Biology subjects

Burt, A.

Publications and source records attributed to Burt, A..

53 records · Page 3Linked to original sources

bioRxiv2021

Montage electron tomography of vitrified specimens

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 15 [A] were observed in the montaged tilt-series, and diverse cellular features were evident in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

biophysics↗

bioRxiv2021

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ( homing) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations. Author summaryGene drives have the potential to be applied as novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism - most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.

genetics↗

bioRxiv2021

Alternative architecture of the E. coli chemosensory array

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

biochemistry↗

bioRxiv2021

Variation in Placental microRNA Expression Associates with Familial Cardiovascular Disease

In the United States, cardiovascular disease is the leading cause of death, and the rate of maternal mortality remains among the highest of any industrialized nation. Maternal cardiometabolic health throughout gestation and postpartum is representative of placental health and physiology. Both proper placental functionality and placental microRNA expression are essential to successful pregnancy outcomes, and both are highly sensitive to genetic and environmental sources of variation. While placental pathologies, such as preeclampsia, are associated with maternal cardiovascular health and may contribute to the developmental programming of cardiovascular disease, the role of more subtle alterations to placental function and microRNA expression in this relationship remains poorly understood. To develop a more comprehensive understanding of how cardiometabolic health influences placental microRNA expression, and how this shapes placental functionality, we performed small RNA sequencing to investigate microRNA in the placentae from the Rhode Island Child Health Study (n=230). We modeled microRNA counts on maternal family history of cardiovascular disease using negative binomial generalized linear models, and identified microRNAs that were differential expressed (DEmiRs) at a false discovery rate (FDR) less than 0.10. Utilizing parallel mRNA sequencing data and bioinformatic target prediction software, we identified potential mRNA targets of these DEmiRs. We identified 9 DEmiRs, with predicted targets of those miRNA enriched overwhelmingly in the TGF{beta} signaling pathway but also in pathways involving cellular metabolism and immunomodulation. Overall, we identified a robust association existing between familial cardiovascular disease and placental microRNA expression which may be implicated in both placental insufficiencies and the developmental programming of cardiovascular disease.

genetics↗

bioRxiv2021

Tools enabling flexible approaches to high-resolution subtomogram averaging

Cryo-electron tomography and subtomogram averaging are increasingly used for macromolecular structure determination in situ. Here we introduce a set of computational tools and resources designed to enable flexible approaches to subtomogram averaging. In particular, our tools simplify metadata handling, increase automation, and interface the Dynamo software package with the Warp-Relion-M pipeline. We provide a framework for ab initio and geometrical approaches to subtomogram averaging combining tools from these packages. We illustrate the power of working within the framework enabled by our developments by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles, and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we establish an open and collaborative online platform for sharing knowledge and tools related to cryo-electron tomography data processing. To this platform, we contribute a comprehensive guide to obtaining state-of-the-art results from EMPIAR-10164.

biophysics↗

bioRxiv2021

Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control.

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

genomics↗

bioRxiv2021

Double drives and private alleles for localised population genetic control

Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations. Author summarySome disease vectors, invasive species, and other pests cannot be satisfactorily controlled with existing interventions, and new methods are required. Synthetic gene drive systems that are able to spread though populations because they are inherited at a greater-than-Mendelian rate have the potential to form the basis for new, highly efficient pest control measures. The most efficient such strategies use natural gene flow to spread a construct throughout a species range, but if control is only desired in a particular location then these approaches may not be appropriate. As some of the most promising gene drive designs use nucleases to target specific DNA sequences, it ought to be possible to exploit sequence differences between target and non-target populations to restrict the spread and impact of a gene drive. In this paper we propose using two-construct "double drive" designs that exploit pre-existing sequence differences between target and non-target populations. Our approaches maintain the efficiencies associated with only small release rates being needed and can work if the differentiated locus is selectively neutral and if the differentiation is far from complete, and therefore expand the range of options to be considered in developing genetic approaches to control pest species.

synthetic biology↗

bioRxiv2020

Direct information estimation from cryo-EM Movies with CARYON

While cryo-EM with modern direct electron detectors has proven incredibly powerful, becoming a dominant technique in structural biology, the analysis of cryo-EM images is significantly complicated by their exceptionally low signal-to-noise ratio, limiting the accuracy of the parameterisation of the physical models required for successful classification and reconstruction. Micrographs from modern direct electron detectors are typically collected as dose-fractionated multi-frame movies to allow the recording of separated individual electron impacts. These detectors improve electron detection and allow for both inter-frame motion correction, and dose-dependent image filtering, lessening the overall impact of effects deleterious to the recovery of high-resolution information. In this study we measured the information content at each spatial frequency in cryo-EM movies as it accrues during the course of an exposure. We show that, as well as correction for motion and radiation damage, the use of the information within movies allows substantially improved direct estimation of the remaining key image parameters required for accurate 3D reconstruction: the image CTF and spectral SNR. We are developing "CARYON" {insert contrived acronym here}, as a LAFTER-family filter for cryo-EM movies based upon such measurements. CARYON is intended to provide the best parameter estimation and filtration possible for a single complete, or large sub-section from a, movie micrograph without the use of a previously refined density. We demonstrate its utility in both single-particle and tomographic cryo-EM data processing.

