Biology subjects

Stewart, P.

Publications and source records attributed to Stewart, P..

9 recordsLinked to original sources

bioRxiv2026

Multi-Modal Kinome Profiling Discovers Mesenchymal-Like Polarity Networks that Underly Directed Hepatocellular Carcinoma Cell Migration

Metastasis and associated therapy resistance remain the principal drivers of cancer related death, and there is a pressing need for a deeper mechanistic understanding and anti-metastatic therapies. For patients that suffer from hepatocellular carcinomas (HCCs), which are the most common primary liver cancers, frequent systemic metastasis results in bleak 5-year survival prognoses of only 4%. To metastasize, carcinoma cells must acquire an invasive phenotype, which typically requires switching from an epithelial-like apical-basal polarity to the front-rear polarity of mesenchymal-like cells. Signaling cues that originate in the tumor microenvironment can activate cellular morphogenic programs that drive polarity switching, like the epithelial-mesenchymal transition (EMT). Protein kinases control most cell signaling pathways and are highly actionable drug targets; however, systematic studies determining the kinases that underly the epithelial-mesenchymal polarity switch (EMPS) are lacking. We developed an assay platform that integrates mass spectrometry (MS)-based kinome profiling, broadly capturing kinase network activity, with chemical genetic screening using selective kinase inhibitors and quantitative phase imaging (QPI), serving as the phenotypic readout. Applying this approach that we dubbed morphokin-MS, to epithelial-like HCC cell lines that we induced to undergo EMPS identified a conserved network of 12 kinases that contributed to HCC cell polarity switching and directed cell migration; MS-based kinome profiling of 17 HCC patient tumors showed that these kinase are frequently upregulated in human tumors. morphokin-MS also revealed that death-associated protein kinase 3 (DAPK3) is one of the principal drivers of the EMPS and directed HCC cell migration. Thus, our mechanistic studies revealed that DAPK3 forms a complex with DAPK1 and filamin-A inter-acting protein 1-like (FILIP1L), which act as scaffold proteins that recruit DAPK3 to the centrosome. Pharmaco-logical and genetic inhibition of the DAPK1-DAPK3-FILIP1L complex blocked centrosome repositioning and microtubule polarization toward the leading edge of mesenchymal-like HCC cells, directed cell migration, and invasion. Our morphokin-MS method and comprehensive kinome profiling data will serve as a valuable resource for the cancer research community; our discovery of an inducible mesenchymal-like DAPK1-DAPK3-FILIP1L polarity complex that controls centrosome positioning in motile HCC cells may lead to the development of novel therapeutics for combatting cancer metastasis.

cancer biology↗

bioRxiv2025

Increased attentive use leads to more idiosyncratic functional connections

Experience is thought to modify neural connections to adapt the network to be more optimal for the environment. Given the brains complexity, multiple network changes could each move the system toward optimality. Standard methods ignore this multiplicity and examine each connection independently; these studies have often shown considerable inter-individual variability and modest effects (1). Here, we take a different strategy, determining how a whole-brain connection pattern differs from the typical pattern, that is, how idiosyncratic the pattern is. We examined how the idiosyncrasy of whole brain connection patterns varies with frequency of the use of that part of cortex for attention-demanding tasks, focusing on central versus peripheral vision in healthy individuals (where individuals use central vision more frequently for attention-demanding tasks). We found that the whole-brain pattern of functional connections to the cortical representations of central vision is idiosyncratic, whereas patterns of connections to representations of peripheral vision were very similar person-to-person. In a second set of analyses, we examined the brains of people with central vision loss who use a portion of peripheral vision (called the preferred retinal locus) more frequently for attention-demanding tasks in their daily lives. The cortical representation of the preferred retinal locus exhibits more idiosyncratic connections, compared to a control brain region, or compared to the same brain region in matched control participants with healthy vision. These results are consistent with the hypothesis that increased attentive use of a brain area results in idiosyncratic patterns of whole brain connections. Significance StatementWe found that increased attentive use of a brain region results in more idiosyncratic patterns of connections of that region to the rest of the brain. Our findings support the view that V1 retains the capacity for plasticity well beyond the critical period and that these adaptations are idiosyncratic to the individuals experiences. This approach suggests that tailoring personalized rehabilitation plans for individuals with retinal diseases may be more effective than a one size fits all approach. More generally, it offers a promising framework for investigating brain plasticity in both typical and clinical populations, especially in the context of sensory loss and compensatory adaptation.

