Biology subjects

Thomas, S. E.

Publications and source records attributed to Thomas, S. E..

5 recordsLinked to original sources

bioRxiv2026

Highly Efficient Lentiviral Transduction of Human iPSC-Derived Microglia and Macrophages

BackgroundMicroglia have become a cell type of interest in the neurodegenerative field given both genetic and pathological evidence for their role in disease development and progression. There has been a rapid growth of studies using iPSC-derived microglial models to understand the molecular mechanisms driving these neurological diseases. However, it remains difficult to transduce myeloid cells effectively which is critical when aiming to study the role of disease associated genes and pathways. Current methods require exposure to multiple viruses which is not suitable for all experimental paradigms. We have therefore sought and characterised a high efficiency promoter and plasmid design to allow high transduction efficacy with a single lentivirus. ResultsUsing the spleen focus-forming virus (SFFV) promoter in combination with central polypurine tract (cPPT) and Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) plasmid elements gave significantly higher transduction efficiency and transgene expression than was achieved with commonly used promoters CMV and EF1. This could then be further improved if required to over 90% transduction efficiency with the removal of lentivirus restriction factor SAM and HD domain-containing protein 1 (SAMHD1) by adding VPX. ConclusionsOur findings allow for a simpler, more efficient and streamlined approach to transgene expression in iPSC-derived microglia and macrophages using only a single lentivirus. This minimises potential unintended side effects such as additional cellular activation and increased cell death.

neuroscience↗

bioRxiv2024

Controlling heterologous protein synthesis through a plant RNA ThermoSwitch

Plants have evolved sophisticated mechanisms to adapt to temperature fluctuations, including transcriptional, post-transcriptional, and post-translational processes. Recent discoveries highlight RNA ThermoSwitches, cis-acting elements in several plant mRNAs that regulate protein synthesis based on temperature changes. These mechanisms, first identified in Arabidopsis thaliana, offer a promising tool for biotechnology by enabling temperature-sensitive control of protein expression. This study demonstrates, for the first time, the feasibility the application of plant RNA ThermoSwitches in Agrobacterium-mediated transient expression systems, presenting a novel method for controlled gene expression in plants. This system is particularly advantageous due to its homogeneous nature and independence from chemical inducers or suppressors.

plant biology↗

bioRxiv2021

Predicted structural mimicry of spike receptor-binding motifs from highly pathogenic human coronaviruses

Viruses often encode proteins that mimic host proteins in order to facilitate infection. Little work has been done to understand the potential mimicry of the SARS-CoV-2, SARS-CoV, and MERS-CoV spike proteins, particularly the receptor-binding motifs, which could be important in determining tropism of the virus. Here, we use structural bioinformatics software to characterize potential mimicry of the three coronavirus spike protein receptor-binding motifs. We utilize sequence-independent alignment tools to compare structurally known or predicted three-dimensional protein models with the receptor-binding motifs and verify potential mimicry with protein docking simulations. Both human and non-human proteins were found to be similar to all three receptor-binding motifs. Similarity to human proteins may reveal which pathways the spike protein is co-opting, while analogous non-human proteins may indicate shared host interaction partners and overlapping antibody cross-reactivity. These findings can help guide experimental efforts to further understand potential interactions between human and coronavirus proteins. HighlightsO_LIPotential coronavirus spike protein mimicry revealed by structural comparison C_LIO_LIHuman and non-human protein potential interactions with virus identified C_LIO_LIPredicted structural mimicry corroborated by protein-protein docking C_LIO_LIEpitope-based alignments may help guide vaccine efforts C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/441187v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1f09454org.highwire.dtl.DTLVardef@19a5557org.highwire.dtl.DTLVardef@158d3fdorg.highwire.dtl.DTLVardef@c59511_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2021

A Fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine, appears to be attractive target for anti-Tuberculosis (TB) drug discovery. Starvation of M. tuberculosis deleted for either argB or argF genes led to rapid sterilization of these strains in mice while Chemical inhibition of ArgJ with Pranlukast was also found to clear chronic M. tuberculosis infection in animal models. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is explored using a fragment-based approach. We reveal several hits for these enzymes validated with biochemical and biophysical assays, and X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential of more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

biochemistry↗

bioRxiv2019

Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification

Translational frameshift errors are often deleterious to the synthesis of functional proteins as they lead to the production of truncated or inactive proteins. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and has been identified as a therapeutic target for several bacterial infections. Here we validate TrmD as a target in Mycobacterium abscessus and describe the application of a structure-guided fragment-based drug discovery approach for the design of a new class of inhibitors against this enzyme. A fragment library screening followed by structure-guided chemical elaboration of hits led to the development of compounds with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and Mycobacterium tuberculosis. The compounds were further active in macrophage infection models against Mycobacterium leprae and M. abscessus suggesting the potential for novel broad-spectrum mycobacterial drugs.

biochemistry↗