Biology subjects

Brennan, E.

Publications and source records attributed to Brennan, E..

15 recordsLinked to original sources

bioRxiv2026

Beyond presence and absence: a survey of TOR and AMPK across 210 eukaryotes reveals unusually long coccidian AMPKγ

Energy and nutrient regulation in eukaryotes depends on AMP-activated protein kinase (AMPK) and target of rapamycin (TOR), yet their evolution is usually described by which components are present or absent. We surveyed 10 TOR- and AMPK-related targets across 210 eukaryotic species and paired that inventory with sequence- and structure-level measurements of the proteins that remain. Across 2100 species-by-target cells, 1255 were coded present, 683 absent and 162 not assessable, with repertoires differing among lineages. Phylogenetically corrected analyses recovered associations among a subset of component absences, especially within canonical complexes, although correlated non-detection in incomplete proteomes remains an alternative explanation. Sequence features varied substantially. In TOR, N-terminal HEAT-region divergence was associated with absence of several TOR-complex partners in discrete-state analyses. In AMPK, the activation-loop threonine was retained in every species with a readable site, while other features varied strongly: {beta} carbohydrate-binding-module conservation covaried with C-terminal length under the primary phylogenetic model, and AMPK{gamma} showed pronounced lineage-associated length variation. In the focal coccidian-group cohort, {gamma} sequences had a median length of 909 residues versus 396 in other sampled copies; the median was 924 among the nine sequences with a tree-based orthology determination, while two Eimeria sequences remain provisional candidates. Low primary nucleotide-contact-site identity was shared more broadly across sampled Apicomplexa. Together, these analyses reveal lineage-structured variation in TOR and AMPK at three levels: component repertoires, sequence features of retained proteins, and associations among components. Signaling architecture cannot be inferred from presence and absence alone.

evolutionary biology↗

bioRxiv2026

NOTCH3 Modulation of Extracellular Matrix, Cytoskeletal Organisation and Metabolic Functions in Human Vascular Smooth Muscle Cells

NOTCH3 is a transmembrane receptor highly expressed in vascular mural cells where it contributes to blood vessel formation and homeostasis. NOTCH3 expression declines in the vasculature with aging, and dysregulated NOTCH3 signalling is implicated in pulmonary arterial hypertension, cancer progression and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). RNA-based approaches targeting NOTCH3 are emerging as potential therapeutic strategies, however, the consequences of NOTCH3 suppression in mature vascular smooth muscle cells (VSMCs) remain incompletely understood. Here, we investigated the molecular and functional effects of siRNA-mediated NOTCH3 knockdown in human aortic smooth muscle cells. Transfection with NOTCH3-targeting siRNA efficiently suppressed NOTCH3 transcript and protein levels. Quantitative proteomics revealed remodelling of extracellular matrix (ECM), cytoskeletal and metabolic pathways, with enrichment of collagen biosynthesis and inhibition of glycolytic signalling. Specifically, NOTCH3 knockdown increased ECM components, including COL3A1, elevated F-actin, and upregulated the actin regulator, CTTN. In parallel, glycolytic capacity was reduced, accompanied by decreased expression of the glycolytic enzyme ENO2. Despite reduced VEGFA and alteration in angiogenic signalling proteins, endothelial network formation in co-cultures, as well as VSMC proliferation and migration remained unaffected. Finally, NOTCH3 interactome analysis revealed key collagen and actin-regulating proteins. These findings identify NOTCH3 as an important regulator of ECM homeostasis, cytoskeletal organisation, and glycolytic metabolism. The preservation of primary cellular functions despite molecular remodelling highlights the adaptive capacity of VSMCs. These findings demonstrate that therapeutic modulation of NOTCH3 may alter vascular cell biology which warrants consideration during development of RNA-based therapeutics for CADASIL and other NOTCH3-associated diseases.

molecular biology↗

bioRxiv2025

Genetic Drivers of Sensitivity or Resistance to RAS(ON) Multi-Selective Inhibitors in NRAS-Mutated Melanoma

