking. The extant variability across technologies and datasets poses challenges in formulating standardized evaluation metrics. In this study, we established a collection of reference tissues and regions characterized by well-defined histological architectures, and used them to generate data to compare six sST methods. We highlighted molecular diffusion as a variable parameter across different methods and tissues, significantly impacting the effective resolutions. Furthermore, we observed that spatial transcriptomic data demonstrate unique attributes beyond merely adding a spatial axis to single-cell data, including an enhanced ability to capture patterned rare cell states along with specific markers, albeit being influenced by multiple factors including sequencing depth and resolution. Our study assists biologists in sST platform selection, and helps foster a consensus on evaluation standards and establish a framework for future benchmarking efforts that can be used as a gold standard for the development and benchmarking of computational tools for spatial transcriptomic analysis.

genomics↗

bioRxiv2023

An optimised protocol for quality control of gene therapy vectors using Nanopore direct RNA sequencing

Despite recent advances made towards improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine Nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analysed lentiviral vectors, which account for most of the detected RNA fragments. Finally, we show that these insights can be used for optimization of lentiviral vector design. In summary, Nanopore direct RNA sequencing is a powerful tool for the quality control and optimisation of lentiviral vectors, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

genomics↗

bioRxiv2023

An immunohistochemical atlas of necroptotic pathway expression

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo. HighlightsO_LI13 automated immunohistochemistry protocols for detecting the necroptotic pathway C_LIO_LINecroptotic pathway expression is confined to fast-cycling immune barriers C_LIO_LINecroptotic pathway expression changes at sites of immunoinflammatory challenge C_LIO_LIImmunodetection of necrosomes in IBD patients is a putative new diagnostic tool C_LI

cell biology↗

bioRxiv2023

scPipe: An extended preprocessing pipeline for comprehensive single-cell ATAC-Seq data integration in R/Bioconductor

scPipe is a flexible R/Bioconductor package originally developed to analyse platform-independent single-cell RNA-Seq data. To expand its preprocessing capability to accommodate new single-cell technologies, we further developed scPipe to handle single-cell ATAC-Seq and multi-modal (RNA-Seq and ATAC-Seq) data. After executing multiple data cleaning steps to remove duplicated reads, low abundance features and cells of poor quality, a SingleCellExperiment object is created that contains a sparse count matrix with features of interest in the rows and cells in the columns. Quality control information (e.g. counts per cell, features per cell, total number of fragments, fraction of fragments per peak) and any relevant feature annotations are stored as metadata. We demonstrate that scPipe can efficiently identify "true" cells and provides flexibility for the user to fine-tune the quality control thresholds using various feature and cell-based metrics collected during data preprocessing. Researchers can then take advantage of various downstream single-cell tools available in Bioconductor for further analysis of scATAC-Seq data such as dimensionality reduction, clustering, motif enrichment, differential accessibility and cis-regulatory network analysis. The scPipe package enables a complete beginning-to-end pipeline for single-cell ATAC-Seq and RNA-Seq data analysis in R.

bioinformatics↗

bioRxiv2023

Flexiplex: A versatile demultiplexer and search tool for omics data

The process of analyzing high throughput sequencing data often requires the identification and extraction of specific target sequences. This could include tasks such as identifying cellular barcodes and UMIs in single cell data, and specific genetic variants for genotyping. However, existing tools which perform these functions are often task-specific, such as only demultiplexing barcodes for a dedicated type of experiment, or are not tolerant to noise in the sequencing data. To overcome these limitations, we developed Flexiplex, a versatile and fast sequence searching and demultiplexing tool for omics data, which is based on the Levenshtein distance and thus allows imperfect matches. We demonstrate Flexiplexs application on three use cases, identifying cell line specific sequences in Illumina short-read single cell data, and discovering and demultiplexing cellular barcodes from noisy long-read single cell RNA-seq data. We show that Flexiplex achieves an excellent balance of accuracy and computational efficiency compared to leading task-specific tools. Flexiplex is available at https://davidsongroup.github.io/flexiplex/.

