Biology subjects

Stewart, E.

Publications and source records attributed to Stewart, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Sex-Specific Stress Outcomes Following Acute and Chronic Social Defeat Stress

Stressful experiences are a major risk factor for the development of neuropsychiatric disorders in vulnerable individuals. Identifying features that can distinguish stress-susceptible from resilient individuals remains a central goal in stress neurobiology. Due to the routine exclusion of female subjects from stress research or the use of male-defined criteria in behavioral characterization of subjects post-stress, female stress responses and vulnerability profiles remain poorly understood. This work aimed to gain a better understanding of sex-specific stress responses to social defeat stress. Here, we used a novel paired male-female social defeat paradigm and tracked subjects longitudinally in a ''two-hit'' design to evaluate responses to both acute social defeat stress (ASDS) and chronic social defeat stress (CSDS), and to determine how prior stress exposure influences subsequent vulnerability. We found that both ASDS and CSDS induces changes in social avoidance/approach behavior in males but not females, as measured by a classic social interaction ratio. Prior ASDS exposure enhanced susceptibility to subsequent CSDS most clearly in males, with females displaying specific changes in corner zone occupancy. Furthermore, we observed divergent anxiety profiles between males and females in a post-stress behavioral battery. Finally, females exhibited specific stress-related locomotor responses following ASDS. Together, these findings suggest that females may adopt distinct behavioral strategies following social defeat stress.

neuroscience↗

bioRxiv2026

Multi-hit STAG2 mutations define a high-risk subset of MDS and reveal convergent evolutionary targeting of cohesin

STAG2 is the most frequently mutated cohesin gene in myeloid neoplasms, yet the significance of multiple mutations within this X-linked tumor suppressor remains unknown. We analyzed a cohort of 1,967 adult patients with myeloid neoplasms and identified 233 cases (12%) harboring STAG2 mutations, including 38 cases (16%) with multiple STAG2 hits. Patients with multi-hit STAG2 mutations exhibited increased multilineage dysplasia compared with single-hit cases and experienced inferior overall survival, an effect driven primarily by patients with myelodysplastic syndromes (MDS). To investigate the molecular basis of recurrent STAG2 acquisition, we performed long-read sequencing in representative cases with phaseable STAG2 mutations. In the informative case examined, distinct truncating STAG2 mutations did not co-occur on the same DNA molecule, supporting independent acquisition rather than stepwise allelic inactivation. Cohort-level variant allele frequency patterns were consistent with recurrent evolutionary targeting of STAG2 across related clonal populations. Together, these findings support a model in which multi-hit STAG2 mutations arise through convergent evolution and define a biologically distinct, adverse-risk subset of MDS.

cancer biology↗

bioRxiv2026

Guide RNA binding induces reverse transcription bias during RT-qPCR analysis of RNA-targeting CRISPR/Cas systems

RNA-targeting CRISPR systems are commonly evaluated by RT-qPCR, but guide RNA binding can confound these measurements. We show that crRNA alone produces apparent knockdown without reducing target RNA abundance, whereas RNA sequencing remains unbiased and reveals guide-associated transcriptomic perturbations. A simple RNA denaturation step before reverse transcription restores accurate RT-qPCR quantification, providing practical guidance for RNA-targeting CRISPR analysis and guide design.

genomics↗

bioRxiv2026

Pathway incompatibility between NF-κB and RAS signaling constrains oncogenicity in B-cell leukemia

