Biology subjects

Whitehead, M.

Publications and source records attributed to Whitehead, M..

6 recordsLinked to original sources

bioRxiv2025

Scalable production of immune-silent circular RNAs for efficient protein expression

Messenger RNA-based therapeutics enable transient protein expression but are limited by its instability. Circular RNA (circRNA) offers enhanced stability, yet scalable production of high-purity circRNA remains challenging. Here, we report an oligo (dT) matrix-based purification strategy for tunable, scalable circRNA production at room temperature. By adjusting poly (A) length, circRNAs are positioned at the midpoint of the oligo (dT) elution profile, enabling one-step separation from both weakly and strongly bound RNA contaminants. We demonstrate that this approach scales linearly with comparable yields for small- and large-scale purifications. In scale-up experiments, 331 mg of highly pure circRNA was recovered in a single run. Incorporating alkaline phosphatase and RNase R digestion prior to chromatography efficiently removes immunogenic contaminants, yielding circRNAs with undetectable immune activation and robust translation in human cells. This workflow provides a practical platform for scalable production of high-quality circRNAs.

bioengineering↗

bioRxiv2025

Discovery of late intermediates in methylenomycin biosynthesis active against drug-resistant Gram-positive bacterial pathogens

The methylenomycins are highly functionalized cyclopentanone antibiotics produced by Streptomyces coelicolor A3(2). A biosynthetic pathway to the methylenomycins has been proposed based on sequence analysis of the proteins encoded by the methylenomycin biosynthetic gene cluster and incorporation of labelled precursors. However, the roles played by putative biosynthetic enzymes remain experimentally uninvestigated. Here, the biosynthetic functions of enzymes encoded by mmyD, mmyO, mmyF and mmyE were investigated by creating in-frame deletions in each gene and investigating the effect on methylenomycin production. No methylenomycin-related metabolites were produced by the mmyD mutant, consistent with the proposed role of MmyD in an early biosynthetic step. The production of methylenomycin A, but not methylenomycin C, was abolished in the mmyF and mmyO mutants, consistent with the corresponding enzymes catalyzing epoxidation of methylenomycin C, as previously proposed. Expression of mmyF and mmyO in a S. coelicolor M145 derivative engineered to express mmr, which confers methylenomycin resistance, enabled the resulting strain to convert methylenomycin C to methylenomycin A, confirming this hypothesis. A novel metabolite (pre-methylenomycin C), which readily cyclizes to form the corresponding butanolide (pre-methylenomycin C lactone), accumulated in the mmyE mutant, indicating the corresponding enzyme is involved in introducing the exomethylene group into methylenomycin C. Remarkably, both pre-methylenomycin C and its lactone precursor were one to two orders of magnitude more active against various Gram-positive bacteria, including antibiotic-resistant Staphylococcus aureus and Enterococcus faecium isolates, than methylenomycins A and C, providing a promising starting point for the development of novel antibiotics to combat antimicrobial resistance.

biochemistry↗

bioRxiv2025

Parallel adaptation to geothermally-warmed habitats due to common structural variation and functional developmental pathways

Climate change is causing rapid increases in temperature which drives genomic changes tied to adaptation. However, predicting the outcomes of climate change presents challenges as the anticipated conditions have yet to be experienced by natural populations. Modelling and lab experiments suggest that natural populations will experience shifts in life history, physiology, phenology, and ecology, but the underlying genomic mechanisms involved are unknown. However, some contemporary natural populations experience habitat warming through geothermal activity and can provide valuable insights into evolutionary responses. Geothermally warmed habitats should impose strong selection on ectotherms compared to ambient habitats as they increase metabolic demands, alter developmental processes, and offer novel ecological conditions. We leveraged Icelandic threespine sticklebacks (Gasterosteus aculeatus) from populations that have adaptively diverged along a geothermal/ambient habitat axis. We obtained 173,485 single nucleotide polymorphisms (SNPs) across four independent instances of population divergence using whole genome sequencing. While the majority of genomic differentiation between geothermal/ambient ecotypes was non-parallel, the MAPK signalling pathway appeared across all ecotype pairs. We also identified a putative inversion located on chromosome XXI which appears to drive parallel genomic differentiation between geothermal and ambient ecotypes. Candidate genes within the putative inversion correspond to metabolic adaptations, including regulation of appetite and fat content. Appetite level showed strong heritable divergence between ecotypes, while the rate of weight loss during starvation and fat levels differed between ecotypes. Overall, both polygenic adaptation and parallel structural variation appeared to be key genomic mechanisms for adaptation to geothermally warmed environments. While allelic divergence was largely unique across populations, it resulted in similar functional phenotypic outcomes. Thus, structural and allelic variation both operate to facilitate adaptation to warming environments. Therefore, while management from a genomic perspective will play a role in mitigating the effects of climate change, this study suggests that consideration of functional molecular pathways will be key to conservation but with precise changes being difficult to predict due to the highly polygenic nature of thermal adaptation.

evolutionary biology↗

bioRxiv2023

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, can invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis. Here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the {beta}1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery preventing cellular spreading and adhesion. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI on the sEV surface. In vitro assays identified collagen VI as playing a key role in cell adhesion and invasion directionality. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate directional invasion, most likely by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction force to enable VSMC movement within the complex vascular ECM network. Figure AbstractVascular smooth muscle cells sense fibronectin via {beta}1 integrin and secrete small extracellular vesicles loaded with collagen VI. These extracellular vesicles are entrapped in the extracellular matrix and induce formation of peripheral focal adhesions presenting adhesion complex ECM proteins including collagen VI, LGALS3BP, EDIL3 and TGFBI. Focal adhesions anchor the extracellular matrix to actin fibrils in the cell. Contraction of the actin fibrils generates the mechanical force for directional cell invasion through the matrix. This figure was created with BioRender (https://biorender.com/). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/551257v3_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1355004org.highwire.dtl.DTLVardef@1186e9forg.highwire.dtl.DTLVardef@106ad30org.highwire.dtl.DTLVardef@1543518_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

The Ecology of Viruses in Urban Rodents with a Focus on SARS-CoV-2

Wild animals are naturally infected with a range of viruses, some of which may be zoonotic for humans. During the human COIVD pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this we have sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralisation ability, that are consistent with rats being exposed to SARS-CoV-2 and / or exposed to other viruses that result in cross-reactive antibodies.

ecology↗

bioRxiv2021

Characterisation of the transcriptional dynamics underpinning the function, fate, and migration of the mouse Anterior Visceral Endoderm

During early post-implantation development of the mouse embryo, the Anterior Visceral Endoderm (AVE) differs from surrounding visceral endoderm (VE) in its migratory behaviour and ability to restrict primitive streak formation to the opposite side of the egg cylinder. In order to characterise the molecular basis for the unique properties of the AVE, we combined single-cell RNA-sequencing of the VE prior to and during AVE migration, with high-resolution imaging, short-term lineage labelling, phosphoproteomics and pharmacological intervention. This revealed the transient nature of the AVE, the emergence of heterogeneities in AVE transcriptional states relative to position of cells, and its prominence in establishing gene expression asymmetries within the spatial constraints of the embryo. We identified a previously unknown requirement of Ephrin- and Semaphorin-signalling for AVE migration. These findings point to a tight coupling of transcriptional state and position in the AVE and reveal molecular heterogeneities underpinning its migratory behaviour and function.

developmental biology↗