Biology subjects

O'Connell, R.

Publications and source records attributed to O'Connell, R..

8 recordsLinked to original sources

bioRxiv2026

Lipoproteins modulate the uptake and biological functionof cationic cell-penetrating peptides across the animal lineage.

Cationic cell-penetrating peptides (+CPPs) are pervasive killer peptides produced by all animals and found in innate immune systems, venoms and neurodegenerative diseases. Despite their ubiquity, the mechanisms underlying their uptake remain opaque and intensely debated. Here, we interrogate +CPPs spanning 600 million years of animal evolution and identify endocytosis as a conserved/convergent uptake mechanism across cell types and organisms, at physiological concentrations. By combining multiplex imaging, genetic screening, cryo-electron tomography, and biophysical methods, we uncover that +CPPs seemingly universally enter eukaryote cells via 'hitchhiking' on lipoproteins. We further show that the interaction of +CPPs with lipoproteins modulates their antimicrobial function. Combined, our findings establish a unified molecular framework describing a pan-eukaryote cell penetrance mechanism. As hyperlipidemia is a common comorbidity, our findings have direct implications for human health. Lastly, the insights gleaned from this work highlight design principles that may inform the future engineering of peptide-based therapeutics and delivery vehicles.

cell biology↗

bioRxiv2023

Efficient multiple gene knock-out in Colletotrichum higginsianum via CRISPR-Cas9 ribonucleoprotein and URA3- based marker recycling

Colletotrichum higginsianum is a hemibiotrophic pathogen that causes anthracnose disease on crucifer hosts, including Arabidopsis thaliana. Despite the availability of genomic and transcriptomic information and the ability to transform both organisms, identifying C. higginsianum genes involved in virulence has been challenging due to their recalcitrance to gene targeting and redundancy of virulence factors. To overcome these obstacles, we developed an efficient method for multiple gene disruption in C. higginsianum by combining CRISPR-Cas9 and URA3-based marker recycling systems. Our method significantly increased the efficiency of gene knock-out via homologous recombination by introducing genomic DNA double-strand breaks. We demonstrated the applicability of the URA3-based marker recycling system for multiple gene targeting in the same strain. Using our technology, we successfully targeted two melanin biosynthetic genes, SCD1 and PKS1, which resulted in deficiency in melanisation and pathogenicity in the mutants. Our findings demonstrate the effectiveness of our developments in analysing virulence factors in C. higginsianum, thus accelerating research on plant-fungus interactions.

microbiology↗

bioRxiv2023

Clec12a tempers inflammation while restricting expansion of a colitogenic commensal

SUMMARYRegulation of the microbiota is critical to intestinal health yet the mechanisms employed by innate immunity remain unclear. Here we show that mice deficient in the C-Type-lectin receptor, Clec12a developed severe colitis, which was dependent on the microbiota. Fecal-microbiota-transplantation (FMT) studies into germfree mice revealed a colitogenic microbiota formed within Clec12a-/- mice that was marked by expansion of the gram-positive organism, Faecalibaculum rodentium. Treatment with F. rodentium was sufficient to worsen colitis in wild-type mice. Macrophages within the gut express the highest levels of Clec12a. Cytokine and sequencing analysis in Clec12a-/- macrophages revealed heighten inflammation but marked reduction in genes associated with phagocytosis. Indeed, Clec12a-/- macrophages are impaired in their ability to uptake F. rodentium. Purified Clec12a had higher binding to gram-positive organisms such as F. rodentium. Thus, our data identifies Clec12a as an innate immune surveillance mechanism to control expansion of potentially harmful commensals without overt inflammation.

immunology↗

bioRxiv2023

Metabolomic Phenotypes Reflect Patient Sex and Injury Status: A Cross-Sectional Analysis of Human Synovial Fluid

