Biology subjects

Booth, A.

Publications and source records attributed to Booth, A..

8 recordsLinked to original sources

bioRxiv2026

A methylome-guided toolkit for genetic engineering of the fast-growing cyanobacterium UTEX 3222

Cyanobacterium aponinum UTEX 3222 is a fast-growing marine cyanobacterium that reaches high cell densities, making it an attractive chassis strain for low-cost biomanufacturing. However, past efforts to genetically engineer UTEX 3222 have been unsuccessful. We hypothesized that the complex restriction modification systems in the UTEX 3222 genome constitute a major barrier to genetic transformation. Using a combined methylation avoidance and methylation mimicking approach, we overcame this barrier and successfully transformed UTEX 3222 by natural transformation, generating a large panel of fully segregated axenic strains. Transformation method was also established for a novel shuttle vector that replicates in UTEX 3222. A large-scale promoter screen identified promoters which exhibited strong expression at high cell density. These advances establish the foundation for developing UTEX 3222 as a scalable platform for photosynthetic biomanufacturing.

synthetic biology↗

bioRxiv2026

Systematic Evaluation of Signal Peptide-Driven Protein Secretion in the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.

bioengineering↗

bioRxiv2025

EGFR-targeted and MMP-activated membranolytic peptides derived from Polybia paulista MP1 kill cancer cells specifically in vitro and reduce tumour growth in vivo

Membranolytic peptides have demonstrated potential as cancer therapeutics, although targeting to cancer cells and reducing toxicity associated with activity in normal cells remain unmet challenges. We have investigated the membranolytic peptide MP1, from Polybia paulista, in order to assess ways to reduce its non-specific toxicity and thereby improve its characteristics as a cancer therapeutic. Using a panel of human breast cell lines and cell survival assays, we show that C-terminal addition of an EGFR binding sequence, with or without a linking MMP-2 cleavage sequence, generally reduced the efficacy of the peptides relative to wildtype MP1, as determined by increases in IC50 values. Critically, cell lines that show the highest sensitivities to these fusion peptides (MDA-MB-468, MDA-MB-231) expressed the highest EGFR and/or MMP-2 levels, demonstrating that these additions direct the cytotoxic activity to cells expressing these biomarkers. MMP-2 inhibition significantly reduced the cell-killing activity of peptides containing MMP-2 cleavage sites, further demonstrating targeting to this biomarker. Fusion peptides significantly induced apoptosis and reduced survival in EGFR/MMP-2 high cancer cells, while sparing EGFR/MMP-2 low cells in the context of standard tissue culture and 3D-spheroids. Finally, systemic treatment with the EGFR and MMP-2 cleavage fusion significantly reduced tumour size in MDA-MB-468 xenograft models, confirming in vivo efficacy against cancer cells and acceptable systemic toxicity. We present this EGFR-MMP-MP1 peptide as a novel cancer therapeutic for further pre-clinical and clinical development.

cancer biology↗

bioRxiv2025

Determinants of membrane sensitivity to the peptide MP-1 (Polybia paulista)

The membrane-disrupting peptide Mastoparan-1 (MP-1), derived from the wasp Polybia paulista is known to possess antimicrobial properties, and exhibits enhanced activity against a number of cancer cell lines relative to healthy cells. Due to the mechanism of action of MP-1 it is likely that differences in plasma membrane lipid composition arising from cancer associated mutations, such as localisation of phosphatidylserine (PS) lipids to the outer leaflet of the plasma membrane, are involved in driving that enhanced activity. Rapid screening of MP-1 mutants in a combined approach using model membrane and cell-based biological assays, has led to the identification of a number of derivative peptides with enhanced selectivity for cancer-like membrane models and breast cancer cell lines and provided insights into the mechanism of membrane disruption and cell death. Notably, the morphology of the membrane perturbations observed by Atomic Force Microscopy (AFM) and activity in cell model systems can change considerably in response to single-point mutations in the MP-1 sequence, indicating a complex structure-activity relationship.

biophysics↗

bioRxiv2020

The influence of phosphatidylserine localisation and lipid phase on membrane remodelling by the ESCRT-II/ESCRT-III complex

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.

biophysics↗

bioRxiv2019

Multispectral Imaging Differentiates Unique Macrophage Profiles in Patients with Distinct Chronic Liver Diseases

Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared to stellate cells, the role of intrahepatic macrophages in the development of fibrosis remains ill defined. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues since it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preserves architecture and the in vivo hepatic milieu. We analyzed resident Kupffer cells (CD68+), monocyte-derived macrophages (Mac387+), pro-fibrogenic macrophages (CD163+), and co-expression of pro-inflammatory (CD14) and anti-inflammatory (CD16) markers in liver biopsies from patients with hepatitis C virus (HCV) and different stages of fibrosis. Liver biopsies with advanced fibrosis showed increased accumulation of CD163+, MAC387+ and CD68+ macrophages in the portal tracts when compared to those with minimal fibrosis. Imaging software generated t-distributed stochastic neighbor embedding (t-SNE) plots and phenotype matrices that facilitated comparison of macrophage profiles. These included monocyte-derived (CD68+/Mac387+) and pro-fibrotic/anti-inflammatory (CD163+/CD16+) phenotypes. We established that the utility of this platform could be extended to liver biopsies from patients with other chronic liver diseases including nonalcoholic steatohepatitis and autoimmune hepatitis. Each disease exhibited a unique profile after spectral imaging analysis and this platform holds the potential to identify patients predisposed to progressive liver disease based on the macrophage composition. In summary, spectral imaging is a powerful tool that enables analysis of macrophage profiles in different types of chronic liver diseases and has potential to change the manner in which we evaluate liver biopsies leading to more personalized treatment strategies.

pathology↗

bioRxiv2018

In vitro membrane remodelling by ESCRT-II/ESCRT-III is regulated by negative feedback from membrane tension

Artificial cells can shed new light on the molecular basis for life and hold potential for new chemical technologies. Inspired by how nature dynamically regulates its membrane compartments, we aim to repurpose the endosomal sorting complex required for transport (ESCRT) to generate complex membrane architectures as suitable scaffolds for artificial cells. Purified ESCRT-III components perform topological transformations on giant unilamellar vesicles (GUVs) to create complex \"vesicles-within-a-vesicle\" architectures resembling the compartmentalisation in eukaryotic cells. Thus far, the proposed mechanisms for this activity are based on how assembly and disassembly of ESCRT-III on the membrane drives deformation. Here we demonstrate the existence of a negative feedback mechanism from membrane mechanics that regulates ESCRT-III activity. ILV formation removes excess membrane area, increasing tension, which in turn suppresses downstream ILV formation. This mechanism for in vitro regulation of ESCRT-III activity may also have important implications for its in vivo functions.

synthetic biology↗

bioRxiv2016

Transcriptomics reveals patterns of sexually dimorphic gene expression in an avian hypothalamic-pituitary-gonadal (HPG) axis

The hypothalamic-pituitary-gonadal (HPG) axis is a key biological system required for reproduction and associated sexual behaviors to occur. Each of these tissues, the hypothalamus in the brain, the pituitary gland, and the gonads (testes and ovaries), has specialized and sometimes sex-specific functions that promote, amongst other things, reproductive processes. To fully characterize the transcript community of each aspect of the HPG axis, as well as to uncover potentially important sex-specific differences, we characterized patterns of gene expression in sexually mature male and female rock dove (Columba livia) hypothalamus, pituitary, and gonads. We describe patterns of gene expression amongst tissues as well as patterns of sex-biased gene expression which may underlie some of the fundamental differences between male and female reproductive behavior. We report greater sex-biased differential expression in the pituitary (233 genes out of 15,102 genes) as compared to the hypothalamus (1 gene out of 15,102 genes), with multiple genes overexpressed in the male pituitary gland being related to locomotion, and multiple genes over-expressed in the female pituitary gland being related to reproduction, growth and development. These genes may be associated with facilitating the different roles the HPG system plays in sex-specific reproductive behavior, including life-history strategies characterized by short-term payoffs in males (i.e. locomotion) and longer-term payoffs in females (i.e. development and reproduction). In addition, we report novel patterns of sex-biased expression in genes involved in reproduction-associated processes. Gonadotropin-releasing hormone, Progesterone, and Androgen receptor were upregulated in the female pituitary as compared to the male pituitary. We also found greater expression of Prolactin in the female pituitary as compared to the male pituitary, but it was more highly expressed in the male hypothalamus as compared to the female hypothalamus. The Prolactin receptor was also more highly expressed in the male hypothalamus and pituitary gland as compared to corresponding female tissues. Finally, we discovered greater expression of Arginine Vasopressin Receptor 1A in the male pituitary as compared to the female pituitary. Using a more global analysis, we discovered other interesting sex-biased patterns in genes not originally targeted for investigation. These genes include like Betacellulin (BTC), which was upregulated in the female pituitary and compared to males, and Ecto-NOX Disulfide-Thiol Exchanger 1 (ENOX1), which was upregulated in the male pituitary as compared to females. These genes may play important, though currently unknown, roles in reproductive physiology and behavior. In conclusion, we reveal patterns of tissue specific and sexually dimorphic gene expression in the HPG axis, highlighting the need for sex parity in transcriptomic studies and providing new lines of investigation of the mechanisms of reproductive function.

neuroscience↗