neuroscience↗

bioRxiv2025

EnrichMET: R Package for Integrated Pathway and Network Analysis for Metabolomics

Advances in metabolomics have significantly improved our understanding of cellular processes by enabling the identification of hundreds of metabolites in a single experiment. These developments provide valuable insights into complex metabolic networks. While efforts have been made to develop pathway enrichment analysis (PEA), existing implementation often require multiple steps, rely on web-based interfaces, or depend on R packages configuration that may affect reproducibility and ease of use. To overcome these limitations, we introduce EnrichMet, an R package for fast, flexible, and reproducible pathway enrichment analysis. EnrichMet modules support over-representation analysis of pathways, metabolite set enrichment analysis (MetSEA), and network-based pathway analysis. The package streamlines the workflow by combining curated pathway information from the Kyoto Encyclopedia of Genes and Genomics (KEGG) and employs Fishers Exact Test to identify significantly enriched pathways. Benchmark analyses show that enrichment on sample data completes in approximately 3 seconds. EnrichMet offers both a command-line and a user-friendly Shiny interface, enabling accessibility for users with or without programming experience. Through case studies on experimental metabolomics datasets, we demonstrated that EnrichMet delivers accurate and comprehensive pathway enrichment results while minimizing computational time and simplifying user interaction. Furthermore, its flexible framework supports extensions to other data types and knowledge bases beyond KEGG, as illustrated through a lipidomics case study. By unifying performance, reproducibility, usability, and visualization within a single package, EnrichMet facilitates deeper insights and promotes efficient, transparent, and reproducible research practices. Availability and implementation(https://github.com/biodatalab/enrichmet.git)

bioinformatics↗

bioRxiv2025

Ubiquitin-Proteasome System Dysregulation in Alzheimer's Disease Impacts Protein Abundance

Alzheimers disease (AD) is a relentlessly progressive, fatal neurodegenerative disorder that results in widespread protein dysfunctions. However, the full extent of aberrant proteomic changes in AD and their impact remains unknown, in part, because of the challenges of comprehensively measuring the proteome. Here, we used plexDIA, an approach that provides deep proteomic coverage and high throughput by parallelizing the acquisition of peptides and samples, to characterize proteomic changes in AD. Using human dorsolateral prefrontal cortex tissue, we identified 281 differentially abundant proteins in AD. By systematically analyzing compartment and protein complex-specific shifts in protein abundance, we identified an AD-specific decrease in levels of the 20S proteasome, the catalytic core of the cells primary protein degradation pathway. This alteration was accompanied by widespread decreases in proteasome subunit stoichiometries. Many proteasome substrate proteins were negatively correlated with 20S levels and increased in AD, suggesting that reduced 20S levels leads to abnormal protein accumulation. By analyzing proteins increased in AD, we identify key properties of such proteins: They have fast degradation rates, they contain signal sequences that allow them to be targeted for proteasomal degradation, and they are targeted by quality control pathways that recognize mislocalized proteins. Changes in these gene products at the protein and mRNA levels were highly discordant, providing additional evidence for increased protein abundance driven by impaired clearance. We also identified coherent sets of ubiquitin system enzymes, proteins that target substrates for proteasomal degradation, whose levels robustly discriminate AD from non-AD samples. One subset exhibited consistent increases in AD, while another, which contained the tau E3 ligase Cul5, exhibited consistent decreases, revealing complex changes in the ubiquitin system in AD. Taken together, our results suggest that decreased ubiquitin-proteasome system capacity and impaired clearance of short-lived and mislocalized proteins contribute substantially to proteopathic burden in AD.

