Biology subjects

Browne, J.

Publications and source records attributed to Browne, J..

4 recordsLinked to original sources

bioRxiv2025

A robust platform for recombinant production of animal venom toxin modulators of ion channels

Background and PurposePeptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions. Experimental Approach13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca2+ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding. Key ResultsMonovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK) toxins from spiders, and -toxins from scorpions, substantially surpassing the performance of unfused toxins or bivalent Fc-toxin fusions which gave low or no yield. Snake toxins targeting nicotinic acetylcholine receptors retained high single digit nanomolar inhibitory potency. Spider and scorpion toxins targeting the voltage-gated Na+ channel Nav1.7 retained pharmacological function and selectivity across a panel of five Nav subtypes, albeit with reduced potencies that did not exceed [~]70 nM. Conclusions and ImplicationsWe present a strategy for straightforward robust production of pure, monodisperse, and functional animal venom-derived toxins. This lowers the barrier to toxin production in a standard laboratory setting for follow-on engineering purposes.

pharmacology and toxicology↗

bioRxiv2025

On-cell Saturation Transfer Difference (STD) NMR on ion channels: characterizing negative allosteric modulator binding interactions of P2X7.

P2X7 receptors are important drug targets involved in pathologies ranging from psychiatric disorders to cancer. Being membrane embedded receptors, they are challenging for structural characterisation and at present we only have a small number of X-ray and cryoEM structures for P2X7 bound to antagonists. We demonstrate that Saturation Transfer Difference (STD) NMR on live mammalian cells (on-cell STD NMR) overexpressing P2X7 receptors allows further structural insight on the complexes of P2X7 with two potent negative allosteric modulators, namely AZ10606120 and JNJ-47465567, via the determination of the binding epitope mapping of the interactions e.g. the main region of contact between the ligand and the binding pocket. This approach, reported for the first time on membrane-embedded ion channels, in combination with molecular docking, allows us to propose the first NMR-validated 3D molecular models for two antagonists as bound to human P2X7 receptors, and to correlate the structural knowledge acquired with the pharmacology data. We highlight the transformative potential of this application to aid drug design efforts in a less resource-demanding fashion than X-ray crystallography and cryo-EM and we envisage on-cell STD NMR to fast become an asset for structure-activity-relationship studies helping knowledge-based development of efficient drugs targeting P2X7 and other ion channels/membrane-embedded proteins.

pharmacology and toxicology↗

bioRxiv2023

Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity.

Hair follicles provide an easily accessible tissue for interrogating gene expression in mammals. RNAlater(R) is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8{degrees}C overnight before transfer to -20{degrees}C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater(R), and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from three horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater(R) solution. Test group A was stored at 4{degrees}C for 24-h, then decanted and stored at -20{degrees}C. Test groups B and C were stored at 4{degrees}C and 19{degrees}C (room temperature) respectively for 7 days, then decanted and stored at -20{degrees}C. RNA was isolated from all samples and RNA quantity and quality were measured. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml;P = 0.4293) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.3193) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater(R) at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.

molecular biology↗

bioRxiv2019

Mycobacterium riyadhense clinical isolates provide insights into ancestry and adaptive evolution in tuberculosis

Current evolutionary scenarios posit the emergence of Mycobacterium tuberculosis, the deadliest bacterial pathogen for humans globally, from an environmental saprophyte through a cumulative process of genome adaptation. Mycobacterium riyadhense is a novel non-tuberculous mycobacterium (NTM) that is being increasingly isolated from human clinical cases with tuberculosis (TB)-like symptoms in various parts of the world. We provide evidence here that M. riyadhense is likely a missing link in our understanding of the evolution of M. tuberculosis. To elucidate the genomic hallmarks that define the evolutionary relationship between M. riyadhense and other mycobacterial species, including members of the Mycobacterium tuberculosis complex (MTBC), eight clinical isolates of M. riyadhense were sequenced and analyzed. We show, among other features, that M. riyadhense shares a large number of conserved orthologues with the MTBC; contains linear and circular plasmids carrying type IV and type VII secretion systems; and shows expansion of toxin/anti-toxin pairs. We conclude that M. riyadhense is an emerging mycobacterial pathogen that shares a common ancestor with members of the MTBC and that can serve as an experimental model to study the evolution and pathogenesis of tubercle bacilli.\n\nAuthor summaryMycobacterium tuberculosis is one of the most prolific infectious killers in humans and is a member of the Mycobacterium tuberculosis complex (MTBC) - a group of genetically related pathogens that cause tuberculosis (TB) in mammalian species. It is postulated that MTBC has evolved from a free-living environmental ancestor into an obligate pathogen. In this evolutionary context, a comprehensive understanding of the genomic hallmarks of the free-living environmental ancestors of the MTBC is of particular scientific interest for better understanding of the evolution of the MTBC. Mycobacterium riyadhense is a novel environmental mycobacterium, first isolated in 2009 in a hospital in Riyadh, that is increasingly being isolated from clinical cases with typical tuberculosis (TB)-like symptoms in humans. In this study, we report the characterization of eight clinical isolates of M. riyadhense, compare their genomes to members of the MTBC, and provide a comprehensive insight into the adaptive changes associated with the evolution of the MTBC from environmental mycobacteria. We show that M. riyadhense is one of the closest known environmental mycobacteria related to the MTBC, and we provide several lines of molecular evidence that M. riyadhense is likely the missing link in the evolution of M. tuberculosis. It shares a common ancestor with members of the MTBC that have evolved through a process of genome reduction, expansion of toxin/antitoxin (T/A) gene systems, and ultimately host adaptation.

evolutionary biology↗