Biology subjects

Bowen, T.

Publications and source records attributed to Bowen, T..

5 recordsLinked to original sources

bioRxiv2026

Structure-Led Exploration of the Metagenome Yields Novel RNA-Guided Nucleases with Broad PAM Diversity

Compact RNA-guided nucleases with favorable targeting properties are challenging to discover due to their low natural abundance. Here, we develop a structure-led search strategy -leveraging predicted protein folds and sequence-independent similarity metrics -to systematically identify extremely low-homology compact RNA-guided nucleases across vast metagenomic datasets with high computational efficiency. Homology clustering resolved these proteins into distinct groups, for which we performed comprehensive PAM profiling and evaluated editing efficiency in eukaryotic cells. This structure-guided discovery revealed a previously undiscovered landscape of compact nuclease subtypes that exhibit extensive protospacer-adjacent motif (PAM) diversity, expanding the targeting potential of compact editors. Comparative analysis across the novel RNA-guided nuclease families demonstrates that compact systems are not intrinsically limited to highly constrained PAMs but instead have a broad and previously unknown breadth of genome targeting capabilities, comparable to that of Cas9 and far exceeding common transposon- derived systems. Additionally, this search revealed that a compact transposon-associated motif (TAM) is a prerequisite for the emergence of a CRISPR-Cas system from ancestral transposons, before protein domain expansions increase the target length and specificity constraints. These results enrich the catalog of RNA-guided nuclease architectures and contribute validated compact genome editing tools with broad and diverse PAM recognition, which may have therapeutic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/714800v2_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@19ccdc9org.highwire.dtl.DTLVardef@18a5027org.highwire.dtl.DTLVardef@1945836org.highwire.dtl.DTLVardef@2b5bae_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Machine Learning-Driven Optimization of Specific, Compact, and Efficient Base Editors via Single-Round Diversification

Base editing shows great potential in research and clinical applications. Current iterations of the deaminases used to create precise single-nucleotide changes via base editing exhibit undesirable effects, including off-targeting, off-base editing, and bystander editing. Current deaminases are derived from either larger eukaryotic deaminases, which exhibit high levels of Cas-independent DNA targeting, or from evolved variants of the smaller E. coli TadA protein (ecTadA), which exhibits off-base editing. To overcome the limitations inherent to using a single protein sequence for engineering, we diversified newly identified TadA orthologs by DNA shuffling to yield millions of training sequences for measuring base editor efficiency. We trained generative models on the performance data from the pools of variants and drew on information-theoretic insights to efficiently explore the sequence space to generate diverse and high-performing deaminases. From a single round of diversification, we created a small set of novel and specific cytosine and adenosine deaminases that were markedly distinct in sequence from published base editor deaminases. We found that our model created deaminases generally outperform those we identified through typical directed evolution. The novel compact deaminases identified here show high on-base activity, comparable to the leading published base editors, and with demonstrably lower off-base activity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/666807v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@cd25c1org.highwire.dtl.DTLVardef@69ad98org.highwire.dtl.DTLVardef@412e4org.highwire.dtl.DTLVardef@1744100_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2024

The Hyaluronan Synthase isoforms direct distinct patterns of fibroblast activation linked to fibrosis progression versus resolution following renal ischaemia

