Biology subjects

Ryan, A.

Publications and source records attributed to Ryan, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Ligand binding remodels the oligomeric state and active site of Klebsiella pneumoniae azoreductase

Azo dyes are widespread in the environment, and humans ingest a range of these dyes present in food and drugs. The human gut microbiome significantly impacts the fate of ingested azo dyes primarily through the reduction of the azo bond (R-N=N-R'). Flavin-dependent azoreductases have been considered the key enzymatic drivers of this process. To challenge this paradigm, we constructed a sequence similarity network of known azoreductases and focused on AzoA of Klebsiella pneumoniae, a central representative within the gut microbiome cluster. Unexpectedly, K. pneumoniae AzoA displayed relatively low in vitro activity against a range of azo compounds. While the physiological implications remain unclear, these findings are consistent with the possibility that non-enzymatic mechanisms contribute substantially to azo compound reduction in the gut. Furthermore, structural characterization of AzoA, as a holoenzyme and in its substrate-bound state, revealed a previously unrecognized ligand-coupled mechanism involving remodeling of the active site and oligomeric state.

biochemistry↗

bioRxiv2026

The liver compensates for zonal loss of glucose 6-phosphatase to prevent glycogen storage disease

The liver is organized into spatial zones with distinct metabolic roles, but how this architecture contributes to disease remains unclear. Glucose production is concentrated in periportal hepatocytes and depends on G6PC1; loss of this enzyme causes glycogen storage disease type Ia (GSD1a). We tested whether G6PC1 loss in specific zones, including its primary periportal location, is sufficient to cause disease. Unexpectedly, loss of G6pc1 in any single zone caused local glycogen accumulation but did not produce the systemic metabolic abnormalities or liver tumors seen after whole-liver deletion. Instead, disease developed only after near-complete loss of G6pc1 across the entire liver. These findings show that organ-wide compensation preserves metabolic homeostasis and suppresses tumorigenesis despite localized disruption. TeaserSpatial G6pc1 loss causes local glycogen storage without systemic metabolic disease.

physiology↗

bioRxiv2026

A multi-organ spatial metabolomic atlas of exercising mice reveals neuronal Complex I as a convergent and sufficient axis for tau pathology reduction in PS19

We constructed a spatially resolved metabolomic atlas of long-term exercise across six major organs in wild-type mice: brain, heart, lung, liver, kidney, and skeletal muscle, cataloguing 224 metabolic features and revealing coordinated inter-organ remodeling. Surprisingly, the brain showed particularly pronounced region-specific adaptation. Because pathological tau associates with synaptic mitochondria from early stages of tauopathy, we extended this multi-organ spatial metabolomic approach to PS19 mice and found that exercise reduced over 70% of observable tau pathology in PS19 hippocampus and restored the mitochondrial-related metabolome. Integrated proteomic and spatial metabolomic analyses identified NADH dehydrogenase Complex I as the convergent node. To test this finding biologically, we expressed the yeast NADH dehydrogenase, Ndi1, in PS19 neurons in the absence of exercise. This increased cerebral antioxidants, restored shuttle-linked metabolites, and reduced tau pathology. Increasing NADH dehydrogenase activity through NDI1 reproduces the core anti-tau and metabolic effects of exercise. These findings provide a molecular mechanism for how exercise may prevent or slow tau pathology accumulation, complementing the human-cohort literature linking exercise to delayed cognitive decline.

neuroscience↗

bioRxiv2026

Optical Spectral Fingerprinting Enables Sensitive Detection of Anthracycline Chemotherapeutics in Synthetic Clinical Biofluids

