Biology subjects

Kirkpatrick, J.

Publications and source records attributed to Kirkpatrick, J..

11 recordsLinked to original sources

bioRxiv2026

Single-Cell Proteomics Reveals Heterogeneous and Bimodal Proteome Responses to DNA Damage

DNA damage response (DDR) involves coordinated activation of repair, checkpoint, and stress-adaptive signalling pathways following genomic toxicity. Bulk-cell proteomics, however, averages divergent responses across neighbouring cells, obscuring the characterisation of biologically meaningful proteome remodelling during genotoxic stress. This is particularly relevant to DDR, where a subset of cells undergo apoptosis, resulting in the sampling of dead cells that confound downstream biological interpretation. Here, we applied single-cell proteomics (SCP) to investigate the proteome response of U-2 OS cells to 6-thioguanine (6TG)-induced DNA damage. By filtering individual cells according to predefined morphological-, sampling- and biological-criteria, to ensure a matched comparison between control and DNA-damaged populations; 6TG treatment was found to activate canonical DDR, including mismatch repair and base excision repair, together with secondary stress responses linked to oxidative stress, mitochondrial dysfunction, inflammatory signalling and proteostasis. Using SCP, we were able to probe neighbouring cell-to-cell heterogeneity, revealing globally increased protein abundance variability following DNA-damage. Importantly, mismatch repair protein MSH3 exhibited amongst the largest abundance increases, concomitant with the greatest reduction in cell-to-cell abundance heterogeneity across all quantified proteins, highlighting it as the most robust and reproducible responder to 6TG-induced DNA damage. Furthermore, analysis of protein-abundance heterogeneity revealed proteins that exhibit bifurcated abundance states across neighbouring cells, with DNA damage inducing either the emergence or loss of these bimodal abundance distributions. Proteins linked to gene expression and protein synthesis were driven towards bifurcation into discrete high-low protein abundance states across neighbouring cells following DNA-damage. Conversely, 6TG abolished the intrinsic high-low abundance bimodality of several core-signalling, stress-signalling and trafficking proteins, suggesting their coordinated homogenisation in response to genotoxic stress. Collectively, these findings demonstrate the power of SCP to resolve the coordinated, yet heterogeneous, cellular response to DNA damage within a population of genetically identical cells.

cell biology↗

bioRxiv2025

A 3D in vitro model of the human hepatobiliary junction

Cholestasis, or disruption in bile flow, is a common yet poorly understood feature of many liver diseases and injuries. Despite this, many engineered human tissue models of liver disease fail to recapitulate physiological bile flow. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction, the interface between hepatocytes and cholangiocytes often disrupted in liver disease that is required for directing bile excreted by hepatocytes into the biliary ductal system. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes into adult hepatobiliary organoids (aHBOs) that structurally connect and functionally transport bile. aHBOs directionally transport bile from hepatocyte bile canaliculi to cholangiocyte-lined ductules, which we visualize through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using fluorescent bile acid analogs and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs recapitulate features of biliary dysfunction that mimics the cholestasis and ischemia-reperfusion injury that complicates liver transplant. Our findings suggest that 1) a reversible reduction in hepatocyte canalicular function under hypoxia, followed by 2) selective cholangiocyte death upon reoxygenation, are processes that potentially contribute to biliary dysfunction upon ischemic injury. This human-derived, scalable platform provides a phenotypically-relevant in vitro model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.

bioengineering↗

bioRxiv2025

Probing site-specific RNA dynamics by solid-state NMR spectroscopy

Knowledge of site-specific dynamics of biomolecules is necessary to understand their specific functions. Solid-state NMR is a uniquely powerful technique that can provide both structural and motional information about complex biomolecules. In recent years, nucleic acids have become increasingly studied by ssNMR, but as yet few RNA or DNA structures are available, with ssNMR-derived dynamics data on RNA rarer still. Here, we report the first systematic ssNMR study of 15N T1 relaxation in RNA using straightforward nucleotide-type-specific and uniform labeling schemes. We observe clear correlation of the measured 15N T1 relaxation time-constants with different structural elements in the RNA molecule, reflecting the distinct characteristics of their underlying motions. We anticipate that this novel approach can be further developed to provide a detailed and comprehensive picture of RNA dynamics in large biomolecular machines.

biophysics↗

bioRxiv2024

A-Islands: A plant dataset for biodiversity research and species monitoring on Australian islands

Australias coastline is fringed by more than 8,000 continental islands. These islands feature a diverse array of landforms, rock and soil types and geological origins. Some of these islands are among the least invaded, most pristine habitats in Australia and support high plant diversity. Here, we present a new Australia-wide curated dataset for plant species occurrences on islands. Combining information from 1,349 species lists and floras, A-Islands includes data on >6,500 plant species from 844 islands ranging in size from 18 m2 to 4,400 km2, exhibiting different degrees of isolation from the mainland, and spanning all major Australian climate zones. Of these, 251 islands have been repeatedly sampled up to 11 times making it possible to investigate temporal compositional change. A-Islands is open access and will be continuously updated. Its simple data structure consisting of three comma separated files allows easy integration with other Australian and global plant-occurrence databases and can serve as a repository for island research in Australia. Knowing which species occur on Australia's islands will provide opportunities for future research, including studying changes in biodiversity and species-turnover within and among archipelagos, tests of classical island biogeography theory and as a baseline for ecological monitoring and conservation.