BackgroundThe stroma plays a key role during renal development and in regeneration after injury. However, following injury, the stroma expands driving progressive fibrosis. Hyaluronan (HA) is a glycosaminoglycan that is absent in healthy kidney stroma but highly expressed in disease. To understand strategies to modulate stromal HA towards therapeutic advantage, this study compares HA and HA Synthase (HAS) enzyme expression in kidney development, health, disease, and recovery. MethodsRats underwent ischaemia reperfusion injury (IRI) with/without ischaemic preconditioning (IPC) and kidneys histologically analysed. Kidneys from C57BL/6 embryos and HAS1/3-/- mice were also analysed and parallel mechanistic cell studies performed using primary human fibroblasts. ResultsIn health, stromal HA was absent from the renal cortex. HAS1 was expressed in some epithelial cells, whilst HAS2 was not expressed. Following IRI there was increased stromal HA in areas of chronic fibrosis, alongside increased HAS2 (but not HAS1) expression. In contrast, during development prominent stromal HA matrices were evident in areas of tubular generation, with strong HAS1 (not HAS2) expression. Following IPC+IRI, stromal HA and HAS2 were attenuated; whilst HAS1+ cells expanded but were distinct from -SMA+ myofibroblasts. Cell studies demonstrated that HAS1+ fibroblasts had a functionally distinct phenotype, with enhanced migration and FAP expression but attenuated -SMA, EDA-FN and COL1A1 expression, whereas HAS2+ fibroblasts demonstrated a classic -SMA+ contractile myofibroblast phenotype with high EDA-FN and COL1A1. ConclusionsHA is a key regulator of stromal fibroblast heterogeneity, with HAS1 and HAS2 defining phenotypically distinct populations that may influence divergent renal outcomes following injury. SIGNIFICANCE STATEMENTHyaluronan (HA) is a matrix glycosaminoglycans that is absent in healthy kidney cortex but demonstrates increased expression in the renal stroma during progressive fibrosis. This study makes comparisons of HA accumulation, localisation, and HA Synthase (HAS) protein expression during kidney development, in health, following ischaemic kidney injury and during renal recovery. The study identifies that different HAS isoenzymes (HAS1 and HAS2) mediate distinct functional fibroblast phenotypes in vitro and are localised in distinct stromal localisations and cell sub-populations in vivo. The data provides interesting insights into HA dependent regulation of fibroblast stromal heterogeneity and identifies the novel finding that HAS1 defines cell populations that are associated with kidney recovery following ischaemic injury and are protective against progressive renal fibrosis.

pathology↗

bioRxiv2023

Sex-specific Proximal Tubular Cell differentiation pathways identified by single-nucleus RNA

BackgroundPostpartum kidney growth is substantial but proliferation and differentiation pathways underpinning nephron elongation are not well defined. Here we performed sequential characterization of mouse kidney transcriptomics at the single cell level to address this. MethodsSingle nuclear RNA sequencing (snRNA-seq) was performed on kidney tissue from male and female mice at 1, 2, 4 and 12 weeks of age using the 10x Chromium platform. ResultsUnbiased clustering was performed on 68,775 nuclei from 16 animals. 31 discrete cellular clusters were seen, which were identified through comparison of their gene expression profiles to canonical markers of kidney cell populations. High levels of proliferation were evident at early time points in some cell types, especially tubular cells, but not in other cell types, for example podocytes. Proliferation was especially evident in Proximal Tubular Cells (PTCs) which are the most abundant cell type in the adult kidney. Uniquely when compared to other kidney cell types, PTCs demonstrated sex-specific expression profiles at late, but not early, time points. Mapping of PTC differentiation pathways using techniques including trajectory and RNA Velocity analyses delineated increasing PTC specialization and sex-specific phenotype specification. ConclusionOur single-cell transcriptomics data characterise cellular states observed during kidney growth. We have identified PTC differentiation pathways that lead to sex-specific tubular cell phenotypes.

cell biology↗

bioRxiv2020

Discovery of the world's highest-dwelling mammal

Environmental limits of animal life are invariably revised upwards when the animals themselves are investigated in their natural habitats. Here we report results of a scientific mountaineering expedition to survey the high-altitude rodent fauna of Volcan Llullaillaco in the Puna de Atacama of northern Chile, an effort motivated by video documentation of mice (genus Phyllotis) at a record altitude of 6205 m. Among numerous trapping records at altitudes >5000 m, we captured a specimen of the yellow-rumped leaf-eared mouse (Phyllotis xanthopygus rupestris) on the very summit of Llullaillaco at 6739 m. This summit specimen represents an altitudinal world record for mammals, far surpassing all specimen-based records from the Himalayas and elsewhere in the Andes. This discovery suggests that we may have generally underestimated the altitudinal range limits and physiological tolerances of small mammals simply because the worlds highest summits remain relatively unexplored by biologists.

evolutionary biology↗