Biology subjects

Edwards, K. J.

Publications and source records attributed to Edwards, K. J..

7 recordsLinked to original sources

bioRxiv2024

A general approach to reduce off-target radioactivity in vivo via Tetrazine-Knock-Out (TKO)

Monoclonal antibodies have had a remarkable impact on cancer therapy due to their high target specificity. However, their large molecular weight results in slow blood clearance, which can take weeks to clear from circulation. As companion nuclear imaging and diagnostic tools, these characteristics force delayed imaging and the use of isotopes with long half-lives such as 89Zr. For optimal clinical application, it is desirable that radioimmunoconjugates remain in the blood for just enough time to accumulate adequately in target tissues, while non-targeted or circulating radioactivity is ideally rapidly excreted from the body to maximize imaging contrast and minimize radiation dose to healthy tissues. We addressed the current challenges of antibody-based imaging by developing rituximab radioimmunoconjugates that accumulate sufficient activity for tumor imaging within 24 h of administration, while clearing circulating radioactivity via administration of a small molecule clearing agent. Rituximab, an anti-CD20 monoclonal antibody, is used as standard first-line therapy for diffuse large B-cell lymphoma. CD20 is expressed by 95% of B-lymphocytes and their malignant counterparts, making it a therapeutic target for B-cell malignancies. We attached 125I, 68Ga, and 89Zr to rituximab using a "clickable" linker containing trans-cyclooctene and tested the ability of tetrazines to induce the inverse electron demand Diels-Alder reaction (iEDDA) after antibody administration. This "tetrazine-knock-out" (TKO) approach liberates the radioactivity from rituximab in the bloodstream, resulting in its rapid renal excretion which enhances target-to-background ratios, and minimizes off-target radiation exposure. Due to the internalization of the radioimmunoconjugate in CD20+ tumor cells, no substantial clearance was observed from Raji xenografts. We characterized different leaving groups, several cellular models and antibodies with distinct internalizaing properties. The TKO approach opens opportunities to use radiolabeled antibodies for low-abundance or heterogeneously expressed biologic targets and may allow radioimmunotherapy (RIT) for targets traditionally untenable due to dose-limiting toxicities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/596510v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11bc2f0org.highwire.dtl.DTLVardef@19b4f72org.highwire.dtl.DTLVardef@17cee32org.highwire.dtl.DTLVardef@fa4491_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Development of a Next Generation SNP Genotyping Array for Wheat

High throughput genotyping arrays have provided a cost effective, reliable and interoperable system for genotyping hexaploid wheat and its related germplasm pool. Existing, highly cited arrays including our 35K Axiom Wheat Breeders genotyping array and the Illumina 90K iSelect array were designed based on a limited amount of varietal sequence diversity and with imperfect knowledge of SNP positions. Recent progress in sequencing wheat varieties and landraces has given us access to a vast pool of SNP diversity, whilst technological improvements in array design has allowed us to fit significantly more probes onto a 384-well format Axiom array than was previously possible. Here we describe a novel High Density Axiom genotyping array, the Triticum aestivum Next Generation array (TaNG), largely derived from whole genome skim sequencing of 204 elite wheat lines and 111 wheat landraces taken from the Watkins "Core Collection". We use a novel "minimal marker" optimisation approach to select up to six SNPs in each 1.5 MB region of the wheat genome with the highest combined varietal discrimination potential. A design iteration step allowed us to test and replace skim-sequence derived SNPs which failed to convert to reliable Axiom markers, resulting in a final design, designated TaNG1.1 with 43,372 SNPs derived from a haplotype-optimised combination of novel SNPs, DArTAG-derived and legacy wheat Axiom markers. We show that this design has an even distribution of SNPs across chromosomes and sub-genomes compared to previous arrays and can be used to generate genetic maps with a significantly higher number of distinct bins than our previous Axiom array. We also demonstrate the improved performance of TaNG1.1 for Genome Wide Association Studies (GWAS) and its utility for Copy Number Variation (CNV) analysis. The array is commercially available, and the marker annotations, initial genotyping results and software used to generate the optimised marker sets are freely available.

genomics↗

bioRxiv2023

Microbial abundance and diversity in 64-74 Ma subseafloor igneous basement from the Louisville Seamount Chain