ecology↗

bioRxiv2024

EstroGene2.0: A multi-omic database of response to estrogens, ER-modulators, and resistance to endocrine therapies in breast cancer

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Understanding the molecular mechanisms is thus key to optimize the existing drugs and to develop new ER-modulators. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities (https://estrogeneii.web.app/). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed substantial diversity in response to different classes of ER-modulators including SERMs, SERDs, SERCA and LDD/PROTAC. Notably, endocrine resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signaling, which is recapitulated clinically. Furthermore, dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of genome-edited versus ectopic overexpression model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancers response to endocrine therapies and explore resistance mechanisms.

cancer biology↗

bioRxiv2023

A nanoparticle priming agent reduces cellular uptake of cell-free DNA and enhances the sensitivity of liquid biopsies

Liquid biopsies are enabling minimally invasive monitoring and molecular profiling of diseases across medicine, but their sensitivity remains limited by the scarcity of cell-free DNA (cfDNA) in blood. Here, we report an intravenous priming agent that is given prior to a blood draw to increase the abundance of cfDNA in circulation. Our priming agent consists of nanoparticles that act on the cells responsible for cfDNA clearance to slow down cfDNA uptake. In tumor-bearing mice, this agent increases the recovery of circulating tumor DNA (ctDNA) by up to 60-fold and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. We envision that this priming approach will significantly improve the performance of liquid biopsies across a wide range of clinical applications in oncology and beyond.

genomics↗

bioRxiv2022

m6A reader Pho92 is recruited co-transcriptionally and couples translation efficacy to mRNA decay to promote meiotic fitness in yeast

N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

biochemistry↗

bioRxiv2021

Identification of RNA base pairs and complete assignment of nucleobase resonances by proton-detected solid-state NMR spectroscopy at 100 kHz MAS

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid-state NMR (ssNMR) is applicable to high molecular-weight complexes and does not require crystallization; thus, it is well-suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA-protein complex by ssNMR using conventional 13C- and 15N-detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H-detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin-systems together with their site-specific base pair pattern using sub-milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein-RNA complexes of any size.

biophysics↗

bioRxiv2019

Late life metformin treatment limits cell survival and shortens lifespan by triggering an aging-associated failure of energy metabolism.

The diabetes drug metformin is to be clinically tested in aged humans to achieve health span extension, but little is known about responses of old non-diabetic individuals to this drug. By in vitro and in vivo tests we found that metformin shortens life span and limits cell survival when provided in late life, contrary to its positive early life effects. Mechanistically, metformin exacerbates aging-associated mitochondrial dysfunction towards respiratory failure, aggravated by the inability of old cells to upregulate glycolysis in response to metformin, leading to ATP exhaustion. The beneficial dietary restriction effect of metformin on lipid reserves is abrogated in old animals, contributing to metabolic failure, while ectopic stabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo. The toxicity is also suspended in nematodes carrying diabetes-like insulin receptor insufficiency and showing prolonged resilience to metabolic stress induced by metformin. In sum, we uncovered an alarming metabolic decay triggered by metformin in late life which may limit its benefits for non-diabetic elderly patients. Novel regulators of life extension by metformin are also presented. HighlightsO_LILate life metformin treatment limits cell survival and shortens lifespan. C_LIO_LIMetformin exacerbates aging-associated mitochondrial dysfunction causing fatal ATP exhaustion. C_LIO_LIOld cells fail to upregulate glycolysis as a compensatory response to metformin. C_LIO_LIThe dietary restriction (DR) mimetic response to metformin is abrogated in old animals. C_LIO_LIPKA and not AMPK pathway instigates the early life DR response to metformin. C_LIO_LIStabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo. C_LI

physiology↗

bioRxiv2018

Noninvasive lung cancer detection via pulmonary protease profiling

Lung cancer is the leading cause of cancer-related death and patients most commonly present with incurable metastatic disease. National guidelines recommend screening for high-risk patients with low-dose computed tomography (LDCT), but this approach has limitations including high false positive rates. Activity-based nanosensors (ABNs) detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease activity. Here, we demonstrate the translational potential of ABNs by coupling ABN multiplexing with intrapulmonary delivery to detect early-stage lung cancer in an immunocompetent, genetically engineered mouse model (GEMM). The design of the multiplexed panel of sensors was informed by comparative transcriptomic analysis of human and mouse lung adenocarcinoma data sets and in vitro cleavage assays with recombinant candidate proteases. When employed in a Kras and Trp53 mutant lung adenocarcinoma mouse model, this approach confirmed the role of metalloproteases in lung cancer and enabled accurate early detection of disease, with 92% sensitivity and 100% specificity.

cancer biology↗

bioRxiv2017

Species comparison of liver proteomes reveals enhanced lipid metabolism, reduced mitochondrial respiration and enhanced expression of detoxifying enzymes in the long-lived naked mole-rat.

Mammals display wide range of variation in their lifespan. Investigating the molecular networks that distinguish long-from short-lived species has proven useful to identify determinants of longevity. Here, we compared the liver of long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omic approach. We found that NMRs livers display a unique expression pattern of mitochondrial proteins that result in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMR and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel longevity pathway and validated it experimentally in the nematode C. elegans.

systems biology↗