Biology subjects

Bryan, N.

Publications and source records attributed to Bryan, N..

5 recordsLinked to original sources

bioRxiv2025

MICRO-TAG enzyme complementation enables quantification of cellular drug-target engagement in temperature series

Drug discovery for challenging drug targets necessitates the proteomic complexities of the cellular milieu for contextual target folding and function. Conventional biophysical methods for assessing drug interaction with a target are often not sufficiently suited for drug discovery as they impose acellular environment on the target and rely on recombinant purified protein material. In contrast, cell target engagement offers a powerful paradigm for drug discovery, through measurement of transitions in the thermodynamic state of a target protein, as it engages with drug molecules in the cell. Split-enzyme cell target engagement methods offer scaled utility during early drug discovery. Here, we describe a novel highly sensitive and scalable fluorescence-based cell target engagement method that leverages complementation of split-RNase S. This offers a unique combination of procedural and biophysical advantages, enabling its seamless integration with various instruments and applications designed for fluorescence detection. Most importantly, this new method allows for quantitation of cell target engagement in programmable temperature series format, consistent with conventional thermal shift assays, rather than at a single melting temperature. We demonstrate the sensitivity and versatility of this approach for drug discovery using targets MAPK1, KRAS, and UBE2N.

biochemistry↗

bioRxiv2024

Phylogenomics of novel clones of Aeromonas veronii recovered from a freshwater lake reveals unique biosynthetic gene clusters

Aquatic ecosystems are important reservoirs for clinically relevant pathogens and antimicrobial resistance genes, thus present a significant risk to global health. Here, we assessed the phylogenomics of Aeromonas veronii (A. veronii) recovered from Lake Wilcox in Ontario using a combination of morphological, biochemical, and whole-genome sequencing (WGS) techniques. Eleven distinct bacterial colonies were isolated and identified as A. veronii (n=9), and two other Aeromonas species (A. caviae and A. allosaccharophila), with significant discrepancies noted between biochemical and WGS identification methods. Of note, 67% (n=6/9) of A. veronii isolates were human pathogens (Pathogenicity score [≥] 0.50). The genomic analysis revealed high genetic diversity among the A. veronii isolates, including the discovery of 41 novel alleles and seven new sequence types (ST) suggesting the lake as a reservoir for multiple human pathogenic clones of this bacterium. The comparison of the newly isolated and sequenced A. veronii with 214 A. veronii genomes revealed significant genetic diversity and suggests potential broad geographical dissemination of strains. Chromosomal genes (OXA-912 and cphA [cphA3, cphA4, cphA7]) genes encoding resistance to {beta}-lactamases were detected in all isolates. Human and non-human pathogenic strains of A. veronii differed in their virulence gene content, with type III secretion systems being associated with human pathogenic isolates. Mobilome analysis revealed the absence of plasmids in A. veronii isolates and the presence of 13 intact the great majority of which were P22-like (Peduoviridae) phages, and nine different insertion sequence families. Novel biosynthetic gene clusters were identified and characterized, indicating the potential for unique secondary metabolite production in A. veronii with different pathogenic potential. Overall, this study underscores the importance of continuous surveillance of aquatic ecosystems for the presence of pathogens, contributing to our understanding of their evolution, potential for human pathogenicity, and the ecological roles of their genetic elements.

microbiology↗

bioRxiv2024

Sex Hormones Alter Pseudomonas aeruginosa Iron Acquisition and Virulence Factors

Urinary Tract Infections (UTI) are one of the most widespread infections in healthcare and community settings worldwide. Pseudomonas aeruginosa is the third most common pathogen associated with catheter-associated UTI (CAUTI). P. aeruginosa infections are highly resistant and difficult to treat and it is currently classified as priority 1 by the World Health Organisation. In vitro studies of microbes typically employ laboratory media. The inadequacy of nutrient-rich media in simulating the physiological environment has led to the development of multiple media that mimic human body fluids, including Artificial Urine Medium (AUM). By studying growth and in vitro biofilm assays along with proteomics, we sought to establish whether UTI P. aeruginosa respond differently in laboratory media, AUM and urine. To further probe the impact of environmental influences, sex hormones estradiol, progesterone and testosterone were added at physiologically relevant concentrations. The proteomic profiles were then compared between hormone supplemented AUM and urine. Our findings indicate that bacterial responses in standard laboratory media, AUM and urine were distinct. Increased proteins associated with iron acquisition mechanisms were similar in both AUM and urine. However, differences were observed in other virulence and iron pathways, such as phenazine production. Treatment with hormones decreased the abundance of P. aeruginosa proteins involved in iron acquisition. Individual hormones exhibited specific bacterial alterations. The presence of estradiol increased protein abundance of the Pseudomonas Quinolone Signal (PQS) quorum sensing system. This study suggests that P. aeruginosa pathogenesis in UTI infections may be influenced by the presence of specific hormones in the host. Understanding the individual role of host factors could contribute to a personalised treatment approach based on the potential impact on infection susceptibility and outcome.

microbiology↗

bioRxiv2022

Multiomic deep delve of synthesis and secretion processes in a model peptidergic system

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus (SON) whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganisation of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and become hyperphosphorylated following stimulation. By multi-omic integration of transcriptome and proteomic data we identify changes to proteins present in afferent inputs to this nucleus. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/494122v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@adc36org.highwire.dtl.DTLVardef@ff7d0corg.highwire.dtl.DTLVardef@16abd16org.highwire.dtl.DTLVardef@a41e3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein which plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen high throughput screening (HTS) assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we discovered that suramin, a negatively charged antiparasitic drug potently inhibits the HMGA2-DNA interaction. Our results also show that the inhibition is through suramin binding to the AT-hooks of HMGA2, therefore blocking its DNA binding capacity. Furthermore, we demonstrate that suramin can induce brain tumor stem cells differentiation into cells with neurite-like structures, a process triggered by disrupting HMGA2-DNA interactions. Since suramin has strong antitumor and anti-metastasis activities, our discovery suggests that HMGA2 and HMGA2-like proteins may be the cellular target of this century-old drug.

biochemistry↗