Most patients with advanced BRAF or NRAS-driven melanoma receive front-line immunotherapy. However, if immunotherapy fails, BRAF-mutated patients have effective second-line therapies, whereas NRAS-mutated patients lack pathway-targeted options. Recently, RAS(ON) multi-selective inhibitors like RMC-7977, and the investigational agent daraxonrasib, were described that, in partnership with cyclophilin-A (CYPA), inhibit RAS[GTP] signaling. Both compounds demonstrate potent anti-proliferative activity against NRAS-mutated melanoma cell lines and robust anti-tumor activity against preclinical melanoma models. However, in preclinical models, resistance to RMC-7977 monotherapy arose through mutations in Ppia (encoding CYPA) or Map2k1 (encoding MEK1). Moreover, two clinical case studies in patients with NRAS-mutated melanoma treated with daraxonrasib demonstrated clear anti-tumor activity in one patient, but progressive disease in another with co-occurring NRAS and MAP2K1 mutations at baseline. These findings support the potential for daraxonrasib in treatment of patients with NRAS-mutated melanoma, and reveal candidate mechanisms of monotherapy resistance, underscoring the need for combination therapies to improve outcomes. SIGNIFICANCEThere are no pathway-targeted therapies for patients with NRAS-mutated melanoma. Here we demonstrate that direct pharmacological inhibition of RAS[GTP] with RMC-7977 or daraxonrasib (RMC-6236) has profound inhibitory effects in preclinical models of NRAS-mutated melanoma. Furthermore, we identify mechanisms of resistance to RMC-7977 through mutational inactivation of CYPA or mutational activation of MEK1.

cancer biology↗

bioRxiv2025

The allelopathic vitamin B1 antagonist bacimethrin impacts microbial gene expression in a hypereutrophic watershed dominated by cyanobacterial blooms

Freshwater cyanobacterial harmful algal blooms (cyanoHABs), often dominated by Aphanizomenon, Dolichospermum, and Microcystis, are intensifying in eutrophic watersheds globally. A potential control on bacterioplankton dynamics in these systems is the availability of the essential metabolic cofactor thiamin (vitamin B1) and presence of the allelopathic thiamin antagonist, bacimethrin, that competitively inhibits thiamin-requiring enzymes. We examined dissolved concentrations of thiamin chemical congeners and bacimethrin, 16S-amplicon based microbiome compositions, prokaryotic mRNA-based metatranscriptomes, and reference genomes in hypereutrophic Upper Klamath Basin before and during seasonal cyanoHABs. Our objective was to connect bacterioplankton community compositions and gene expression patterns with thiamin congener and bacimethrin availability under different cyanoHAB conditions. Bacimethrin was present in all samples at nearly equimolar concentrations to the thiamin precursor, HMP, suggesting that similar mechanisms influence the availability of both compounds. Additionally, bacimethrin concentrations were positively correlated with cyanoHAB species abundance (cells mL-1) and the expression of microbial thiamin biosynthesis genes. Samples with high cyanoHAB abundances displayed elevated transcription of genes for thiamin biosynthesis, the pentose phosphate pathway, and photosynthesis. Bacterioplankton unable to synthesize thiamin and thus vulnerable to bacimethrin allelopathy, such as Limnohabitans spp., showed reduced gene expression when cyanoHAB abundances were high. Reference genomes of cyanoHAB and many picocyanobacteria strains contained complete thiamin biosynthesis gene pathways, implicating these taxa as major thiamin sources. These results suggest that bacimethrin provides a competitive advantage to bacterioplankton that do not require exogenous vitamin B1 by eliminating the risk of bacimethrin uptake with vitamin B1 transporters, potentially facilitating cyanoHAB dominance in Upper Klamath Basin and broader eutrophic watersheds.

microbiology↗

bioRxiv2025

Tissue scarring provides a biomechanical framework to promote mammalian bile duct regeneration through the activation of integrin-SRC/FAK signalling.