B-cell acute lymphoblastic leukemia (B-ALL) arises from lymphoid precursors that fail to complete normal B-cell maturation and frequently harbor activating mutations in the RAS-ERK pathway that promote leukemic survival. These cells remain dependent on precursor B-cell receptor (pre-BCR)-associated signaling programs, which oncogenic RAS can functionally mimic. While oncogenesis is often attributed to cooperating mutations, mutually exclusive genetic alterations suggest that certain oncogenic pathways may be functionally incompatible. In our previous work, we proposed the concept of pathway incompatibility, in which co-activation of specific oncogenic signaling programs antagonizes their oncogenic activity and suppresses tumorigenesis. Here, we examined the interaction between RAS signaling and canonical NF-kB activity in B-ALL within this framework. Genomic analysis revealed underrepresentation of cases harboring concurrent RAS and NF-kB pathway alterations. Functionally, activation of canonical NF-kB induced apoptotic depletion of RAS-pathway-mutated B-ALL cells, whereas activation of the non-canonical pathway conferred a growth advantage. Mechanistically, canonical NF-kB suppressed pre-BCR-associated signaling programs and increased expression of mature B-cell receptor (BCR) components, indicating a developmental signaling state incompatible with oncogenic RAS activity. Consistent with this framework, enforced oncogenic RAS signaling was poorly tolerated in BCR-positive lymphoma cells unless BCR expression was disrupted. Pharmacologic activation of NF-kB reduced ERK signaling and selectively impaired viability of RAS-pathway-mutated B-ALL cells, with enhanced effects in combination with ERK inhibition. Together, these findings identify canonical NF-kB signaling as a developmental context-dependent constraint on RAS-driven leukemogenesis and support pathway incompatibility as a determinant of the oncogenicity of signaling pathways.

cancer biology↗

bioRxiv2025

Microglial plasticity across development mediates infantile amnesia

Infantile amnesia, the inability to recall episodic memories formed during early childhood, is a hallmark of postnatal brain development. Yet the underlying mechanisms remain poorly understood. This work aimed to gain a better mechanistic understanding of infantile amnesia. Microglia, specialized macrophages of the central nervous system, are known to play an important role in synaptic refinement during postnatal development and have recently been implicated in memory related functions. Here, we identified microglia as key regulators of memory accessibility in infancy. We profiled dynamic changes in microglial morphology across the postnatal window that parallelled the onset of infantile forgetting. We found that pharmacological inhibition of microglial activity during a specific postnatal window prevents infantile amnesia for a contextual fear memory, implicating microglia as active modulators of infant memory persistence. Using activity-dependent tagging of infant encoded engram cells, we demonstrated that microglial inhibition alters engram size and engram reactivation in the amygdala and results in changes in microglia-engram cell interactions. Furthermore, we characterized a relationship between microglial dysfunction and the lack of infantile amnesia in maternal immune activation offspring. Together, these findings reveal a novel role for microglia in regulating infant memory retrieval and suggest that microglial dysfunction may contribute to altered memory trajectories in neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Did Iron Suppress Eukaryote Emergence and Early Radiation?

The last eukaryotic common ancestor (LECA) is widely thought to have been an oxygen-respiring organism, arising through endosymbiosis when a free-living bacterium became the mitochondrion. Owing to the mitochondrions central metabolic task of oxidative phosphorylation, oxygen availability has long been a hypothesized driver of eukaryogenesis. However, this hypothesis is challenged by a temporal disconnect, spanning several hundred million years, between the earliest geochemical evidence for oxygen in the environment ([~]3.2-2.5 Ga) and the oldest widely accepted eukaryotic fossils ([~]1.7 Ga). Notably, the earliest candidate eukaryotes appear contemporaneous with the cessation of major iron deposits and rise of sulfide- and sulfate-rich marine sediments in coastal environments. Here, we integrate Proterozoic surface geochemical records with the microbial biochemistry of iron to examine potential environmental constraints on early eukaryotic evolution. Iron bioavailability exerts complex and often antagonistic effects on both aerobic and anaerobic microbial lineages that contributed to LECA. Elevated iron levels likely disrupted cellular homeostasis, particularly by destabilizing labile iron pools and promoting oxidative damage to bacterial lipids. The programmed cell death pathways known as ferroptosis, which is widespread among eukaryotic lineages, may trace its origins to iron-rich conditions in Archaean and Paleoproterozoic seawater and LECA. Our findings challenge oxygen-centered paradigms of eukaryogenesis and reframes the long-recognized temporal gap as a consequence of iron-mediated physiological constraints.

evolutionary biology↗

bioRxiv2025

SLFN11 Loss-Induced Chemoresistance is Associated with Overexpression of Glycerophospholipid Biosynthesis in Ewing Sarcoma.