bioinformatics↗

bioRxiv2023

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

AbstractThe Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

genomics↗

bioRxiv2023

Metabolism regulates muscle stem cell self-renewal by connecting the microenvironment and histone acetylation

Skeletal muscle contains a resident population of somatic stem cells capable of both self-renewal and differentiation. The signals that regulate this important decision have yet to be fully elucidated. Here we use metabolomics and mass spectrometry imaging (MSI) to identity a state of localized hyperglycaemia following skeletal muscle injury. We show that committed muscle progenitor cells exhibit an enrichment of glycolytic and TCA cycle genes and that extracellular monosaccharide availability regulates intracellular citrate levels and global histone acetylation. Muscle stem cells exposed to a reduced (or altered) monosaccharide environment demonstrate reduced global histone acetylation and transcription of myogenic determination factors (including myod1). Importantly, reduced monosaccharide availability was linked directly to increased rates of asymmetric division and muscle stem cell self-renewal in regenerating skeletal muscle. Our results reveal an important role for the extracellular metabolic environment in the decision to undergo self-renewal or myogenic commitment during skeletal muscle regeneration.

cell biology↗

bioRxiv2023

Restrander: rapid orientation and QC of long-read cDNA data

In transcriptomic analyses, it is helpful to keep track of the strand of the RNA molecules. However, the Oxford Nanopore long-read cDNA sequencing protocols generate reads that correspond to either the first or second-strand cDNA, therefore the strandedness of the initial transcript has to be inferred bioinformatically. Reverse transcription and PCR can also introduce artefacts which should be flagged in data pre-processing. Here we introduce Restrander, a lightning-fast and highly accurate tool for restranding and quality checking long-read cDNA sequencing data. Thanks to its C++ implementation, Restrander was faster than Oxford Nanopore Technologies existing tool Pychopper, and correctly restranded more reads due to its strategy of searching for polyA/T tails in addition to primer sequences from the reverse transcription and template-switch steps. We found that restranding improved the process of visualising and exploring data, and increased the number of novel isoforms discovered by bambu, particularly in regions where sense and antisense transcripts co-occur. The artefact detection implemented in Restrander quantifies reads which do not have the correct 5 and 3 ends, a feature which is useful in quality control for library preparation. Restrander is pre-configured for all major cDNA protocols, and can be customised with user-defined primers. Restrander is available at https://github.com/jakob-schuster/restrander

bioinformatics↗

bioRxiv2023

Dividing out quantification uncertainty allows efficient assessment of differential transcript expression

Differential expression analysis of RNA-seq is one of the most commonly performed bioinformatics analyses. Transcript-level quantifications are inherently more uncertain than gene-level read counts because of ambiguous assignment of sequence reads to transcripts. While sequence reads can usually be assigned unambiguously to a gene, reads are very often compatible with multiple transcripts for that gene, particularly for genes with many isoforms. Software tools designed for gene-level differential expression do not perform optimally on transcript counts because the read-to-transcript ambiguity (RTA) disrupts the mean-variance relationship normally observed for gene level RNA-seq data and interferes with the efficiency of the empirical Bayes dispersion estimation procedures. The pseudoaligners kallisto and Salmon provide bootstrap samples from which quantification uncertainty can be assessed. We show that the overdispersion arising from RTA can be elegantly estimated by fitting a quasi-Poisson model to the bootstrap counts for each transcript. The technical overdispersion arising from RTA can then be divided out of the transcript counts, leading to scaled counts that can be input for analysis by established gene-level software tools with full statistical efficiency. Comprehensive simulations and test data show that an edgeR analysis of the scaled counts is more powerful and efficient than previous differential transcript expression pipelines while providing correct control of the false discovery rate. Simulations explore a wide range of scenarios including the effects of paired vs single-end reads, different read lengths and different numbers of replicates.

bioinformatics↗

bioRxiv2023

FGF independent MEK1/2 signalling is essential for male germline development in mice