Anthracycline chemotherapeutics are commonly used as frontline treatments for a wide array of cancers. However, their administration to patients results in substantial side effects, primarily cardiotoxicity, as well as myelosuppression and gastrointestinal toxicity. Current clinical management of such side effects is solely based on a lifetime dosage limit, which inhibits their anti-tumor efficacy. Many individualized factors, including age, family history of cardiovascular disease, treatment regimen, and other co-morbidities influence drug pharmacology. Despite this heterogeneity, there is no method for determining actual organ or tumor exposure to the treatment in an individual. Here, we developed an optical nanosensor array for four anthracyclines--doxorubicin, daunorubicin, epirubicin, and idarubicin. We used single-walled carbon nanotubes as the signal transducer due to their tunable near-infrared fluorescence. We screened twelve distinct ssDNA sequences paired with seven SWCNT (n,m) species at increasing concentrations of each of the four anthracyclines. The spectral responses were then used to develop machine learning-based classification models to identify different anthracycline types and concentrations. The optimized extreme gradient boosting model was able to classify high levels of each anthracycline with 100% accuracy. Concentration-based classification by PCA was performed for each anthracycline, distinguishing low ([≤] 5 {micro}M) and high (> 5 {micro}M) concentrations. Finally, we validated the sensor performance using synthetic urine and sweat. Our findings demonstrate the potential of carbon nanotube-based sensor array to measure the pharmacokinetics of anthracyclines in patients with the goal of enhancing anti-tumor efficacy and monitoring off-target toxicities.

bioengineering↗

bioRxiv2026

Selective and robust dopamine detection is enabled by aptamer-SWCNT optical sensors in physiological media

Monitoring dopamine in complex biological environments is essential for understanding neurological disorders and disease diagnosis, though it presents a unique chemical challenge. In this work, we rationally designed several single-walled carbon nanotube (SWCNT)-based near-infrared fluorescent sensors for dopamine using ssDNA aptamers as selective molecular recognition elements. The performance of three dopamine-selective aptamer-SWCNT hybrids and sensitive but non-selective (GT)10-SWCNT constructs were evaluated and compared for their magnitude of response, sensitivity, and selectivity to dopamine. We performed these studies in buffer, in complex media with noradrenaline and serotonin, and in synthetic cerebrospinal fluid. We evaluated sensor constructs alone, with heat + divalent cation addition, and with four different molecular passivation agents. Ultimately, sensors passivated with bovine serum albumin (BSA) demonstrated strong selectivity for dopamine relative to noradrenaline, serotonin, and ascorbic acid, with a greater magnitude of response compared to (GT)10-SWCNT. Concentration-response curves in PBS, in a serotonin and noradrenaline solution, and artificial cerebrospinal fluid (aCSF) revealed dynamic ranges between 30 and 200 nM, and we found that the response occurs within five minutes. Together, these results demonstrate that dopamine aptamer-SWCNT sensors enable more selective and robust optical detection in complex biological environments.

bioengineering↗

bioRxiv2026

The impact of intermittent palatable food consumption on microbiota structure in male and female rats

The gut microbiome plays a vital role in metabolism, behavior, and overall health, with diet being a key factor shaping its composition. This study examines the impact of intermittent palatable food (PF) consumption on microbiota structure in male and female rats, focusing on feeding preferences and sex differences. Rats were characterized as high preferring (HP) or low preferring (LP) based on PF intake, and microbial analyses were conducted across different gastrointestinal regions, including the colon, feces, cecum, and cecal contents. However, microbiota composition varied with significant differences in Firmicutes, Bacteroidetes, and Actinobacteria abundances. Sex-based differences were evident particularly in fecal and cecal samples, where Proteobacteria and Actinobacteria populations varied between males and females within the same feeding groups. Our findings support the notion that dietary habits and microbiota composition may form a feedback loop, reinforcing food preferences through gut-brain axis signaling. While alpha diversity remained unchanged, beta diversity analysis indicated subtle, but significant differences in microbial community structures based on sex and feeding behavior. This research provides novel insights into the interplay between diet, gut microbiota, and behavior, while emphasizing the importance of considering sex as a variable in microbiome studies. Understanding these relationships may inform dietary interventions aimed at optimizing microbiota composition to improve metabolic and mental health outcomes linked to diet-induced microbiota shifts. ImportanceThe gut microbiome plays a critical role in various bodily functions, from the brains protective mechanisms to dietary behaviors and food choices. In this study, we sought to broaden the understanding of how various levels of high fat and high carbohydrate diet consumption alter gut microbiota, and ultimately, shape food preference behaviors. We assessed preference behaviors by categorizing rats into high-preference and low-preference groups based on their consumption of high calorie, palatable food, then analyzed their gut bacterial composition, comparing diet preference groups and examining whether sex differences were reflected in the results. The findings from this research hold significant implications for understanding the complex interplay between diet, microbiota, and behavior. Understanding the bidirectional relationship between feeding behavior and microbiota may provide novel insights into the mechanisms underlying eating disorders and metabolic dysregulation.

