Biology subjects

Burke, M.

Publications and source records attributed to Burke, M..

22 records · Page 2Linked to original sources

bioRxiv2023

Characterization and super-resolution imaging of small tau aggregates in human samples.

Hyperphosphorylation and aggregation of the microtubule binding protein tau plays a key role in the development of Alzheimers disease. While the structure of the filamentous aggregates formed in humans has recently been determined to atomic resolution, there is far less information available about the smaller aggregate precursors, thought to be the most neurotoxic. To address this gap, we have developed a single molecule pull-down (SiMPull) able to detect tau aggregates in clinically relevant human samples. This method enables the detection and characterisation of individual tau aggregates, as opposed to averaged features obtained from traditional bulk techniques. We report the number, size and shape of individual aggregates measured via super-resolution microscopy, revealing disease-specific differences in tau aggregate morphology. By adapting the assay to simultaneously detect multiple phosphorylation sites in individual aggregates, we were also able to derive compositional profiles for pathological modifications present in individual aggregates. We demonstrate that tau aggregates in Alzheimers disease are significantly more likely to contain both the AT8 and T181 pathological phosphorylation markers, rather than only one. Together, tau SiMPull identified distinct subpopulations of large, modified tau aggregates that were invisible to traditional methodologies. These morphological and compositional differences distinguish samples taken from disease cohorts, offering to illuminate underlying disease mechanisms, and providing a foundation for novel diagnostic strategies.

biochemistry↗

bioRxiv2021

Crossing design shapes patterns of genetic variation in synthetic recombinant populations of Saccharomyces cerevisiae

"Synthetic recombinant" populations have emerged as a useful tool for dissecting the genetics of complex traits. They can be used to derive inbred lines for fine QTL mapping, or the populations themselves can be sampled for experimental evolution. In latter application, investigators generally value maximizing genetic variation in constructed populations. This is because in evolution experiments initiated from such populations, adaptation is primarily fueled by standing genetic variation. Despite this reality, little has been done to systematically evaluate how different methods of constructing synthetic populations shape initial patterns of variation. Here we seek to address this issue by comparing outcomes in synthetic recombinant Saccharomyces cerevisiae populations created using one of two strategies: pairwise crossing of isogenic strains or simple mixing of strains in equal proportion. We also explore the impact of the varying the number of parental strains. We find that more genetic variation is initially present and maintained when population construction includes a round of pairwise crossing. As perhaps expected, we also observe that increasing the number of parental strains typically increases genetic diversity. In summary, we suggest that when constructing populations for use in evolution experiments, simply mixing founder strains in equal proportion may limit the adaptive potential.

genomics↗

bioRxiv2020

Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

Convenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2019

Age-dependent changes in electrophysiology and calcium handling: implications for pediatric cardiac research

RationaleThe heart continues to develop and mature after birth and into adolescence. Accordingly, cardiac maturation is likely to include a progressive refinement in both organ morphology and function during the postnatal period. Yet, age-dependent changes in cardiac electrophysiology and calcium handling have not yet been fully characterized.\n\nObjectiveThe objective of this study, was to examine the relationship between cardiac maturation, electrophysiology, and calcium handling throughout postnatal development in a rat model.\n\nMethodsPostnatal rat cardiac maturation was determined by measuring the expression of genes involved in cell-cell coupling, electrophysiology, and calcium handling. In vivo electrocardiograms were recorded from neonatal, juvenile, and adult animals. Simultaneous dual optical mapping of transmembrane voltage and calcium transients was performed on isolated, Langendorff-perfused rat hearts (postnatal day 0-3, 4-7, 8-14, adult).\n\nResultsYounger, immature hearts displayed slowed electrical conduction, prolonged action potential duration and increased ventricular refractoriness. Slowed calcium handling in the immature heart increased the propensity for calcium transient alternans which corresponded to alterations in the expression of genes encoding calcium handling proteins. Developmental changes in cardiac electrophysiology were associated with the altered expression of genes encoding potassium channels and intercalated disc proteins.\n\nConclusionUsing an intact whole heart model, this study highlights chronological changes in cardiac electrophysiology and calcium handling throughout postnatal development. Results of this study can serve as a comprehensive baseline for future studies focused on pediatric cardiac research, safety assessment and/or preclinical testing using rodent models.

physiology↗