Following chronic injury, the adult mammalian bile duct regenerates by forming new branches, essentially replumbing the ductular system to overcome blockages and breaks. To regenerate effectively, biliary epithelial cells (BECs) receive a range of pro-mitogenic signals from myofibroblasts, which concurrently deposit a collagen-rich scar around the duct as it regrows. Despite epithelial regeneration and scarring occurring side-by-side, whether the deposition of scar tissue regulates ductular regeneration per se remains unclear. By inducing ductular fibrosis and regeneration in vivo, we show that the formation of collagen-I-rich scars around regenerating ducts changes the local biomechanical properties of these tissues, promoting the growth of ducts. Critically, this changing structural landscape is perceived by a spatially restricted population of biliary epithelial cells which forms a "leading-tip" of integrin-2-high cells. This leading-tip undergoes partial-EMT-type reprogramming, allowing it to become migratory and coordinate ductular regeneration. We show that this process is directly driven through an integrin-2-SRC/FAK signalling axis; thereby connecting epithelial regeneration directly to the changing fibrotic environment in chronic ductular disease. HighlightsO_LIChronic liver disease results in the formation of stiff, collagen scars around ducts. C_LIO_LINew ducts acquire high levels of integrin-2 which is spatially localised to a "leading-tip", which loses epithelial features. C_LIO_LIIntegrin-2{beta}1-SRC/FAK signalling regulates ductular migration by linking changes in the bio-structural composition of the liver with ductular cells. C_LI

cell biology↗

bioRxiv2025

Impaired Macrophage DNase Secretion Promotes Neutrophil Extracellular Trap Mediated Defective Efferocytosis in Atherosclerosis

Neutrophil extracellular traps (NETs) drive atherosclerosis progression and are associated with adverse clinical outcomes like myocardial infarction and stroke. While the triggers of NETosis in atherosclerotic plaque are well-characterized, the mechanisms underlying NET degradation and clearance remain unclear. Moreover, the impact of impaired NET clearance on atherosclerosis progression have not been elucidated. Here we show that macrophages are critical for the release of DNases, which degrade NETs. We identified endoplasmic reticulum (ER) stress mediated activation of PERK-ATF4 pathway as a key driver of impaired macrophage DNase secretion, leading to delayed NET clearance and their persistence. Elevated NET levels trigger cleavage of the efferocytosis receptor Mertk leading to defective macrophage efferocytosis and exacerbation of plaque necrosis. Human atherosclerotic plaques and Ldlr-/- mice treated with ISRIB, a PERK inhibitor, show enhanced DNase secretion and clearance of NETs. Together, the identification of key mechanisms of NET clearance in atherosclerosis offers new therapeutic strategies to stabilize plaques.

pathology↗

bioRxiv2024

CDH3-AS1 antisense RNA enhances P-cadherin translation and acts as a tumor suppressor in melanoma

Thousands of regulatory noncoding RNAs (ncRNAs) have been annotated; however, their functions in gene regulation and contributions to cancer formation remain poorly understood. To gain a better understanding of the influence of ncRNAs on gene regulation during melanoma progression, we mapped the landscape of ncRNAs in melanocytes and melanoma cells. Nearly half of deregulated genes in melanoma are ncRNAs, with antisense RNAs (asRNAs) comprising a large portion of deregulated ncRNAs. CDH3-AS1, the most significantly downregulated asRNA, overlaps the CDH3 gene, which encodes P-cadherin, a transmembrane glycoprotein involved in cell adhesion that was also reduced in melanoma. Overexpression of CDH3-AS1 increased cell aggregation and reduced xenograft tumor growth, mimicking the tumor-suppressive effects of CDH3. CDH3-AS1 interacted with CDH3 mRNA and enhanced P-cadherin protein levels. Interestingly, secondary structures at the CDH3 5 end regulated P-cadherin translation, and ribosome profiling revealed that CDH3-AS1 promotes ribosome occupancy at the CDH3 mRNA. Notably, ribosome occupancy was generally increased in mRNAs having cognate asRNA that are complementary to the 5UTR. Taken together, this study revealed the CDH3-AS1-mediated enhancement of P-cadherin translation, underscoring the broader potential of asRNAs as regulators of protein-coding genes and their role in diseases like melanoma.

molecular biology↗

bioRxiv2024

The over-activated peritoneal immune environment in endometriosis is characterised by a lack of PD-1 inhibition.