microbiology↗

bioRxiv2026

High-throughput SWCNT NIR-II screening enables cell and in vivo applications

Diagnostic sensor development and clinical translation lag, in part, due to a lack of rapid, high-throughput screening methodology. Optimization of high-throughput sensor development and deployment will likely help in expediting tools toward the clinic. To address this issue, we optimized high-throughput screening parameters using a near-infrared (NIR-II) plate reader attached to an external probe for in vivo testing. We assessed spectroscopy parameters to improve speed and precision in screening a single-walled carbon nanotube (SWCNT)-based optical sensor. To do so, we assessed the appropriate well plate specifications, including laser power, excitation wavelength, exposure time, and focal height parameters for SWCNT-based optical sensor development. We also used the plate reader to screen fluorescent SWCNT which were endocytosed by a macrophage cell line. We then performed NIR probe spectroscopy to assess SWCNT embedded within a methylcellulose hydrogel. Finally, we used the NIR probe to measure SWCNT center wavelength and intensity from live immunocompetent mice. We anticipate that this framework may be broadly applicable to the development of near infrared nanosensors with the potential for more rapid clinical diagnostic translation.

bioengineering↗

bioRxiv2026

Screening Molecular Recognition Element-Based SWCNT Optical Sensors for the Inflammatory Cytokine TNF-α

TNF- (Tumor Necrosis Factor) is a proinflammatory cytokine that amplifies inflammatory response and promotes leukocyte recruitment. TNF- is primarily produced by activated macrophages, among others, in response to infection, inflammation, or tissue damage. Given its central role in normal and abnormal immune responses, it is the target of several therapeutics, such as adalimumab and etanercept. TNF- is also a prognostic and diagnostic biomarker associated with Rheumatoid Arthritis, Alzheimers disease, Multiple Sclerosis, several kidney diseases, several cancers, Type 2 diabetes, sepsis, and others. Spatial quantification of TNF- in disease models can also be a powerful tool to understand the contributions of inflammatory processes to disease progression. Single-walled carbon nanotubes (SWCNT) are cylindrical carbon lattices that emit distinct near-infrared bandgap photoluminescence. In this work, we evaluated three aptamer-based sensor constructs, plus an additional two iterations of one aptamer sequence, and two antibody-based sensor constructs for TNF- that use SWCNT near-infrared photoluminescence signal transduction. Several, but not all, of these aptamer and antibody-based sensors sensitively and selectively detected TNF- in serum in a physiologically relevant range, and we found that their sensing was improved by both passivation and incorporating an exogenous quencher onto the aptamer sequence. Specifically, we found that modification of one aptamer sequence with a Black Hole Quencher induced selective detection in serum when passivated with poly-L-Lysine. This study highlights the importance, and challenges, of translating previously-validated molecular recognition elements to new detection conditions, in this case on the surface of SWCNT and in challenging serum conditions. It also validated a lead sensor construct that builds upon constructs that failed in serum. We anticipate that the sensors evaluated here will have utility in both the diagnosis and study of inflammation-driven chronic disease, while the sensor assessment framework will help drive the broader field of molecularly specific diagnostics.

bioengineering↗

bioRxiv2025

SpoIIE drives asymmetric cell division in B. subtilis by sequential modulation of the cytokinesis machinery

