Biology subjects

Wood, C.

Publications and source records attributed to Wood, C..

35 records · Page 2Linked to original sources

bioRxiv2022

Fluorescence fluctuation based super resolution microscopy, basic concepts for an easy start.

Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of about half of the wavelength of the detected light, i.e., within the range of 200 to 300 nm. The Fluorescence Fluctuation based Super Resolution Microscopy (FF-SRM) encompases a collection of image analysis techniques which rely on the statistical processing of temporal variations of fluorescence to reduce the uncertainty about the fluorophore positions within a sample, hence, bringing spatial resolution down to several tens of nm. The FF-SRM is known to be suitable for live-cell imaging due to its compatibility with most fluorescent probes and lower instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are revisited. Their operational parameters are explained and guidance for its selection is provided.

bioinformatics↗

bioRxiv2022

Biological misinterpretation of transcriptional signatures in tumour samples can unknowingly undermine mechanistic understanding and faithful alignment with preclinical data

Precise mechanism-based gene expression signatures (GESs) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signalling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumour samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumour stroma percentage can directly influence GESs, undermining the intended mechanistic signalling. Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single cell RNAseq and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely-used GESs to be influenced, or confounded, by stromal content in tumour tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumour models not fully recapitulating the stromal and immune microenvironment. As such, efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

cancer biology↗

bioRxiv2021

Nanoscopic resolution within a single imaging frame

Mean-Shift Super Resolution (MSSR) is a principle based on the Mean Shift theory that extends spatial resolution in fluorescence images, beyond the diffraction limit. MSSR works on low- and high-density fluorophore images, is not limited by the architecture of the detector (EM-CCD, sCMOS, or photomultiplier-based laser scanning systems) and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image series, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other analytical super resolution image approaches. Altogether, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

bioinformatics↗

bioRxiv2021

Genome-wide identification of sexual-reproduction genes in fission yeast via transposon-insertion sequencing

Many genes required for sexual reproduction remain to be identified. Moreover, many of the genes that are known have been characterized in distinct experiments using different conditions, which complicates understanding the relative contributions of genes to sex. To address these challenges, we developed an assay in Schizosaccharomyces pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wildtype. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

genetics↗

bioRxiv2021

Improving RNA branching predictions: advances and limitations

Minimum free energy prediction of RNA secondary structures is based on the Nearest Neighbor Thermodynamics Model. While such predictions are typically good, the accuracy can vary widely even for short sequences, and the branching thermodynamics are an important factor in this variance. Recently, the simplest model for multiloop energetics -- a linear function of the number of branches and unpaired nucleotides -- was found to be the best. Subsequently, a parametric analysis demonstrated that per family accuracy can be improved by changing the weightings in this linear function. However, the extent of improvement was not known due to the ad hoc method used to find the new parameters. Here we develop a branch-and-bound algorithm that finds the set of optimal parameters with the highest average accuracy for a given set of sequences. Our analysis shows that the previous ad hoc parameters are nearly optimal for tRNA and 5S rRNA sequences on both training and testing sets. Moreover, cross-family improvement is possible but more difficult because competing parameter regions favor different families. The results also indicate that restricting the unpaired nucleotide penalty to small values is warranted. This reduction makes analyzing longer sequences using the present techniques more feasible.

bioinformatics↗

bioRxiv2020

The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death

Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The similar Wtf4antidote protein also forms aggregates but is sequestered within or near vacuoles and is mostly benign. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate sequestration, as mutations that disrupt either of these processes results in cell death. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy gametes.

cell biology↗

bioRxiv2019

Engineering a functional NifDK polyprotein resistant to mitochondrial degradation

To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

synthetic biology↗

bioRxiv2019

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death

NR4A is a nuclear receptor protein family whose members act as sensors of cellular environment and regulate multiple processes such as metabolism, proliferation, migration, apoptosis, and autophagy. Since the ligand binding domains of these receptors have no cavity for ligand interaction, their function is most likely regulated by protein abundance and post-translational modifications. In particular, NR4A1 is regulated by protein abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic translocation), and acts both as a transcription factor and as a regulator of other interacting proteins. SUMOylation is a post-translational modification that can affect protein stability, transcriptional activity, alter protein-protein interactions and modify intracellular localization of target proteins. In the present study we evaluated the role of SUMOylation as a posttranslational modification that can regulate the activity of NR4A1 to induce autophagy-dependent cell death. We focused on a model potentially relevant for neuronal cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic cell death. We observed that a triple mutant in SUMOylation sites has reduced SUMOylation, increased transcriptional activity, altered intracellular distribution, and more importantly, its ability to induce autophagic cell death is impaired.\n\nSummary StatementThe modification of the nuclear receptor NR4A1 by SUMO regulates its transcriptional activity, intracellular localization and is required to induce autophagic cell death.