BackgroundEndometriosis is characterised by chronic inflammation in the peritoneal cavity causing acute and chronic pelvic pain, largely explained by dysregulation in the immune environment within peritoneal fluid. The activation status of the peritoneal immune cells is unclear. In addition, a comparison with the status of the systemic immune system is desirable to explore avenues of diagnosis and treatment of endometriosis-related inflammation and pain. Objective(s)To investigate the immune environment in endometriosis in peritoneal fluid and blood by full-spectrum flow cytometry with a focus on activation and inhibition of immune cells. Study DesignThis was an observational study in patients undergoing laparoscopy for diagnosis or treatment of peritoneal endometriosis or for unrelated conditions; PF was collected from n=5 endometriosis patients and n=4 controls, blood from n=4 endometriosis patients and n=3 controls. Data were analysed for statistical significance using ANOVA, the Kruskal-Wallis or Mann-Whitney U test, with a p-value below 0.05 considered significant. ResultsWe observed a prevailing of myeloid immune cells in the peritoneal fluid as opposed to lymphoid cells in the blood. The main differences between endometriosis and control samples, however, were found in the smaller compartments, i.e., in lymphoid populations in peritoneal fluid and myeloid populations in blood. PD-1 levels in peritoneal fluid endometriosis samples were significantly lower than in controls (p<0.05). Conclusion(s)The immune checkpoint PD-1 could be a new angle of treating endometriosis-related inflammation and pain in women suffering from this chronic and intractable condition. TweetableLack of PD-1 plays a role in endometriosis-related inflammation. AJOG at a glanceO_LIThe inflammatoru immune environment in endometriosis needs investigating as it iis causative of the pain, i.e., the predominant symptom. C_LIO_LIWe found a lack of PD-1 expression on peritoneal fluid cells in endometriosis compared to controls. C_LIO_LIThis could explain the persistent inflammation and open avenues of treatment. C_LI

immunology↗

bioRxiv2024

Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

Lung structural cells, including epithelial cells and fibroblasts, form barriers against pathogens and trigger immune responses following infections such as influenza A virus. This response leads to the recruitment of innate and adaptive immune cells required for viral clearance. Some of these recruited cells remain within the lung following infection and contribute to enhanced viral control following subsequent infections. There is growing evidence that structural cells can also display long-term changes following infection or insults. Here we investigate long-term changes to mouse lung epithelial cells, fibroblasts, and endothelial cells following influenza virus infection and find that all three cell types maintain an imprint of the infection, particularly in genes associated with communication with T cells. Lung epithelial cells from IAV-infected mice display functional changes by more rapidly controlling influenza virus than cells from naive animals. This rapid anti-viral response and increased expression of molecules required to communicate with T cells demonstrates sustained and enhanced functions following infection. These data suggest lung structural cells could be effective targets for vaccines to boost durable protective immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/604410v5_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@2774e0org.highwire.dtl.DTLVardef@6a39e6org.highwire.dtl.DTLVardef@1ff5863org.highwire.dtl.DTLVardef@103df12_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILung epithelial cells, fibroblasts, and blood endothelial cells maintain an inflammatory imprint of influenza A virus (IAV) infection for at least 40 days post-infection. C_LIO_LIIn vivo re-infection leads to a more spatially restricted anti-viral response compared to primary IAV-infected animals. C_LIO_LIT cells are not required for enhanced viral control early after re-infection in vivo C_LIO_LIEx vivo lung epithelial cells from IAV-infected mice more rapidly control IAV than cells from naive animals in the absence of immune cells. C_LI

immunology↗

bioRxiv2023

Lung influenza virus specific memory CD4 T cell location and optimal cytokine production are dependent on interactions with lung antigen-presenting cells