To form a dormant spore, Bacillus subtilis and related endospore-forming bacteria divide asymmetrically to generate daughter cells of unequal size, the smaller of which becomes the spore. The transmembrane protein SpoIIE repositions the cell division machinery and controls cytokinesis during sporulation, but the molecular basis for the precise placement of the asymmetrical division site to the quarter cell point is unknown. Here, we applied live-cell fluorescence microscopy techniques to reveal that SpoIIE localizes with the treadmilling components of the cell division machinery. We found that SpoIIE opposes the inhibitory activity of the MinCD complex, which prevents assembly of Z-rings near the cell poles. Cells expressing a variant of SpoIIE with its transmembrane region replaced by an unrelated transmembrane anchor assembled condensed Z-rings that were unable to initiate constriction. This reveals a new function of SpoIIE and a possible checkpoint licensing cytokinesis downstream of Z-ring condensation. Potentially explaining the role of SpoIIE in cytokinesis, we demonstrated that SpoIIEs transmembrane region interacts with DivIB, an enigmatic structural component of the cell wall synthesis complex required for cytokinesis during sporulation. Finally, we found that FtsZ filaments are unusually short during sporulation, which requires the transmembrane domain of SpoIIE. Together, these results demonstrate that SpoIIE sequentially influences polar divisome assembly at distinct steps to drive asymmetric cell division.

microbiology↗

bioRxiv2025

Multiplexed Molecularly-Specific SWCNT Cytokine Sensing is Enabled and Enhanced by Amine-Functionalized DNA Aqueous Two-Phase Extraction

Single-walled carbon nanotubes (SWCNT) are versatile building blocks for optical sensors. Their carbon lattice structure, denoted by chiral (n,m) indices, determines the wavelength of their near infrared fluorescence emission. Commercially available SWCNT are polydisperse, containing many (n,m) species with overlapping fluorescence peaks, leading to spectral congestion. Aqueous two-phase extraction (ATPE) has emerged as a valuable tool to sort SWCNT according to their chiral structure and promises improved nanosensor performance as well as unique opportunities for multiplexing. Previous applications of ATPE have enabled ratiometric sensors, but simultaneous detection of multiple analytes using distinct SWCNT chiralities has yet to be realized. This hurdle is largely due to the contrasting surface chemistries needed for ATPE sorting and those needed for biosensing applications. In this work, we introduce a method for purifying multiple chiralities of SWCNT which can be functionalized with molecularly specific antibodies to create a multiplexed sensor platform. We utilize aminated DNA (DNA-NH2), which serves as both a conduit for ATPE sorting and an intermediate linker for later antibody conjugation, to realize this goal. We demonstrate that chemically modified DNA enables ATPE sorting, supporting the broad future utility of the technique. We further show that chirality-sorted SWCNT-DNA-NH2 serve as excellent base constructs for antibody conjugation, forming highly specific sensors with enhanced optical properties (compared to unsorted SWCNT sensors). Importantly, one batch of monochiral base constructs can be functionalized with different antibodies towards different biological applications. Finally, we show that we can sort and functionalize two separate SWCNT chiralities and combine them together to create a multiplexed sensor platform where each peak responds distinctly to its target. There are vast opportunities for expansion of this work through the use of alternative DNA sequences to enable sorting, various SWCNT chiralities, and virtually any commercially available antibody for molecular recognition.

bioengineering↗

bioRxiv2025

Small pangenome of Candida parapsilosis reflects overall low intraspecific diversity