neuroscience↗

bioRxiv2025

Increasing mass spectrometry throughput using time-encoded sample multiplexing

Liquid chromatography-mass spectrometry (LC-MS) can enable precise and accurate quantification of analytes at high-sensitivity, but the rate at which samples can be analyzed remains limiting. Throughput can be increased by multiplexing samples in the mass domain with plexDIA, yet multiplexing along one dimension will only linearly scale throughput with plex. To enable combinatorial-scaling of proteomics throughput, we developed a complementary multiplexing strategy in the time domain, termed timePlex. timePlex staggers and overlaps the separation periods of individual samples. This strategy is orthogonal to isotopic multiplexing, which enables combinatorial multiplexing in mass and time domains when paired together, and thus multiplicatively increased throughput. We demonstrate this with 3-timePlex and 3-plexDIA, enabling the multiplexing of 9 samples per LC-MS run, and 3-timePlex and 9-plexDIA exceeding 500 samples / day with a combinatorial 27-plex. Crucially, timePlex supports sensitive analyses, including of single cells. These results establish timePlex as a methodology for label-free multiplexing and combinatorial scaling of the throughput of LC-MS proteomics. We project this combined approach will eventually enable an increase in throughput exceeding 1,000 samples / day. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/655515v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c1d799org.highwire.dtl.DTLVardef@1319339org.highwire.dtl.DTLVardef@1b8bf4borg.highwire.dtl.DTLVardef@16d9e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Impact of deprivation and preferential usage on functional connectivity between early visual cortex and category-selective visual regions

Human behavior can be remarkably shaped by experience, such as the removal of sensory input. Many studies of conditions such as stroke, limb amputation, and vision loss have examined how the removal of input changes brain function. However, an important question has yet to be answered: when input is lost, does the brain change its connectivity to preferentially use some remaining inputs over others? In individuals with healthy vision, the central portion of the retina is preferentially used for everyday visual tasks, due to its ability to discriminate fine details. However, when central vision is lost in conditions like macular degeneration, peripheral vision must be relied upon for those everyday tasks, with certain portions receiving "preferential" usage over others. Using resting-state fMRI collected during total darkness, we examined how deprivation and preferential usage influence the intrinsic functional connectivity of sensory cortex by studying individuals with selective vision loss due to late stages of macular degeneration. We found that cortical regions representing spared portions of the peripheral retina, regardless of whether they are preferentially used, exhibit plasticity of intrinsic functional connectivity in macular degeneration. Cortical representations of spared peripheral retinal locations showed stronger connectivity to MT, a region involved in processing motion. These results suggest that long-term loss of central vision can produce widespread effects throughout spared representations in early visual cortex, regardless of whether those representations are preferentially used. These findings support the idea that connections to visual cortex maintain the capacity for change well after critical periods of visual development. HighlightsO_LIPortions of early visual cortex representing central vs. peripheral vision exhibit different patterns of connectivity to category-selective visual regions. C_LIO_LIWhen central vision is lost, cortical representations of peripheral vision display stronger functional connections to MT than central representations. C_LIO_LIWhen central vision is lost, connectivity to regions selective for tasks that involve central vision (FFA and PHA) are not significantly altered. C_LIO_LIThese effects do not depend on which locations of peripheral vision are used more. C_LI

neuroscience↗

bioRxiv2021

Fronto-parietal Network Segregation Predicts Maintained Cognition in the Cognitively Healthy Oldest-old (85+): evidence for dedifferentiation

Functional connections among groups of brain areas give insight into the brains organization. The cognitive effects of aging may relate to the brains large-scale organization. Examining the relationship between individual differences in brain organization and cognitive function in healthy older adults can help us understand how these networks support healthy cognitive aging. We investigated functional network segregation in 146 cognitively healthy participants aged 85+ in the McKnight Brain Aging Registry. We found that the segregation of the cortical association system and the segregation of individual networks within that system [the fronto-parietal network (FPN), cingulo-opercular network (CON) and default mode network (DMN)], were strong predictors of overall cognition and processing speed. We also provide a healthy oldest-old (85+) cortical parcellation that can be used in future work in this age group. This study shows that network segregation of the oldest-old brain is closely linked to cognitive performance. This work adds to the growing body of knowledge about differentiation in the aged brain by demonstrating that cognitive ability is associated with differentiated functional networks in very old individuals experiencing successful cognitive aging.

neuroscience↗

bioRxiv2019

11β-Hydroxysteroid Dehydrogenase Type 1 inhibition in Idiopathic Intracranial Hypertension: a double-blind randomized controlled trial.