Influenza A virus (IAV) infection leads to the formation of mucosal memory CD4 T cells that can protect the host. An in-depth understanding of the signals that shape memory cell development is required for more effective vaccine design. We have examined the formation of memory CD4 T cells in the lung following IAV infection of mice, characterising changes to the lung landscape and immune cell composition. IAV-specific CD4 T cells were found throughout the lung at both primary and memory time points. These cells were found near lung airways and in close contact with a range of immune cells including macrophages, dendritic cells, and B cells. Interactions between lung IAV-specific CD4 T cells and MHCII+ cells during the primary immune response were important in shaping the subsequent memory pool. Treatment with an anti-MHCII blocking antibody increased the proportion of memory CD4 T cells found at lung airways but reduced interferon-g expression by IAV-specific immunodominant memory CD4 T cells. The immunodominant CD4 T cells expressed higher levels of PD1 than other IAV-specific CD4 T cells and PD1+ memory CD4 T cells were located further away from MHCII+ cells than their PD1-negative counterparts. This distinction in location was lost in mice treated with anti-MHCII antibody. These data suggest that sustained antigen presentation in the lung impacts on the formation of memory CD4 T cells by regulating their cytokine production and location.

immunology↗

bioRxiv2022

Neuronal Activity Alters Neuron to OPC Synapses

The mechanisms that drive the timing and specificity of oligodendrocyte myelination during development, or remyelination after injury or immune attack are not well understood. Recent work has shown that oligodendrocyte progenitors receive synapses from neurons, providing a potential mechanism for neuronal-glial communication. We hypothesize that these connections are important both for correct myelination of neurons during development and for myelination during neuronal plasticity. We utilized chemogenetic tools and viral monosynaptic circuit tracing to analyze these neuroglial connections and to examine OPC proliferation, myelination, synapse formation, and neuronal-glial connectivity after increasing or decreasing neuronal activity in vivo. We found that increasing neuronal activity increased OPC activation, but not proliferation. We also found that altering neuronal activity altered neuronal-glial synaptic connections: while it did not impact the total number of neuronal inputs, or the number of inhibitory neuronal inputs, it did alter the number of excitatory neuron to OPC connections. We also found that increasing or decreasing neuronal activity impacted the ratio of excitatory and inhibitory synapses. Our data show that neuronal activity affects OPC activation, neuronal synapse formation onto OPCs, as well as the types of neuronal inputs to OPCs, indicating that neuronal activity is important for OPC circuit composition and function.

neuroscience↗

bioRxiv2022

Differences in the regulatory strategies of marine oligotrophs and copiotrophs reflect differences in motility

Aquatic bacteria frequently are divided into lifestyle categories oligotroph or copiotroph. Oligotrophs have proportionately fewer transcriptional regulatory genes than copiotrophs and are generally non-motile/chemotactic. We hypothesized that the absence of chemotaxis/motility in oligotrophs prevents them from occupying nutrient patches long enough to benefit from transcriptional regulation. We first confirmed that marine oligotrophs are generally reduced in genes for transcriptional regulation and motility/chemotaxis. Next, using a non-motile oligotroph (Ca. Pelagibacter st. HTCC7211), a motile copiotroph (Alteromonas macleodii st. HOT1A3), and [14C]L-alanine, we confirmed that L-alanine catabolism is not transcriptionally regulated in HTCC7211 but is in HOT1A3. We then found that HOT1A3 took 2.5-4 min to initiate L-alanine oxidation at patch L-alanine concentrations, compared to <30s for HTCC7211. By modeling cell trajectories, we predicted that, in most scenarios, non-motile cells spend <2 min in patches, compared to >4 mins for chemotactic/motile cells. Thus, the time necessary for transcriptional regulation to initiate prevents transcriptional regulation from being beneficial for non-motile oligotrophs. This is supported by a mechanistic model we developed, which predicted that HTCC7211 cells with transcriptional regulation of L-alanine metabolism would produce 12% of their standing ATP stock upon encountering an L-alanine patch, compared to 880% in HTCC7211 cells without transcriptional regulation.

microbiology↗

bioRxiv2020

Assessment of differentially methylated loci in individuals with end-stage kidney disease attributed to diabetic kidney disease