The aquifer in subseafloor igneous basement is a massive, continuous microbial substrate, yet sparingly little is known about life in this habitat. The work to date has focused largely on describing microbial diversity in young basement (<10 Ma) at oceanic spreading regions and ridge flanks, where the basaltic crust is still porous and fluid flow through it is active. While the prevailing belief used to be that fluid flow through older parts of the seafloor was non-existent, recent heat flow models predict that fluid moves through subseafloor basement >65 Ma, and that seamounts can act as mid-plate conduits for fluids into and out of the subsurface aquifer in older crustal settings. Here we test the hypothesis that microbial life exists in subseafloor basement >65Ma using samples collected from the Louisville Seamount Chain via seafloor drilling. Cell biomass was heterogeneous in nature and ranged from below detection to [~]104 cells cm-3. Bacterial 16S rRNA genes from core samples and enrichment incubations are dominated by lineages putatively carrying out hydrocarbon oxidation and nitrogen, sulfur and metal redox processes. Samples from two different seamounts were statistically different, indicating some degree of biogeography. Archaea were not detected via quantitative polymerase chain reaction, indicating they are rare in the Louisville subsurface. Taken together, the data indicate that microbial life is indeed present in subseafloor igneous basement >65 Ma, which significantly expands the range of the subseafloor biosphere where microbial life is known to exist. Impact StatementThe aquifer in subseafloor igneous basement is the largest continuous microbial substrate on Earth, but it is difficult to access and therefore understudied. We here collected samples from the Louisville Seamount Chain using seafloor drilling to determine if microbial life exists in the >65 Ma subseafloor basement made at these seamounts. A low biomass environment dominated by Bacteria potentially capable of using the Fe and S inherent in subseafloor basalt was detected, including Bacteria that were revived in enrichment experiments. This discovery expands the range of seafloor where confirmed microbial life exists and indicates the interior of seamounts is habitable.

ecology↗

bioRxiv2022

Genome-wide circadian gating of a cold temperature response in bread wheat

Circadian rhythms coordinate the responses of organisms to their daily fluctuating environments, by establishing a temporal program of gene expression. This schedules aspects of metabolism, physiology, development and behaviour according to the time of day. Circadian regulation in plants is extremely pervasive, and is important because it underpins both productivity and seasonal reproduction. Circadian regulation extends to the control of environmental responses through a regulatory process known as circadian gating. Circadian gating is the process whereby the circadian clock regulates the response to an environmental cue, such that the magnitude of response to an identical cue varies according to the time of day of the cue. Here, we show that there is genome-wide circadian gating of responses to cold temperatures in plants. By using bread wheat as an experimental model, we establish that circadian gating is crucial to the programs of gene expression that underlie the environmental responses of a crop of major socioeconomic importance. Furthermore, we identify that circadian gating of cold temperature responses are distributed unevenly across the three wheat subgenomes, which might reflect the geographical origins of the ancestors of modern wheat. One-sentence summaryThere is genome-wide circadian gating of a response to low temperatures in a crop of major socioeconomic importance.

plant biology↗

bioRxiv2022

A wheat chromosome segment substitution line series supports characterisation and use of progenitor genetic variation

Genome-wide introgression and substitution lines have been developed in many plant species, enhancing mapping precision, gene discovery and the identification and exploitation of variation from wild relatives. Created over multiple generations of crossing and/or backcrossing accompanied by marker-assisted selection, the resulting introgression lines are a fixed genetic resource. In this study we report the development of spring wheat chromosome segment substitution lines generated to systematically capture genetic variation from tetraploid (Triticum turgidum ssp dicoccoides) and diploid (Aegilops tauschii) progenitor species. Generated in a common genetic background over four generations of backcrossing, the material is a base resource for the mapping and characterisation of wheat progenitor variation. To facilitate further exploitation the final population was genetically characterised using a high- density genotyping array and a range of agronomic and grain traits assessed to demonstrate the the potential use of the populations for trait localisation in wheat.

genetics↗

bioRxiv2020

Detecting SARS-CoV-2 variants with SNP genotyping

Tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020- and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 76% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with our marker panel at a cost of < {pound}1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

genomics↗

bioRxiv2020

Poly(ADP-ribose) induces α-synuclein aggregation in neuronal-like cells andinteracts with phosphorylated α-synuclein in post mortem PD samples

BackgroundPoly (ADP-ribose) (PAR) is a negatively charged polymer that is biosynthesized by Poly (ADP-ribose) Polymerase-1 (PARP-1) and regulates various cellular processes. Alpha-synuclein (Syn) is an intrinsically disordered protein (IDP) that has been directly implicated with driving the onset and progression of Parkinsons disease (PD). The mechanisms by which Syn elicits its neurotoxic effects remain unclear. Recent findings indicate that one of the key processes driving PD pathology are oligomeric species of Syn. Furthermore, it is well established that the main components of Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients are aggregated hyperphosphorylated (S129) forms of Syn (pSyn). MethodsWe used biochemical and immunofluorescence-based assays to explore if PARP-1 enzymatic product (PAR) drives the conversion of monomeric Syn into aggregated assemblies. We performed quantitative measurements using in situ proximity ligation assays (PLA) on a transgenic murine model of -synucleinopathy (M83-SNCA*A53T) and post-mortem PD/PDD patient samples to characterize PAR-pSyn interactions. Additionally, we used bioinformatic approaches and site-directed mutagenesis to identify PAR-binding regions on fibrillar Syn. ResultsOur studies show that elevated intracellular levels of PAR promote the transition of Syn into higher molecular weight forms. We report that PAR-pSyn interactions are predominant in pathological states. Moreover, we confirm that the interactions between PAR and Syn involve electrostatic forces between negatively charged PAR and lysine residues on the N-terminal region of Syn. ConclusionsPAR plays a critical role in the early stages of monomeric Syn aggregation, thereby attributing to PD pathogenesis. Based on our results, we report that PAR seeds monomeric Syn aggregation and directly interacts with phosphorylated Syn in conditions that are pathologically relevant to PD.

neuroscience↗