Ewing sarcoma (EWS) is an aggressive cancer in adolescents and young adults with frequent relapse rates and poor outcomes in recurrent or metastatic cases. Schlafen family member 11 (SLFN11) gene is associated with the sensitivity to DNA-damaging agents (DDAs). The knockout of SLFN11 is associated with acquired chemoresistance in both cell lines and preclinical models. Here, we aimed to elucidate the metabolic underpinnings of SLFN11-loss associated chemoresistance in patient derived cell lines of EWS. Our integrated transcriptomic and metabolomic analyses revealed downregulation of mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2) gene, which was accompanied by the upregulation of glycerophospholipid (GPL) biosynthesis pathway. Further, therapeutic targeting of lipid synthesis with the glycerol-3-phosphate acyltransferase 1 (GPAT1) inhibitor (FSG67) enhanced the efficacy of the DDA (SN-38) in SLFN11-/- cells. These findings indicate that SLFN11 loss-mediated chemoresistance can be targeted by blocking GPL biosynthesis in addition to DDA administration.

cancer biology↗

bioRxiv2025

Enhanced FLI1 accessibility mediates STAG2-mutant leukemogenesis

Chromatin architecture governs transcriptional output and cell identity; however, how its disruption promotes leukemic transformation remains incompletely understood. Here, we show that loss of the cohesin subunit STAG2 creates a hyper-accessible chromatin landscape that amplifies FLI1 activity and extends its binding to ectopic loci. Using multi-omic analyses in human AML samples, cell lines, and mouse models, we identify chromatin-dependent co-occupancy of FLI1 and Menin, accompanied by amplification of Menin occupancy at non-canonical loci beyond its HOXA/MEIS1 targets. Therapeutically, the aberrant expansion of Menin binding drives an altered response towards Revumenib and activates interferon response pathways, creating a therapeutic vulnerability to Menin inhibition. Functionally, STAG2/NPM1c co-mutation drives a stem cell-like immunophenotype and a fully penetrant leukemia in vivo. Collectively, these findings define a model in which cohesin loss rewires transcription factor occupancy to amplify oncogenic chromatin programs and expose context-specific therapeutic dependencies.

cancer biology↗

bioRxiv2024

Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy--a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RAs overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RAs ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.

cancer biology↗

bioRxiv2024

Metabolic enzymes moonlight as selective autophagy receptors to protect plants against viral-induced cellular damage

AbstractRNA viruses co-opt the host endomembrane system and organelles to build replication complexes for infection. How the host responds to these membrane perturbations is poorly understood. Here, we explore the autophagic response of Arabidopsis thaliana to three viruses that hijack different cellular compartments. Autophagy is significantly induced within systemically infected tissues, its disruption rendering plants highly sensitive to infection. Contrary to being an antiviral defense mechanism as previously suggested, quantitative analyses of the viral loads established autophagy as a tolerance pathway. Further analysis of one of these viruses, the Turnip Crinkle Virus (TCV) that hijack mitochondria, showed that despite perturbing mitochondrial integrity, TCV does not trigger a typical mitophagy response. Instead, TCV and Turnip yellow mosaic virus (TYMV) infection activates a distinct selective autophagy mechanism, where oligomeric metabolic enzymes moonlight as selective autophagy receptors and degrade key executors of defense and cell death such as EDS1. Altogether, our study reveals an autophagy-regulated metabolic rheostat that gauges cellular integrity during viral infection and degrades cell death executors to avoid catastrophic amplification of immune signaling.

plant biology↗

bioRxiv2024

Germinal center BCR maturation in appendicitis reveals a role for antigen-specific adaptive immune responses during disease

