Biology subjects

Norman, M.

Publications and source records attributed to Norman, M..

4 recordsLinked to original sources

bioRxiv2025

PYEAST - Python Enabled Automated Strain Transformaiton

Saccharomyces cerevisiae is a widely used biotechnological workhorse in both academic and industrial settings. One reason for its continued popularity is the extensive legacy of genetic tools, developed over its long history of use, that enable precise manipulation of the S. cerevisiae genome. These tools have enabled extensive genetic characterisation and dramatic re-programming efforts for applications ranging from fundamental research to industrial chemical production. Here we present a digital toolkit called PYEAST (Python Enabled Automated Strain Transformation) that encodes some of the most widely used methods for working with S. cerevisiae and modernizes them to leverage advances in DNA synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/655004v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@4584feorg.highwire.dtl.DTLVardef@1e65caorg.highwire.dtl.DTLVardef@1acbe2borg.highwire.dtl.DTLVardef@1f91abc_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Wheat pollen uptake of CRISPR/Cas9 RNP-PDMAEMA nanoassemblies results in targeted loss of gene function in progeny

The utility of CRISPR in plants has remained limited by the dual difficulties of delivering the molecular machinery to target cells and the use of somatic cell techniques that require tissue culture-based de novo organogenesis. We developed 5-10 nm isodiametric polyplex nanoassemblies, comprising poly [2-(dimethylamino)ethylmethacrylate] PDMAEMA (PD) polycationic linear homopolymers and CRISPR/Cas9 ribonucleoproteins (RNPs), that enable endocytosis-driven RNP uptake into pollen grains. Pollen from wheat plants (genotype Gladius+Sr50), homozygous for monogenic Sr50-mediated resistance to stem rust (Puccinia graminis f. sp. tritici -Pgt), were incubated with RNP/PD nanoassemblies targeting the dominant, Sr50 rust resistance gene. The treated pollen grains were then used to fertilize Gladius+Sr50 florets and the resulting M1 plants were tested for loss of Sr50 function via rust resistance screens. The identification of fully susceptible M1 seedlings indicated that the Sr50 RNPs acted on both alleles, indicating they were transferred via the treated pollen to the zygote. The ability to readily deliver CRISPR RNPs to reproductive cells via biodegradable, polymeric nanocomplexes has significant implications for the efficiency of gene editing in plants.

plant biology↗

bioRxiv2020

Comparison of Extracellular Vesicle Isolation Methods from Human Biofluids using Single Molecule Arrays

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

biochemistry↗

bioRxiv2020

L1CAM is not Associated with Extracellular Vesicles in Human Cerebrospinal Fluid or Plasma

Neuron-derived extracellular vesicles (NDEVs) present a tremendous opportunity to learn about the biochemistry of brain cells in living patients. L1CAM is a transmembrane protein expressed in neurons that is presumed to be found on NDEVs in human biofluids. Previous studies have used L1CAM immuno-isolation from human plasma to isolate NDEVs for neurodegenerative disease diagnostics. We developed a panel of ultrasensitive Single Molecule Array (Simoa) assays for known EV markers, as well as L1CAM, and applied it to study EVs in human plasma and cerebrospinal fluid (CSF). We fractionated plasma and CSF by size exclusion chromatography (SEC) and density gradient centrifugation (DGC) to separate EVs from free proteins. We observed that L1CAM did not elute in the EV fractions, but rather eluted in the free protein fractions. We found that L1CAM is present as a free protein in human plasma and CSF, possibly due to proteolytic cleavage and/or alternative splicing. We further demonstrate that the isoforms found in CSF and plasma are different. These data collectively establish that L1CAM in plasma is not EV associated and should therefore not be used for NDEV isolation. Importantly, the framework and tools described herein will allow for evaluation of other potential candidate markers for isolation of NDEVs.

neuroscience↗