biochemistry↗

bioRxiv2019

High-throughput, image-based flow cytometry and clustering method for phenotyping heterogeneous cell populations

Image-based cell classification has become a common tool to identify phenotypic changes in cell populations. However, this methodology is limited to organisms possessing well characterized species-specific reagents (e.g., antibodies) that allow cell identification, clustering and convolutional neural network (CNN) training. In the absence of such reagents, the power of image-based classification has remained mostly off-limits to many research organisms. We have developed an image-based classification methodology we named Image3C (Image-Cytometry Cell Classification) that does not require species-specific reagents nor pre-existing knowledge about the sample. Image3C combines image-based flow cytometry with an unbiased, high-throughput cell cluster pipeline and CNN integration. Image3C exploits intrinsic cellular features and non-species-specific dyes to perform de novo cell composition analysis and to detect changes in cellular composition between different conditions. Therefore, Image3C expands the use of imaged-based analyses of cell population composition to research organisms in which detailed cellular phenotypes are unknown or for which species-specific reagents are not available. Impact statementImage3C analyzes cell populations through image-based clustering and neural network training, which allows single-cell analysis in research organisms devoid of species-specific reagents or pre-existing knowledge on cell phenotypes.

zoology↗

bioRxiv2018

Cryptic and extensive hybridization between ancient lineages of American crows

Most species and therefore most hybrid zones have historically been described using phenotypic characters. However, both speciation and hybridization can occur with negligible morphological differentiation. The Northwestern Crow (Corvus caurinus) and American Crow (Corvus brachyrhynchos) are sister taxonomic species with a continuous distribution that lack reliable traditional characters for identification. In this first population genomic study of Northwestern and American crows, we use genomic SNPs (nuDNA) and mtDNA to investigate whether these crows are genetically differentiated and the extent to which they may hybridize. We found that American and Northwestern crows have distinct evolutionary histories, supported by two nuDNA ancestry clusters and two 1.1%-divergent mtDNA clades dating to the late Pleistocene, when glacial advances may have isolated crow populations in separate refugia. We document extensive hybridization, with geographic overlap of mtDNA clades and admixture of nuDNA across >1,400 km of western Washington and western British Columbia. This broad hybrid zone consists of late-generation hybrids and backcrosses, not recent (e.g., F1) hybrids. Nuclear DNA and mtDNA clines were both centered in southwestern British Columbia, farther north than previously postulated. The mtDNA cline was narrower than the nuDNA cline, consistent with Haldanes rule but not sex-biased dispersal. Overall, our results suggest a history of reticulate evolution in American and Northwestern crows, consistent with potentially recurring neutral expansion(s) from Pleistocene glacial refugia followed by lineage fusion(s). However, we do not rule out a contributing role for more recent potential drivers of hybridization, such as expansion into human-modified habitats.

evolutionary biology↗

bioRxiv2018

Nanoscale organization of rotavirus replication machineries

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viro-plasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins and viral dsRNA within and around the VPs. The observed viral components are spatially organized as 6 concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6 proteins and the dsRNA. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