BackgroundGermline development provides the founding cells for spermatogenesis and oogenesis in males and females, respectively. Disrupted germline differentiation or compromised testis development can lead to subfertility or infertility and are strongly associated with testis cancer in humans. In mice, SRY and SOX9 induce expression of a range of genes, including Fgf9, that promote Sertoli cell differentiation and testis development. FGF9 is also thought to promote male germline differentiation but the pathway through which it signals is unknown. As FGFs signal through Mitogen-Activated Protein Kinases (MAPKs) in other tissues, we explored whether FGF9 regulates male germline development through MAPK by inhibiting either FGF or MEK1/2 signalling in fetal testis cultures from embryonic day (E)12.5, immediately after gonadal sex determination and testis cord formation, but prior to male germline commitment. ResultsInhibition of MEK1/2 disrupted mitotic arrest, dysregulated a broad range of male germline development genes and prevented the upregulation of key male germline markers DPPA4 and DNMT3L. In contrast, when FGF signalling was inhibited, the male germline specific transcriptional program and the expression of male germline markers DPPA4 and DNMT3L were unaffected, and germ cells entered mitotic arrest normally. While male germline development was not disrupted by FGF inhibition, some genes were commonly altered after 24h of FGF or MEK1/2 inhibition including genes involved in maintenance, germline stem cells, Nodal signalling, proliferation, and germline cancer. ConclusionsTogether, these data demonstrate a novel and essential role for MEK1/2 signalling in male germline differentiation, but a more limited role for FGF signalling. Our data strongly indicate that additional ligands act through MEK1/2 to promote male germline differentiation and highlight a need for further mechanistic understanding of male germline development.

developmental biology↗

bioRxiv2023

Establishment of cell transcriptional identity during seed germination

Germination involves highly dynamic transcriptional programs as the cells of seeds reactivate and express the functions necessary to establish in the environment. Individual cell types have distinct roles within the embryo, so must therefore have cell-type specific gene expression and gene regulatory networks. We can better understand how the functions of different cell types are established and contribute to the embryo by determining how cell-type specific transcription begins and changes through germination. Here we describe a temporal analysis of the germinating Arabidopsis embryo at single-cell resolution. We define the highly dynamic cell-type specific patterns of gene expression and how these relate to changing cellular function as germination progresses. Underlying these are unique gene regulatory networks and transcription factor activity. We unexpectedly discover that most embryo cells transition through the same initial transcriptional state early in germination, after which cell-type specific gene expression is established. Furthermore, our analyses support previous findings that the earliest events leading to the induction of embryo growth take place in the vasculature. Overall, our study constitutes a general framework to characterise Arabidopsis cell states through embryo growth, allowing investigation of different genotypes and other plant species whose seed strategies may differ.

plant biology↗

bioRxiv2022

Integrating defense and leaf economic spectrum traits in a tropical savanna plant

O_LIAllocation to plant defense traits likely depends on resource supply, herbivory, and other plant functional traits such as the leaf economic spectrum (LES) traits. Yet, attempts to integrate defense and resource acquisitive traits remains elusive. C_LIO_LIWe assessed intraspecific correlations between different defense and LES traits in a widely distributed tropical savanna herb, Solanum incanum, a unique model species for studying allocations to physical, chemical, and structural defenses to mammalian herbivory. C_LIO_LIIn a multivariate trait space, the structural defenses - lignin and cellulose - were positively related to the resource conservative traits - low SLA and low leaf N. Phenolic content, a chemical defense, was positively associated with resource acquisitive traits - high SLA and high leaf N - while also being associated with an independent third component axis. Both principal components 1 and 3 were not associated with resource supply and herbivory intensity. In contrast, spine density - a physical defense - was orthogonal to the LES axis and positively associated with soil P and herbivory intensity. C_LIO_LISynthesis: These results suggest a hypothesized "pyramid" of trade-offs in allocation to defense along the LES and herbivory intensity axes. Therefore, future attempts to integrate defense traits with the broader plant functional trait framework needs a multifaceted approach that accounts for unique influences of resource acquisitive traits and herbivory intensity. C_LI

ecology↗

bioRxiv2022

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that comprise distinct phasevarions

ABSTRACTActinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase- variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferase, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase variants results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

microbiology↗

bioRxiv2022

Landscape variation in defense traits along gradients of multiple resources and mammalian herbivory