Treatment options for idiopathic intracranial hypertension are limited. The enzyme 11{beta}-hydroxysteroid dehydrogenase type 1 has been implicated in regulating cerebrospinal fluid secretion, and its activity is associated with alterations in intracranial pressure in idiopathic intracranial hypertension. We assessed therapeutic efficacy, safety and tolerability, and investigate indicators of in vivo efficacy of the 11{beta}-hydroxysteroid dehydrogenase type 1 inhibitor AZD4017 compared to placebo in idiopathic intracranial hypertension. A multicenter, UK, 16-week phase II randomized, double-blind, placebo-controlled trial of 12-weeks treatment with AZD4017 or placebo was conducted. Women aged 18 to 55 years with active idiopathic intracranial hypertension (>25cmH2O lumbar puncture opening pressure and active papilledema) were included. Participants received 400mg twice daily of oral AZD4017 compared to matching placebo over 12-weeks. The outcome measures were initial efficacy, safety and tolerability. The primary clinical outcome was lumbar puncture opening pressure at 12 weeks analysed by intention-to-treat. Secondary clinical outcomes were symptoms, visual function, papilledema, headache and anthropological measures. In vivo efficacy was evaluated in the central nervous system and systemically. 31 subjects (mean age 31.2 (SD=6.9) years and BMI 39.2 (SD=12.6) kg/m2) were randomized to AZD4017 (n=17) or placebo (n=14). At 12 weeks, lumbar puncture pressure was lower in the AZD4017 group (29.7 cmH2O) compared with placebo (31.3 cmH2O), but the difference between groups was not statistically significant (mean difference: -2.8, 95% confidence interval: -7.1-1.5; p=0.2). An exploratory analysis assessing mean change in lumbar puncture pressure within each group found a significant decrease in the AZD4017 group (mean change: -4.3 cmH2O (SD=5.7); p=0.009) but not in the placebo group (mean change: -0.3 cmH2O (SD=5.9); p=0.8). AZD4017 was safe, with no withdrawals related to adverse effects. Nine transient drug-related adverse events were reported. One serious adverse event occurred in the placebo group (deterioration requiring shunt surgery). In vivo biomarkers of 11{beta}-hydroxysteroid dehydrogenase type 1 activity (urinary glucocorticoid metabolites, hepatic prednisolone generation and CSF cortisone to cortisol ratios) demonstrated significant enzyme inhibition. This is the first phase 2 randomized controlled trial in idiopathic intracranial hypertension evaluating a novel therapeutic target. AZD4017 was safe, well-tolerated and inhibited 11{beta}-hydroxysteroid dehydrogenase type 1 activity in vivo. Possible clinical benefits were noted in this small cohort. A longer, larger study would now be of interest.

clinical trials↗

bioRxiv2019

Bubble Trouble: Conquering Microbubble Limitations in Contrast Enhanced Ultrasound Imaging by Nature-Inspired Ultrastable Echogenic Nanobubbles

Advancement of ultrasound molecular imaging applications requires not only a reduction in size of the ultrasound contrast agents (UCAs) but also a significant improvement in the in vivo stability of the shell-stabilized gas bubble. The transition from first generation to second generation UCAs was marked by an advancement in stability as air was replaced by a hydrophobic gas, such as perfluoropropane and sulfur hexafluoride. Further improvement can be realized by focusing on how well the UCAs shell can retain the encapsulated gas under extreme mechanical deformations. Here we report the next generation of UCAs for which we engineered the shell structure to impart much better stability under repeated prolonged oscillation due to ultrasound, and large changes in shear and turbulence as it circulates within the body. By adapting an architecture with two layers of contrasting elastic properties similar to bacterial cell envelopes, our ultrastable nanobubbles (NBs) withstand continuous in vitro exposure to ultrasound with minimal signal decay and have a significant delay on the onset of in vivo signal decay in kidney, liver, and tumor. Development of ultrastable NBs can potentially expand the role of ultrasound in molecular imaging, theranostics, and drug delivery.

bioengineering↗