microbiology↗

bioRxiv2018

SOCIAL STRESS AS A PRIMING MECHANISM FOR THERMAL TOLERANCE DURING A POLEWARD RANGE SHIFT

Cold tolerance plays a critical role in determining species geographical range limits. Previous studies have found that range shifts in response to climate warming are facilitated by cold acclimation capacities, due to increasingly colder and variable weather at high latitudes, and that cold tolerance can also be influenced by social factors. In this study we combined experiments and field studies to investigate the climatic and social factors affecting cold tolerances in range-shifting populations of the female-polymorphic damselfly Ischnura elegans in northeast Scotland. In the field, we observed both environmental (measured via habitat suitability) and social (sex ratio and density) effects on cold tolerance (CTmin). Androchrome females (male-like females) were less susceptible to beneficial social effects on cold tolerance than gynochromes (female-like females), and correspondingly, gynochrome frequency increased at colder, environmentally-limiting sites towards the range margin. Our manipulations of density in the laboratory further provide novel, experimental evidence that social interactions directly impact cold tolerance n this species. These results suggest that reciprocal effects of social environments on thermal acclimation may be an important but commonly overlooked aspect of allee effects which contribute to the formation of range margins. Moreover, our results point to a wider need to consider the role of population and social dynamics to shape both the thermal physiology of individuals and the thermal niches of species.

evolutionary biology↗

bioRxiv2018

The Relationship Between the Cervical Microbiome, HIV Status, and Pre-Cancerous Lesions

Nearly all cervical cancers are causally associated with Human Papillomavirus (HPV). The burden of HPV-associated dysplasias in Sub-Saharan Africa is influenced by HIV. To investigate the role of the bacterial microbiome in cervical dysplasia, cytobrush samples were collected directly from cervical lesions of 144 Tanzanian women. The V4 hypervariable region of the 16S rRNA gene was amplified and deep-sequenced. Alpha diversity metrics; Chao1, PD whole tree, and operational taxonomic Unit (OTU) estimates, displayed significantly higher bacterial richness in HIV positive patients (P = 0.01) than in HIV negative patients. Within HIV positive patients, there was higher bacterial richness in patients with high grade squamous intraepithelial lesions (HSIL; P = 0.13) than those without lesions. The most abundant OTUs associated with high-grade squamous intraepitheilal lesions (HSIL) were Mycoplasmatales, Pseudomonadales, and Staphylococcus. We suggest that a chronic mycoplasma infection of the cervix can contribute to HPV-dependent dysplasia by sustained inflammatory signals.

cancer biology↗

bioRxiv2018

Conserved Roles of C. elegans and Human MANFs in Sulfatide Binding and Cytoprotection

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) is an endoplasmic reticulum (ER) protein that can be secreted and protect dopamine neurons and cardiomyocytes from ER stress and apoptosis. The mechanism of action of extracellular MANF has long been elusive. From a genetic screen for mutants with abnormal ER stress response, we identified the gene Y54G2A.23 as the evolutionarily conserved C. elegans MANF orthologue. We find that MANF binds to the lipid sulfatide, also known as 3-O-sulfogalactosylceramide present in serum and outer-cell membrane leaflets, directly in isolated forms and in reconstituted lipid micelles. Sulfatide binding promotes cellular MANF uptake and cytoprotection from hypoxia-induced cell death. Heightened ER stress responses of MANF-null C. elegans mutants and mammalian cells are alleviated by human MANF in a sulfatide-dependent manner. Our results demonstrate conserved roles of MANF in sulfatide binding and ER stress response, supporting sulfatide as a long-sought lipid mediator of MANFs cytoprotection.

cell biology↗

bioRxiv2017

Sperm chemotaxis is driven by the slope of the chemoattractant concentration field

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis.

biophysics↗

bioRxiv2017

De novo assembly of microbial genomes from human gut metagenomes using barcoded short read sequences

Although shotgun short-read sequencing has facilitated the study of strain-level architecture within complex microbial communities, existing metagenomic approaches often cannot capture structural differences between closely related co-occurring strains. Recent methods, which employ read cloud sequencing and specialized assembly techniques, provide significantly improved genome drafts and show potential to capture these strain-level differences. Here, we apply this read cloud metagenomic approach to longitudinal stool samples from a patient undergoing hematopoietic cell transplantation. The patients microbiome is profoundly disrupted and is eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allows us to predict that particular mobile element integrations result in increased antibiotic resistance, which we further support using in vitro antibiotic susceptibility testing. Thus, we find read cloud sequencing to be useful in identifying strain-level differences that underlie differential fitness.

bioinformatics↗

bioRxiv2017

Geographically structured genetic variation in the Medicago lupulina-Ensifer mutualism

Mutualisms are interspecific interactions affecting the ecology and evolution of species. Patterns of geographic variation in interacting species may play an important role in understanding how variation is maintained in mutualisms, particularly in introduced ranges. One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome-wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.

evolutionary biology↗