1.BackgroundPost-traumatic osteoarthritis (PTOA) is caused by knee injuries like anterior cruciate ligament (ACL) injuries. Often, ACL injuries are accompanied by damage to other tissues and structures within the knee including the meniscus. Both are known to cause PTOA but underlying cellular mechanisms driving disease remain unknown. Aside from injury, patient sex is a prevalent risk factor associated with PTOA. HypothesisMetabolic phenotypes of synovial fluid that differ by knee injury pathology and participant sex will be distinct from each other. Study DesignA cross-sectional study. MethodsSynovial fluid from n=33 knee arthroscopy patients between 18 and 70 years with no prior knee injuries was obtained pre-procedure and injury pathology assigned post-procedure. Synovial fluid was extracted and analyzed via liquid chromatography mass spectrometry metabolomic profiling to examine differences in metabolism between injury pathologies and participant sex. Additionally, samples were pooled and underwent fragmentation to identify metabolites. ResultsMetabolite profiles revealed that injury pathology phenotypes were distinct from each other where differences in endogenous repair pathways that are triggered post-injury were detected. Specifically, acute differences in metabolism mapped to amino acid metabolism, lipid-related oxidative metabolism, and inflammatory-associated pathways. Lastly, sexual dimorphic metabolic phenotypes were examined between male and female participants, and within injury pathology. Specifically, Cervonyl Carnitine and other identified metabolites differed in concentration between sexes. ConclusionsThe results of this study suggest that different injuries (e.g., ligament vs. meniscus), as well as sex are associated with distinct metabolic phenotypes. Considering these phenotypic associations, a greater understanding of metabolic mechanisms associated with specific injuries and PTOA development may yield data regarding how endogenous repair pathways differ between injury types. Furthermore, ongoing metabolomic analysis of synovial fluid in injured male and female patients can be performed to monitor PTOA development and progression. Clinical RelevanceExtension of this work may potentially lead to the identification of biomarkers as well as drug targets that slow, stop, or reverse PTOA progression based on injury type and patient sex.

bioengineering↗

bioRxiv2020

Model-based learning deficits in compulsivity are linked to faulty representations of task structure

Individuals with higher levels of compulsivity exhibit poorer performance on tasks that require model-based planning but the underlying causes have yet to be established. Here, we sought to determine whether these deficits stem from impoverished action-outcome relational knowledge (i.e. issues building an accurate model of the world) and/or an inability to translate models into action. 192 participants performed a two-step reinforcement learning task with concurrent EEG recordings. We found that representations of task-relevant action-outcome associations reflected in reaction time and parietal-occipital alpha-band power were stronger in individuals whose decisions were more model-based, and critically, were weaker in those high in compulsivity. At the time of choice, mid-frontal theta power, a general marker of cognitive control, was also negatively associated with compulsivity, but not model-based planning. These data suggest that model-based planning deficits in compulsive individuals may arise from failures in building an accurate model of the world.

neuroscience↗

bioRxiv2019

Molecular determinants of Arc oligomerization and formation of virus-like capsids

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed self-association mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA. SIGNIFICANCEArc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain.

biochemistry↗

bioRxiv2018

Catecholamine modulation of evidence accumulation during perceptual decision formation; a randomised trial.

Recent behavioural modelling and pupillometry studies suggest that neuromodulatory arousal systems play a role in regulating decision formation but neurophysiological support for these observations is lacking. We employed a randomised, double-blinded, placebo-controlled, crossover design to probe the impact of pharmacological enhancement of catecholamine levels on perceptual decision making. Catecholamine levels were manipulated using the clinically relevant drugs methylphenidate (MPH) and atomoxetine (ATM) and their effects were compared to those of citalopram (CIT), and placebo (PLA). Participants performed a classic EEG oddball paradigm which elicits the P3b, a centro-parietal potential that has been shown to trace evidence accumulation, under each of the four drug conditions. We found that MPH and ATM administration shortened RTs to the oddball targets. The neural basis of this behavioural effect was an earlier P3b peak latency, driven specifically by an increase in its build-up rate without any change in its time of onset or peak amplitude. This study provides neurophysiological evidence for the catecholaminergic enhancement of a discrete aspect of human decision making, i.e. evidence accumulation. Our results also support theoretical accounts suggesting that catecholamines may enhance cognition via increases in neural gain.

neuroscience↗

bioRxiv2017

Reconciling age-related changes in behavioural and neural indices of human perceptual decision making

Ageing impacts on decision making behaviour across a wide range of cognitive tasks and scenarios. Computational modeling has proven highly valuable in providing mechanistic interpretations of these age-related differences; however, the extent to which model parameter differences accurately reflect changes to the underlying neural computations has yet to be tested. Here, we measured neural signatures of decision formation as younger and older participants performed motion discrimination and contrast-change detection tasks, and compared the dynamics of these signals to key parameter estimates from fits of a prominent accumulation-to-bound model (drift diffusion) to behavioural data. Our results indicate marked discrepancies between the age-related effects observed in the model output and the neural data. Most notably, while the model predicted a higher decision boundary in older age for both tasks, the neural data indicated no such differences. To reconcile the model and neural findings, we used our neurophysiological observations as a guide to constrain and adapt the model parameters. In addition to providing better fits to behaviour on both tasks, the resultant neurally-informed models furnished novel predictions regarding other features of the neural data which were empirically validated. These included a slower mean rate of evidence accumulation amongst older adults during motion discrimination and a beneficial reduction in between-trial variability in accumulation rates on the contrast-change detection task, which was linked to more consistent attentional engagement. Our findings serve to highlight how combining human brain signal measurements with computational modelling can yield unique insights into group differences in neural mechanisms for decision making.

neuroscience↗