Appendicitis is one of the most common abdominal emergencies globally, yet little is understood about the inflammatory mechanisms or potential drivers of disease. Neutrophil inflammation and increased cytokine expression such as IL-6 and IL-8 are hallmarks of appendicitis inflammation. However, early histological studies identified increased T and B cell infiltration during appendicitis, providing support for adaptive immune activation as well, although this has never been investigated in depth. We hypothesized that antigen-dependent activation of the adaptive immune response contributes to appendicitis pathology, in addition to the known innate-mediated processes. Via a series of transcriptomic approaches and lymphocyte repertoire analysis in human appendiceal tissue, we identified evidence of antigen-dependent B cell activation. Increased somatic hypermutation in the germinal center and plasma cell compartment was comprised of presumed high-affinity IgG and IgA B cells. We propose that the appendiceal microbiome acts as a source of antigen, as significant microbial dysbiosis was observed during appendicitis. This dysbiosis was characterized by outgrowth of pathobionts such as Parvimonas and oral biofilm-formers such as Fretibacterium and Fusobacterium, in line with previous reports. We also identified potential loss of epithelial barrier integrity via spatial transcriptomic analysis of the appendiceal epithelium, supporting the possibility of microbial invasion into the tissue during appendicitis. This study provides insight into the inflammatory mechanisms of a common disease and helps to define the immune and microbial compartment of an often-ignored organ, the appendix.

immunology↗

bioRxiv2023

Single cell transcriptomic profiling identifies tumor-acquired and therapy-resistant cell states in pediatric rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity and molecular features are shared with human development have not been fully ascribed. Here, we report a single-cell/nucleus RNA sequencing atlas derived from 72 datasets that includes patient tumors, patient-derived xenografts, primary in vitro cultures, and established cell lines. We report four dominant muscle-lineage cell states in RMS: progenitors, proliferative, differentiated, and ground cells. We stratify these RMS cells along the continuum of human muscle development and show that RMS cells share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Indeed, fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that faithfully recapitulates fetal muscle development. We also identify therapy-resistant FN-RMS progenitor cells that share transcriptomic similarity with bipotent skeletal mesenchymal cells, while a subset of fusion-positive (FP) RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in development. Chemotherapy induced upregulation of progenitor signatures in FN-RMS while the neuronal gene programs were retained after therapy in FP-RMS. Taken together, this work identifies new cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.

cancer biology↗

bioRxiv2023

Dominant CIZ1 fragments drive epigenetic instability and are expressed in early stage cancers

CIZ1 is a nuclear matrix protein that is part of the large RNA-dependent supramolecular assembly complexes (SMACs) that form at the inactive X chromosome (Xi) in female cells, and smaller assemblies throughout the nucleus in males and females. It plays a role in maintenance of epigenetic state and gene expression in differentiated cells, via stabilisation of histone post-translational modifications H2AK119ub1 and H3K27me3, added by polycomb repressive complexes (PRC) 1 and 2. Here, we show that expression of the N-terminal replication domain (RD) and C-terminal anchor domain (AD) of human CIZ1 transcript is uncoupled, with consistently elevated AD in early stage breast cancers, and sporadically elevated AD in other common solid tumours. At the protein level CIZ1-Xi SMACs are corrupted in female breast cancers cells, and this is accompanied by elevated AD-encoding transcripts. We modelled the effect of AD fragments in primary murine embryonic fibroblasts and observed dominant-negative interference with CIZ1 SMACs during their assembly in early G1 phase. Mutagenesis identified the matrin 3 homology domain as essential for self-interaction to form stable homodimers in vitro, and as a determinant of its dominant-negative effect in cells, implicating the dimerization interface in CIZ1 SMAC integrity. SMAC disruption was coincident with depletion of PRC1-dependent H2AK119ub1 from Xi chromatin, in a manner abrogated by the PR-deubiquitinase inhibitor PR619, suggesting that CIZ1 SMACs normally stabilise H2AK119ub1 by shielding Xi chromatin from attack by deubiquitinases. Moreover, SMAC disruption was accompanied by changes in gene expression within days. Together, the data suggest that inappropriate expression of CIZ1 AD fragments could drive epigenetic instability in early stage breast cancers by destabilizing the CIZ1 SMACs that normally protect repressed chromatin. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/558821v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@13b1be2org.highwire.dtl.DTLVardef@1a3a16corg.highwire.dtl.DTLVardef@1bac386org.highwire.dtl.DTLVardef@7ac176_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A genome-wide cytotoxicity screen of Cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes ([~]28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function (NKF) and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic NKF proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

microbiology↗

bioRxiv2023

A bench-top dark-root device built with LEGO bricks enables a non-invasive plant root development analysis in soil conditions mirroring nature

