Biology subjects

Clarke, D. J.

Publications and source records attributed to Clarke, D. J..

10 recordsLinked to original sources

bioRxiv2025

A haplotype-resolved, chromosome-scale genome assembly for the southern live oak, Quercus virginiana

Hybridization is a major force driving diversification, migration, and adaptation in Quercus species. While population genetics and phylogenetics have traditionally been used for studying these processes, advances in sequencing technology now enable us to incorporate comparative and pan-genomic approaches as well. Here we present a highly contiguous, chromosome-scale and haplotype-resolved genome assembly for the southern live oak, Quercus virginiana, the first reference genome for section Virentes, as part of the American Campus Tree Genomes (ACTG) program. Originating from a clone of Auburn Universitys historic "Toomers Oak," this assembly contributes to the pool of genomic resources for investigating recombination, haplotype variation, and structural genomic changes influencing hybridization potential in this clade and across Quercus. It also provides insights into the architecture of the putative centromeric regions within the genus. Alongside other oak references, the Q. virginiana genome will support research into the evolution and adaptation of the Quercus genus.

genomics↗

bioRxiv2023

Two-color coincidence single-molecule pull-down for the specific detection of disease-associated protein aggregates

The misfolding and aggregation of protein is a characteristic of many neurodegenerative disorders, including Alzheimers and Parkinsons disease. The wide range of sizes and structures of oligomers and fibrils generated have previously been studied using single-molecule and super-resolution microscopy. These methods, however, tend to rely on the use of either directly labeled protein, or on the addition of non-specific amyloid stains, such as thioflavin-T. This has prevented the characterization of protein aggregate composition in complex biological samples. Here, we have developed a single-molecule two-color aggregate pull-down (STAPull) assay to overcome this challenge by probing immobilized proteins using orthogonally labeled antibodies targeting the same epitope. By looking at colocalized signals, we can eliminate monomeric protein, and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 pM. Furthermore, we show that STAPull can be used in a range of samples, including in human biofluids. STAPull is generally applicable to protein aggregates from a variety of disorders, and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.

neuroscience↗

bioRxiv2022

ReproTox-KG: Toxicology Knowledge Graph for Structural Birth Defects

Birth defects are functional and structural abnormalities that impact 1 in 33 births in the United States. Birth defects have been attributed to genetic as well as other factors, but for most birth defects there are no known causes. Small molecule drugs, cosmetics, foods, and environmental pollutants may cause birth defects when the mother is exposed to them during pregnancy. These molecules may interfere with the process of normal fetal development. To characterize associations between small molecule compounds and their potential to induce specific birth abnormalities, we gathered knowledge from multiple sources to construct a reproductive toxicity Knowledge Graph (ReproTox-KG) with an initial focus on associations between birth defects, drugs, and genes. Specifically, to construct ReproTox-KG we gathered data from drug/birth-defect associations from co-mentions in published abstracts, gene/birth-defect associations from genetic studies, drug- and preclinical-compound-induced gene expression data, known drug targets, genetic burden scores for all human genes, and placental crossing scores for all small molecules in ReproTox-KG. Using the data stored within ReproTox-KG, we scored 30,000 preclinical small molecules for their potential to induce birth defects. Querying the ReproTox-KG, we identified over 500 birth-defect/gene/drug cliques that can be used to explain molecular mechanisms for drug-induced birth defects. The ReproTox-KG is provided as curated tables and via a web-based user interface that can enable users to explore the associations between birth defects, approved and preclinical drugs, and human genes.

bioinformatics↗

bioRxiv2022

Lipidomic analysis reveals differences in Bacteroides species driven largely by plasmalogens, glycerophosphoinositols and certain sphingolipids