Candida parapsilosis is an opportunistic yeast pathogen that can cause life-threatening infections in immunocompromised humans. Whole genome sequencing (WGS) studies of the species have demonstrated remarkably low diversity, with strains typically differing by about 1.5 single nucleotide polymorphisms (SNPs) per 10 kb. However, SNP calling alone does not capture the full extent of genetic variation. Here, we define the pangenome of 372 C. parapsilosis isolates to determine variation in gene content. The pangenome consists of 5,859 genes, of which 48 are not found in the genome of the reference strain. This includes 5,791 core genes (present in [≥] 99.5% of isolates). Four genes, including the allantoin permease gene DAL4, were present in all isolates but were truncated in some strains. The truncated DAL4 was classified as a pseudogene in the reference strain CDC317. CRISPR-Cas9 gene editing showed that removing the early stop codon (producing the full-length Dal4 protein) is associated with improved use of allantoin as a sole nitrogen source. We find that the accessory genome of C. parapsilosis consists of 68 homologous clusters. This includes 38 previously annotated genes, 27 novel paralogs of previously annotated genes and 3 uncharacterised ORFs. Approximately one-third of the accessory genome (24/68 genes) is associated with gene fusions between tandem genes in the major facilitator superfamily (MFS). Additionally, we identified two highly divergent C. parapsilosis strains and find that, despite their increased phylogenetic distance ([~]30 SNPs per 10 kb), both strains have similar gene content to the other 372. ImportanceCandida parapsilosis is a human fungal pathogen, listed in the high priority group by the World Health Organisation. It is an increasing cause of hospital-acquired and drug-resistant infection. Here, we studied the genetic diversity of 372 C. parapsilosis isolates, the largest genomic surveillance of this species to date. We show that there is relatively little genetic variation. However, we identified two more distantly-related isolates from Germany, suggesting that even more sampling may yield more diversity. We find that the pangenome (the cumulative gene content of all isolates) is surprisingly small, compared to other fungal species. Many of the non-core genes are involved in transport. We also find that variations in gene content are associated with nitrogen metabolism, which may contribute to the virulence characteristics of this species.

genomics↗

bioRxiv2024

Viral mimicry redirects immunosuppressed colorectal tumour landscapes towards a proinflammatory and CMS1-like regenerative state

In colorectal cancer (CRC), tumours classifier as consensus molecular subtype 4 (CMS4) have the worst prognosis and derive negligible benefit from chemotherapy. We previously described how repressed interferon-related signalling is associated with increased relapse in CMS4 tumours. Although the viral mimetic poly(I:C) can reduce liver metastasis in vivo, the initial phenotypic changes that underpin its anti-metastatic response remain poorly described, particularly in the immunosuppressed CMS4 tumour microenvironment. Here we characterise lineage-specific anti-metastatic responses induced by poly(I:C), including acute macrophage polarisation and a novel CMS1-like regenerative stem cell state, which drive pro-inflammatory microenvironmental changes in CRC. These insights enabled the development of tractable biomarkers that identify an "immune-warm" patient subset most likely to respond to poly(I:C), enriched for mismatch-repair proficient (pMMR), anti-inflammatory macrophages and CMS4-like features. The viral mimetic poly(I:C) offers a tailored treatment option for CMS4 tumours, by reprogramming stem cell states and activation of an innate-adaptive anti-metastatic response.

cancer biology↗

bioRxiv2024

Investigating Protein Degradability through Site-Specific Ubiquitin Ligase Recruitment

We report targeted protein degradation through the site-specific recruitment of native ubiquitin ligases to a protein of interest via conjugation of E3 ligase ligands. Direct comparison of degradation ability of proteins displaying the corresponding bioconjugation handle at different regions of protein surfaces was explored. We demonstrate the benefit of proximal lysine residues and investigate flexibility in linker length for the design of optimal degraders. Two proteins without known small molecule ligands, EGFP and DUSP6, were differentially degraded when modified at different locations on their protein surfaces. Further, the cereblon-mediated degradation of the known PROTAC target ERR was improved through the recruitment of the E3 ligase to regions different from the known ligand binding site. This new methodology will provide insight into overall protein degradability, even in the absence of a known small molecule ligand and inform the process of new ligand and PROTAC development to achieve optimal protein degradation. Furthermore, this approach represents a new, small molecule-based conditional OFF switch of protein function with complete genetic specificity. Importantly, the protein of interest is only modified with a minimal surface modification (< 200 Da) and does not require any protein domain fusions.

synthetic biology↗

bioRxiv2024

Mitochondrial metabolism is a key determinant of chemotherapy sensitivity in Colorectal Cancer