Variation in defense traits likely depends on access to different resources and risk from herbivory. Plant defense theories have predicted both positive and negative associations between defense traits and access to resources, but relatively few studies have explored intraspecific variation in defense traits along multiple resource and mammalian herbivory risk gradients. We assessed relationships between herbivory intensity, multiple resources, and plant defense traits using a widely distributed tropical savanna herb, Solanum incanum. As independent measures of risk from large mammal herbivores are rare, we used a satellite-based vegetation index to predict herbivory intensity at the landscape scale. We found that the satellite-based estimate of herbivory intensity was positively associated with browser abundance and total soil P, but negatively associated with rainfall. Intraspecific defense traits too varied substantially across sites (n=43) but only variation in spine density was associated with herbivory intensity and plant resources, such that spine density was positively associated with both rainfall and soil P, but bimodally associated with herbivory intensity. Taken together, it suggests that defenses maybe favored either where resources for defense are abundant under low but still present risk (i.e, at high rainfall sites) or where resource-expensive plant tissue is at high risk (i.e, at high soil P sites). This hints at the possibility of a shift from a resource-associated (bottom-up) to an herbivory-associated (top-down) control of allocation to defenses along an environmental gradient. Additionally, the independent effect of soil P on a carbon-based defense, spine density, suggests potential for resources that are not components of defenses to also influence allocation to defense traits. Thus, our study provides evidence for the influence of multiple drivers, resources, and herbivory intensity, on anti-herbivore defenses and their shifting relative importance on allocation to defenses along an environmental gradient.

ecology↗

bioRxiv2022

Modelling group heteroscedasticity in single-cellRNA-seq pseudo-bulk data

Group heteroscedasticity is commonly observed in pseudo-bulk single-cell RNA-seq datasets and when not modelled appro-priately, its presence can hamper the detection of differentially expressed genes. Most bulk RNA-seq methods assume equal group variances which will under- and/or over-estimate the true variability in such datasets. We present two methods that account for heteroscedastic groups, namely voomByGroup and voomWithQualityWeights using a blocked design (voomQWB). Compared to current gold standard methods that do not account for heteroscedasticity, we show results from simulation studies and various experiments that demonstrate the superior performance of both voomByGroup and voomQWB in error control and power when group variances in pseudo-bulk scRNA-seq data are unequal. We recommend the use of either of these methods over established approaches, with voomByGroup having the advantage of accurate variance estimation since group variance trends can take on different "shapes", whilst voomQWB has the advantage of catering to complex study designs.

bioinformatics↗

bioRxiv2022

Benchmarking long-read RNA-sequencing analysis tools using in silico mixtures

The current lack of benchmark datasets with inbuilt ground-truth makes it challenging to compare the performance of existing long-read isoform detection and differential expression analysis workflows. Here, we present a benchmark experiment using two human lung adenocarcinoma cell lines that were each profiled in triplicate together with synthetic, spliced, spike-in RNAs ("sequins"). Samples were deeply sequenced on both Illumina short-read and Oxford Nanopore Technologies long-read platforms. Alongside the ground-truth available via the sequins, we created in silico mixture samples to allow performance assessment in the absence of true positives or true negatives. Our results show that, StringTie2 and bambu outperformed other tools from the 6 isoform detection tools tested, DESeq2, edgeR and limma-voom were best amongst the 5 differential transcript expression tools tested and there was no clear front-runner for performing differential transcript usage analysis between the 5 tools compared, which suggests further methods development is needed for this application.

bioinformatics↗

bioRxiv2022

A method for stabilising the XX karyotype in female mESC cultures

Female mouse embryonic stem cells (mESCs) present differently to male mESCs in several fundamental ways, however complications with their in vitro culture have resulted in an underrepresentation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female mESCs apart from males. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state near indistinguishable from male. If female pluripotency is to be studied effectively in this system, it is critical that high quality cultures of XX mESCs are available. Here we report a method for better maintaining XX female mESCs in culture, which also stabilises the male karyotype, and makes study of female specific pluripotency more feasible.

cell biology↗