biophysics↗

bioRxiv2020

New insights into large tropical tree mass and structure from direct harvest and terrestrial lidar

A large portion of the terrestrial vegetation carbon stock is stored in the above-ground biomass (AGB) of tropical forests, but the exact amount remains uncertain, partly due to the lack of measurements. To date, accessible peer-reviewed data are available for just 10 large tropical trees in the Amazon that have been harvested and directly measured entirely via weighing. Here, we harvested four large tropical rainforest trees (stem diameter: 0.6-1.2m, height: 30-46m, AGB: 3960-18 584 kg) in intact old-growth forest in East Amazonia, and measured above-ground green mass, moisture content and woody tissue density. We first present rare ecological insights provided by these data, including unsystematic intra-tree variations in density, with both height and radius. We also found the majority of AGB was usually found in the crown, but varied from 42-62%. We then compare non-destructive approaches for estimating the AGB of these trees, using both classical allometry, and new lidar-based methods. Terrestrial lidar point clouds were collected pre-harvest, on which we fitted cylinders to model woody structure, enabling retrieval of volume-derived AGB. Estimates from this approach were more accurate than allometric counterparts (mean tree-scale relative error: 3% vs. 15%), and error decreased when up-scaling to the cumulative AGB of the four trees (1% vs. 15%). Further, whilst allometric error increased four-fold with tree size over the diameter range, lidar error remained constant. This suggests error in these lidar-derived estimates is random and additive. Were these results transferable across forest scenes, terrestrial lidar methods would reduce uncertainty in stand-scale AGB estimates, and therefore advance our understanding of the role of tropical forests in the global carbon cycle.

ecology↗

bioRxiv2020

Placental microRNA Expression Associates with Birthweight through Control of Adipokines: Results from Two Independent Cohorts

MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally. In the placenta, the master regulator of fetal growth and development, microRNAs shape the basic processes of trophoblast biology and specific microRNA have been associated with fetal growth. To comprehensively assess the role of microRNAs in placental function and fetal development, we have performed small RNA sequencing to profile placental microRNAs from two independent mother-infant cohorts: the Rhode Island Child Health Study (n=225) and the New Hampshire Birth Cohort Study (n=317). We modeled microRNA counts on infant birthweight percentile (BWP) in each cohort, while accounting for race, sex, parity and technical factors, using negative binomial generalized linear models. We identified microRNAs that were differentially expressed (DEmiRs) with BWP at false discovery rate (FDR) less than 0.05 in both cohorts. hsa-miR-532-5p (miR-532) was positively associated with BWP in both cohorts. By integrating parallel whole transcriptome and small RNA sequencing in the RICHS cohort, we identified putative targets of miR-532. These targets are enriched for pathways involved in adipogenesis, adipocytokine signaling, energy metabolism and hypoxia response, and included Leptin, which we further demonstrated to have decreasing expression with increasing BWP, particularly in male infants. Overall, we have shown a robust and reproducible association of miR-532 with BWP, which could influence BWP through regulation of adipocytokines Leptin and Adiponectin.

genetics↗

bioRxiv2019

Genome variation and population structure among 1,142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii. We analysed whole genomes of 1,142 individual mosquitoes sampled from the wild in 13 African countries, and a further 234 individuals comprising parents and progeny of 11 lab crosses. The data resource includes high confidence single nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We used these data to analyse genetic population structure, and characterise genetic diversity within and between populations. We also illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance, and for accelerating research and development of new vector control tools.

genomics↗

bioRxiv2019

Complete structure of the core signalling unit of the E. coli chemosensory array in an optimised minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays.1,2 Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit (CSU). Here we introduce a novel E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We provide a three-dimensional (3D) map of a complete CSU at ~16 [A] resolution by cryo-electron tomography (cryo-ET) and subtomogram averaging. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields an atomistic model of the membrane-bound CSU and enables spatial localisation of its signalling domains. Our work thus offers a solid structural basis for interpretation of existing data and design of new experiments to elucidate signalling mechanisms within the CSU and larger array.

cell biology↗

bioRxiv2019

The mechanical stability of the worlds tallest broadleaf trees

The factors that limit the maximum height of trees, whether ecophysiological or mechanical, are the subject of longstanding debate. Here we examine the role of mechanical stability in limiting tree height and focus on trees from the tallest tropical forests on Earth, in Sabah, Malaysian Borneo, including the recently discovered tallest tropical tree, a 100.8 m Shorea faguetiana. We use terrestrial laser scans, in situ strain gauge data and finite-element simulations to map the architecture of tall broadleaf trees and monitor their response to wind loading. We demonstrate that a trees risk of breaking due to gravity or self-weight decreases with tree height and is much more strongly affected by tree architecture than by material properties. In contrast, wind damage risk increases with tree height despite the larger diameters of tall trees, resulting in a U-shaped curve of mechanical risk with tree height. The relative rarity of extreme wind speeds in north Borneo may be the reason it is home to the tallest trees in the tropics.