Therapy resistance is attributed to over 80% of cancer deaths per year emphasizing the urgent need to overcome this challenge for improved patient outcomes. Despite its widespread use in colorectal cancer (CRC) treatment, resistance to 5-fluorouracil (5FU) remains poorly understood. Here, we investigate the transcriptional responses of CRC cells to 5FU treatment, revealing significant metabolic reprogramming towards heightened mitochondrial activity. Utilizing CRC models, we demonstrate sustained enhancement of mitochondrial biogenesis and function following 5FU treatment, leading to resistance in both in vitro and in vivo settings. Furthermore, we show that targeting mitochondrial metabolism, specifically by inhibiting Complex I (CI), sensitizes CRC cells to 5FU, resulting in delayed tumour growth and prolonged survival in preclinical models. Additionally, our analysis of patient data suggests that oxidative metabolism signatures may predict responses to 5FU-based chemotherapy. These findings shed light on mechanisms underlying 5FU resistance and propose a rational strategy for combination therapy in CRC, emphasizing the potential clinical benefit of targeting mitochondrial metabolism to overcome resistance and enhance patient outcomes.

cancer biology↗

bioRxiv2024

Integrated multiomic analysis identifies TRIP13 as a mediator of alveolar epithelial type II cell dysfunction in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal progressive lung disease urgently needing new therapies. Current treatments only delay disease progression, leaving lung transplant as the sole remaining option. Recent studies support a model whereby IPF arises because alveolar epithelial type II (AT2) cells, which normally mediate distal lung regeneration, acquire airway and/or mesenchymal characteristics, preventing proper repair. Mechanisms driving this abnormal differentiation remain unclear. We performed integrated transcriptomic and epigenomic analysis of purified AT2 cells which revealed genome-wide alterations in IPF lungs. The most prominent epigenetic alteration was activation of an enhancer in thyroid receptor interactor 13 (TRIP13), coinciding with TRIP13 upregulation. TRIP13 is broadly implicated in epithelial-mesenchymal plasticity and transforming growth factor-{beta} signaling. In cultured human AT2 cells and lung slices, small molecule TRIP inhibitor DCZ0415 prevented acquisition of the mesenchymal gene signature characteristic of IPF, suggesting TRIP13 inhibition as a potential therapeutic approach to fibrotic disease.

bioinformatics↗

bioRxiv2024

Defining focal neuroendocrine differentiation as a transcriptionally distinct form of prostate cancer pathology characterized by the expression of androgen receptors

BackgroundMen with neuroendocrine prostate cancer (NEPC) have a poor prognosis. NEPC is commonly diagnosed by immunohistochemical markers (CHGA, SYP and NCAM1) and genomic features (mutations in RB1, PTEN, TP53). But by pathology, NEPC tumours are variable, leading to a classification of NE subtypes such as small cell and large cell neuroendocrine carcinomas, focal neuroendocrine differentiation (Focal NED), and Amphicrine. We postulated the diversity observed in NEPC pathologies might arise from differences in transcriptional profiles and the aim of this study is to utilize single-cell RNA sequencing to define the transcriptional differences between NEPC subtype pathologies. MethodsGene expression profiles were obtained for 18,632 individual tumour cells from 9 patient-derived xenograft (PDX) models representing five distinct neuroendocrine pathologies of prostate cancer. Integration and clustering of cell-level data demarked transcriptionally distinct sub-populations of cells. Differential gene expression, gene set enrichment and transcriptional factor regulon analysis identified expression signatures unique to specific neuroendocrine pathologies. Copy-number estimated from expression data revealed the clonal structure of PDXs with mixed adenocarcinoma and neuroendocrine pathologies. ResultsSignificant differences were observed in the transcriptional profiles of NEPC pathology subtypes. Focal NED cells maintain AR signaling, similar to the amphicrine subtype but different from small and large cell carcinomas. Cellular sub-populations enriched for expression of KRAS, IL2-STAT5 and TNF-signaling genes were found in focal NED and amphicrine pathologies, but not in small or large cell carcinomas. In contrast, sub-populations enriched for the YAP, Myc and E2F pathways were detected in small cell, large cell and amphicrine tumours, but not in focal NED cells. Each pathology showed unique patterns of master regulator activity as well, further implicating focal NED as a transcriptionally distinct entity. Based on copy number alterations within PDXs of mixed pathology, focal NED cells showed little clonal divergence from neighboring adenocarcinoma cells, whereas cells with small cell neuroendocrine pathology were clonally distinct. ConclusionsNeuroendocrine prostate cancer subtypes can be identified by pathology and our study shows that transcriptional features identified by single-cell RNA-sequencing also distinguish neuroendocrine subtypes pathologies from each other. In particular, our data redefine focal neuroendocrine differentiation as a pathology expressing androgen receptors (AR), exhibiting its distinctive composition of transcriptionally unique sub-populations. These findings advocate for differences in the treatment of NEPC tumors, particularly those displaying focal NED.