A subset of individuals with type 1 diabetes mellitus (T1DM) are predisposed to developing diabetic kidney disease (DKD), which is the most common cause globally of end-stage kidney disease (ESKD). Emerging evidence suggests epigenetic changes in DNA methylation may have a causal role in both T1DM and DKD. The aim of this investigation was to assess differences in blood-derived DNA methylation patterns between individuals with T1DM-ESKD and individuals with long-duration T1DM but no evidence of kidney disease upon repeated testing. Blood-derived DNA from individuals (107 cases, 253 controls and 14 experimental controls) were bisulphite treated before DNA methylation patterns from both groups were generated and analysed using Illuminas Infinium MethylationEPIC BeadChip arrays (n=862,927 sites). Differentially methylated CpG sites (dmCpGs) were identified (false discovery rate adjusted p[≤]x10-8 and fold change {+/-}2) by comparing methylation levels between ESKD cases and T1DM controls at single site resolution. Gene annotation and functionality was investigated to enrich and rank methylated regions associated with ESKD in T1DM. Top-ranked genes within which several dmCpGs were located and supported by in silico functional data, and replication where possible, include; AFF3, ARID5B, CUX1, ELMO1, FKBP5, HDAC4, ITGAL, LY9, PIM1, RUNX3, SEPTIN9, and UPF3A. Top-ranked enrichment pathways included pathways in cancer, TGF-{beta} signalling and Th17 cell differentiation. Epigenetic alterations provide a dynamic link between an individuals genetic background and their environmental exposures. This robust evaluation of DNA methylation in carefully phenotyped individuals, has identified biomarkers associated with ESKD, revealing several genes and implicated key pathways associated with ESKD in individuals with T1DM.

molecular biology↗

bioRxiv2020

The Atlas of Inflammation-Resolution (AIR)

Acute inflammation is a protective reaction by the immune system in response to invading pathogens or tissue damage. Ideally, the response should be localized, self-limited, and returning to homeostasis. If not resolved, acute inflammation can result in organ pathologies leading to chronic inflammatory phenotypes. Acute inflammation and inflammation resolution are complex coordinated processes, involving a number of cell types, interacting in space and time. The biomolecular complexity and the fact that several biomedical fields are involved, make a multi and interdisciplinary approach necessary. This Atlas of Inflammation Resolution (AIR) is a web-based resource capturing the state-of-the-art in acute inflammation and inflammation resolution research. The AIR provides an interface for users to search thousands of interactions, arranged in inter-connected multi-layers of process diagrams, covering a wide range of clinically relevant phenotypes. The AIR serves as an open access knowledgebase, including a gateway to numerous public databases. It is furthermore possible for the user to map experimental data onto the molecular interaction maps of the AIR, providing the basis for bioinformatics analyses and systems biology approaches. By mapping experimental data onto the Atlas, it can be used to elucidate drug action as well as molecular mechanisms underlying different disease phenotypes. For the visualization and exploration of information, the AIR uses the Minerva platform, which is a well-established tool for the presentation of disease maps. The molecular details of the AIR are encoded using international standards. The Atlas of Inflammation Resolution was created as a freely accessible resource, supporting research and education in the fields of acute inflammation and inflammation resolution. The AIR connects research communities, facilitates clinical decision making, and supports research scientists in the formulation and validation of hypotheses.

immunology↗

bioRxiv2019

Extracellular vesicles from monocyte/platelet aggregates modulate human atherosclerotic plaque responses

In atherosclerosis, a chronic disease characterized by lipid accumulation, fibrosis and vascular inflammation, extracellular vesicles (EVs) are emerging as key players in different stages of disease development. Here we provide evidence that EVs released by mixed aggregates of monocytes and platelets in response to TNF- are both CD14+ and CD41+. Tempering platelet activation with Iloprost impacted the quality and quantity of EV produced. Proteomics of EVs from cells activated with TNF- alone or in presence Iloprost revealed distinct proteome, with selective hits like gelsolin. EVs from TNF- stimulated monocytes augmented release of cytokines, and modulated more than 500 proteins by proteomics, when added to human atherosclerotic plaques. In contrast, EVs generated by TNF- and Iloprost produced minimal plaque activation. In conclusion, attenuating platelet activation has an effect on EV composition released from monocyte/platelet aggregates with downstream modulation of their pro-inflammatory actions and contribution to the development and progression of atherosclerosis.

immunology↗