There has been increasing interest in bacterial lipids in recent years due, in part, to their emerging role as molecular signalling molecules. Bacteroides thetaiotaomicron is an important member of the mammalian gut microbiota that has been shown to produce sphingolipids (SP) that pass through the gut epithelial barrier to impact host SP metabolism and signal into host inflammation pathways. B. thetaiotaomicron also produces a novel family of N-acyl amines (called glycine lipids) that are potent ligands of host Toll-like receptor 2 (TLR2). Here, we specifically examine the lipid signatures of 4 species of gut associated Bacteroides. In total we identify 170 different lipids and we report that the range and diversity of Bacteroides lipids is species-specific. Multi-variate analysis reveals that the differences in the lipid signatures are largely driven by the presence/absence of plasmalogens, glycerophosphoinositols and certain SP. Moreover, we show that, in B. thetaiotaomicron, mutations altering either SP or glycine lipid biosynthesis results in significant changes in the levels of other lipids suggesting the existence of compensatory mechanisms required to maintain the functionality of the bacterial membrane. ImportanceBacteroides are important beneficial members of the gut microbiome that produce lipids that can function as cross-kingdom signalling molecules. We describe, for the first time, a comprehensive and qualitative comparison of the lipid signatures of 4 important Bacteroides species. We identify a group of Bacteroides core lipids and uncover species-specific differences in plasmalogen, glycerophospholipid and SP metabolism with more subtle differences observed in glycine lipid production. This data will provide a useful platform for the further characterisation of the lipid-based host-microbe dialogue and the influence of microbial lipids on host health and disease states.

microbiology↗

bioRxiv2022

Structural conversion of α-synuclein at the mitochondria induces neuronal toxicity

Aggregation of -Synuclein (-Syn) drives Parkinsons disease, although the initial stages of self-assembly and structural conversion have not been captured inside neurons. We track the intracellular conformational states of -Syn utilizing a single-molecule FRET biosensor, and show that -Syn converts from its monomeric state to form two distinct oligomeric states in neurons in a concentration dependent, and sequence specific manner. 3D FRET-CLEM reveals the structural organization, and location of aggregation hotspots inside the cell. Notably multiple intracellular seeding events occur preferentially on membrane surfaces, especially mitochondrial membranes. The mitochondrial lipid, cardiolipin triggers rapid oligomerization of A53T -Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial ROS generation, which accelerates the oligomerization of A53T -Syn, and ultimately causes permeabilization of mitochondrial membranes, and cell death. Patient iPSC derived neurons harboring A53T mutations exhibit accelerated oligomerization that is dependent on mitochondrial ROS, early mitochondrial permeabilization and neuronal death. Our study highlights a mechanism of de novo oligomerization at the mitochondria and its induction of neuronal toxicity.

neuroscience↗

bioRxiv2021

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment revealed by Cryo-EM and hydrogen/deuterium exchange mass spectrometry

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications. TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible.

biochemistry↗

bioRxiv2019

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this proposed iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site.

biochemistry↗

bioRxiv2019

Photorhabdus Dam methyltransferase overexpression impairs virulence of the nemato-bacterial complex in insects

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that the P. luminescens Dam plays a role in the pathogenicity of the nemato-bacterial complex.

microbiology↗

bioRxiv2018

Iso-propyl stilbene: A life-cycle signal?

Members of the Gram-negative bacterial genus Photorhabdus are all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode worm Heterorhabditis. All members of the genus produce the small molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal and anticancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response of Photorhabdus itself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology of Photorhabdus bacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

microbiology↗

bioRxiv2018

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

Iron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised the Rhodospirillum rubrum EncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the same distinct annular decamer topology observed in R. rubrum EncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass spectrometr analyses show that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The catalytic role of the EncFtn proteins was confirmed by biochemical assays, and we show that Zn(II) ions inhibit the ferroxidase activity of the EncFtn proteins to varying degrees. Our results represent a further step in the characterisation of the recently discovered EncFtn ferritin-like sub-family, indicating a common structural organisation and catalytic activity, despite diverse host environments.

biochemistry↗