cancer biology↗

bioRxiv2024

Evaluation of Gene Set Enrichment Analysis (GSEA) tools highlights the value of single sample approaches over pairwise for robust biological discovery.

BackgroundGene set enrichment analysis (GSEA) tools can be used to identify biological insights from transcriptional datasets and have become an integral analysis within gene expression-based cancer studies. Over the years, additional methods of GSEA-based tools have been developed, providing the field with an ever-expanding range of options to choose from. Although several studies have compared the statistical performance of these tools, the downstream biological implications that arise when choosing between the range of pairwise or single sample forms of GSEA methods remain understudied. MethodsIn this study, we compare the statistical and biological interpretation of results obtained when using a variety of pre-ranking methods and options for pairwise GSEA and fast GSEA (fGSEA), alongside single sample GSEA (ssGSEA) and gene set variation analysis (GSVA). These analyses are applied to a well-established cohort of n=215 colon tumour samples, using the clinical feature of cancer recurrence status, non-relapse (NR) and relapse (R), as an initial exemplar, in conjunction with the Molecular Signatures Database "Hallmark" gene sets. ResultsDespite minor fluctuations in statistical performance, pairwise analysis revealed remarkably similar results when deployed using a range of gene pre-ranking methods or across a range of choices of GSEA versus fGSEA, with the same well-established prognostic signatures being consistently returned as significantly associated with relapse status. In contrast, when the same statistically significant signatures, such as Interferon Gamma Response, were assessed using ssGSEA and GSVA approaches, there was a complete absence of biological distinction between these groups (NR and R). ConclusionsData presented here highlights how pairwise methods can overgeneralise biological enrichment within a group, assigning strong statistical significance to gene sets that may be inadvertently interpreted as equating to distinct biology. Importantly, single sample approaches allow users to clearly visualise and interpret statistical significance alongside biological distinction between samples within groups-of-interest; thus, providing a more robust and reliable basis for discovery research.

cancer biology↗

bioRxiv2024

3D-Printable Non-invasive Head Immobilization System for Non-Human Primates

A critical contribution to understanding the primate brain is our ability to directly record, manipulate, or image the brain in awake, behaving monkeys. A challenge with carrying out these studies is that typically the monkeys head must be fixed in place. While non-invasive head immobilization systems (NHIS) have been demonstrated to be effective, they have not fully replaced the use of surgically applied headposts. Here, we introduce a novel NHIS, with an option for voluntary engagement, for macaques using a widely available resource: the 3D printer. We designed customized 3D-printable head-immobilization masks for monkeys and a docking system using standard CT scans and user-friendly open-source software packages (FLoRIN, Blender, Python). We examined the efficacy and stability of our mask system for collecting eye fixation data by measuring trial-by-trial gaze precision and accuracy in two mask-immobilized monkeys and three headposted monkeys, including a within-subjects comparison. Our results demonstrate that monkeys stabilized by the mask maintain gaze precision and accuracy comparable with their headposted counterparts. While the headpost outperformed the mask, the difference in precision was 0.03{degrees} of visual angle, and the difference in accuracy was less than 0.2{degrees}, well within the acceptable range for most use-cases. Our process allows for the production of 3D-printable masks based on CT or MRI scans with the flexibility to incorporate design modifications for experimental equipment and resizing, potentially serving as a promising and more easily accessible alternative to headposts and other NHISs for primate neuroscience research.

physiology↗