Roots are the hidden parts of plants, anchoring their above ground counterparts in the soil. They are responsible for water and nutrient uptake, as well as for interacting with biotic and abiotic factors in the soil. The root system architecture (RSA) and its plasticity are crucial for resource acquisition and consequently correlate with plant performance, while being highly dependent on the surrounding environment, such as soil properties and therefore environmental conditions. Thus, especially for crop plants and regarding agricultural challenges, it is essential to perform molecular and phenotypic analyses of the root system under conditions as near as possible to nature (#asnearaspossibletonature). To prevent root illumination during experimental procedures, which would heavily affect root development, dark-root (D-Root) devices (DRDs) have been developed. In this article, we describe the construction and different applications of a sustainable, affordable, flexible, and easy to assemble open-hardware bench-top LEGO(R) DRD, the DRD-BIBLOX (Brick Black Box). The DRD-BIBLOX consists of one or more 3D-printed rhizoboxes which can be filled with soil, while still providing root visibility. The rhizoboxes sit in a scaffold of secondhand LEGO(R) bricks, which allows root development in the dark as well as non-invasive root-tracking with an infrared (IR) camera and an IR light emitting diode (LED) cluster. Proteomic analyses confirmed significant effects of root illumination on barley root and shoot proteome. Additionally, we confirmed the significant effect of root illumination on barley root and shoot phenotypes. Our data therefore reinforces the importance of the application of field conditions in the lab and the value of our novel device, the DRD-BIBLOX. We further provide a DRD-BIBLOX application spectrum, spanning from investigating a variety of plant species and soil conditions as well as simulating different environmental conditions and stresses, to proteomic and phenotypic analyses, including early root tracking in the dark.

plant biology↗

bioRxiv2022

Immune activation state modulates retrieval of infant engrams

Infantile amnesia is possibly the most ubiquitous form of memory loss in mammals. Despite its widespread relevance, little is known about the biological conditions for infantile amnesia to occur and its effect on the engram cells that encode a memory. We investigated how memories are stored in the brain throughout development by integrating engram labeling technology with mouse models of infantile amnesia. Here, we discovered a phenomenon in which male offspring in maternal immune activation models of autism spectrum disorder do not experience infantile amnesia. We rescued the same apparently forgotten infantile memories in mice by optogenetically reactivating dentate gyrus engram cells labeled during complex infant development experiences. Further, we were able to permanently reinstate lost infantile memories by artificially updating the memory engram, demonstrating that infantile amnesia is a reversible process. Our findings suggest that immune activation during development modulates innate, and reversible, forgetting switches that determine whether infantile amnesia will occur.

neuroscience↗

bioRxiv2021

The Myogenesis Program Drives Clonal Selection and Drug Resistance in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single cell and single nucleus RNA-sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show chemotherapy eliminates the most proliferative component with features of myoblasts; after treatment, the quiescent immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, this data serves as a proof-of-concept that targeting each developmental state in RMS is an effective strategy for improving outcomes by preventing disease recurrence.

cancer biology↗

bioRxiv2021

Microtubules Promote the Non-cell Autonomy of MicroRNAs by Inhibiting their Cytoplasmic Loading into ARGONAUTE1 in Arabidopsis

Mobile microRNAs (miRNAs) serve as local and long-distance signals in developmental patterning and stress responses in plants. However, mechanisms governing the non-cell autonomous activities of miRNAs remain elusive. Here, we show that mutations that disrupt microtubule dynamics are specifically defective for the non-cell autonomous actions of mobile miRNAs, including miR165/6 that is produced in the endodermis and moves to the vasculature to pattern xylem cell fates in Arabidopsis roots. We show that KTN1, a subunit of a microtubule-severing enzyme, is required in source and intermediary cells to inhibit the loading of miR165/6 into ARGONUATE1 (AGO1), which is cell-autonomous, to enable the miRNA"s cell exit. Microtubule disruption enhances the association of miR165/6 with AGO1 in the cytosol. These findings suggest that, while cell-autonomous miRNAs load into AGO1 in the nucleus, cytoplasmic AGO1 loading of mobile miRNAs is a key step regulated by microtubules to promote the range of miRNA"s cell-to-cell movement.

plant biology↗