ecology↗

bioRxiv2018

Robust estimation of recent effective population size from number of independent origins in soft sweeps

Estimating recent effective population size is of great importance in characterising and predicting the evolution of natural populations. Methods based on nucleotide diversity may underestimate current day effective population sizes due to historical bottlenecks, whilst methods that reconstruct demographic history typically only detect long-term variations. However, soft selective sweeps, which leave a fingerprint of mutational history by recurrent mutations on independent haplotype backgrounds, holds promise of an estimate more representative of recent population history. Here we present a simple and robust method of estimation based only on knowledge of the number of independent recurrent origins and the current frequency of the beneficial allele in a population sample, independent of the strength of selection and age of the mutation. Using a forward time theoretical framework, we show the mean number of origins is a function of{theta} = 2N and current allele frequency, through a simple equation, and the distribution is approximately Poisson. This estimate is robust to whether mutants pre-existed before selection arose, and is equally accurate for diploid populations with incomplete dominance. For fast (e.g., seasonal) demographic changes compared to time scale for fixation of the mutant allele, and for moderate peak-to-trough ratios, we show our constant population size estimate can be used to bound the maximum and minimum population size. Applied to the Vgsc gene of Anopheles gambiae, we estimate an effective population size of roughly 6 x 107, and including seasonal demographic oscillations, a minimum effective population size greater than 6 x 106 and a maximum less than 3 x 109.

evolutionary biology↗

bioRxiv2018

Epigenome-wide Analysis Identifies Genes and Pathways Linked to Neurobehavioral Variation in Preterm Infants

Background & ObjectivesNeonatal neurobehavioral performance measures, such as the NICU Network Neurobehavioral Scale (NNNS), have been developed to assess the neurobehavioral characteristics of infants and provide insights into future developmental trajectories. The identification of molecular biomarkers of very early life neurobehavioral experiences could lead to better predictions of the long-term developmental outcomes of high-risk infants including preterm infants. To this end, we aimed to examine whether variability in DNA methylation (DNAm) or epigenetic age from surrogate tissues are associated with NNNS profiles in a cohort of infants born less than 30 weeks postmenstrual age (PMA).\n\nMethodsThis study was performed within the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study and included those infants with complete NNNS assessment data and DNAm measured from buccal cells, collected at near term-equivalent age using the Illumina EPIC array (N=536). We tested whether epigenetic age and age acceleration differed between infants based on their NNNS profile classifications. Then we performed an epigenome-wide association study, to test whether DNAm at individual epigenetic loci varied between these NNNS profile groupings. Models were adjusted for recruitment site, infant sex, postmenstrual age, and estimated tissue heterogeneity.\n\nResultsWe found that infants with an optimal NNNS profile had slightly older epigenetic age than other NOVI infants ({beta}1 = 0.201, p-value = 0.026), and that infants with an atypical NNNS profile had differential methylation at 29 CpG sites (FDR < 10%). The genes annotated to these differentially methylated CpGs included PLA2G4E, TRIM9, GRIK3, and MACROD2, which have previously been associated with neurological structure and function, or with neurobehavioral disorders.\n\nConclusionsGreater epigenetic age is associated with optimal NNNS responses while altered DNAm of multiple genes are associated with an atypical neurobehavioral profile at near-term equivalent age. These findings build upon the existing evidence that epigenetic variations in buccal cells may serve as markers of neonatal neurobehavior and might facilitate early identification of children at risk for abnormal developmental outcome.

epidemiology↗

bioRxiv2017

Self-limiting population genetic control with sex-linked genome editors

In male heterogametic species the Y chromosome is transmitted solely from fathers to sons, and is selected for based only on its impacts on male fitness. This fact can be exploited to develop efficient pest control strategies that use Y-linked editors to disrupt the fitness of female descendants. In simple \"strategic\" population models we show that Y-linked editors can be substantially more efficient than other self-limiting strategies and, while not as efficient as gene drive approaches, are expected to have less impact on non-target populations with which there is some gene flow. Efficiency can be further augmented by simultaneously releasing an autosomal X-shredder construct, in either the same or different males. Y-linked editors may be attractive option to consider when efficient control of a species is desired in some locales but not others.

synthetic biology↗

bioRxiv2016

Natural diversity of the malaria vector Anopheles gambiae

The sustainability of malaria control in Africa is threatened by rising levels of insecticide resistance, and new tools to prevent malaria transmission are urgently needed. To gain a better understanding of the mosquito populations that transmit malaria, we sequenced the genomes of 765 wild specimens of Anopheles gambiae and Anopheles coluzzii sampled from 15 locations across Africa. The data reveal high levels of genetic diversity, with over 50 million single nucleotide polymorphisms across the 230 Mbp genome. We observe complex patterns of population structure and marked variations in local population size, some of which may be due at least in part to malaria control interventions. Insecticide resistance genes show strong signatures of recent selection associated with multiple independent mutations spreading over large geographical distances and between species. The genetic variability of natural populations substantially reduces the target space for novel gene-drive strategies for mosquito control. This large dataset provides a foundation for tracking the emergence and spread of insecticide resistance and developing new vector control tools.

genomics↗