Biology subjects

Stephens, R.

Publications and source records attributed to Stephens, R..

11 recordsLinked to original sources

bioRxiv2026

Partitioning of nuclear material into apoptotic fragments through establishment of asymmetric cell death morphology

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

cell biology↗

bioRxiv2025

IL-4 and TGF-β Regulate Inflammatory cytokines and Cellular Infiltration in the Lung in Mouse-adapted SARS-CoV-2 Infection

The pathology of severe COVID-19 is due to a hyperinflammatory immune response persisting after viral clearance. To understand how the immune response to SARS-CoV-2 is regulated to avoid severe COVID-19, we tested relevant immunoregulatory cytokines. TGF-{beta}, IL-10 and IL-4 were neutralized upon infection with mouse-adapted SARS-CoV-2 (CMA3p20), a model of mild disease; and lung inflammation was quantified by histology and flow cytometry at early and late time points. Mild weight loss, and lung inflammation including consolidation and alveolar thickening were evident 3 days post-infection (dpi) and inflammation persisted to 7 dpi. Coinciding with early monocytic infiltrates, CCL2 and granulocyte-colony stimulating factor (G-CSF) were transiently produced 3 dpi, while IL-12 and CCL5 persisted to 7 dpi, modeling viral and inflammatory phases of disease. Neutralization of TGF-{beta}, but not IL-10 or IL-4, significantly increased lung inflammatory monocytes and elevated serum but not lung IL-6. Neutralization of IL-4 prolonged weight loss and increased early perivascular infiltration without changing viral titer. Anti-IL-4 reduced expression of Arg1, a gene associated with alternative activation of macrophages. Neutralizing TGF-{beta} and IL-4 had differential effects on pathology after virus control. Lung perivascular infiltration was reduced 7 dpi by neutralization of IL-4 or TGF-{beta}, and peri-airway inflammation was affected by anti-TGF-{beta}, while alveolar infiltrates were not affected by either. Anti-IL-4 prolonged IL-12 to 7 dpi along with reduced IL-10 in lungs. Overall, the immunoregulatory cytokines TGF-{beta} and IL-4 dampen initial inflammation in this maSARS-CoV-2 infection, suggesting that promotion of immunoregulation could help patients in early stages of disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653138v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14c34org.highwire.dtl.DTLVardef@1322a8dorg.highwire.dtl.DTLVardef@8792aborg.highwire.dtl.DTLVardef@ae513e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells

Acquisition of specific cell shapes and morphologies is a central component of cell fate transitions. Although signaling circuits and gene regulatory networks that regulate pluripotent stem cell differentiation have been intensely studied, how these networks are integrated in space and time with morphological transitions and mechanical deformations to control state transitions remains a fundamental open question. Here, we focus on two distinct models of pluripotency, primed pluripotent stem cells and pre-implantation inner cell mass cells of human embryos to discover that cell fate transitions associate with rapid changes in nuclear shape and volume which collectively alter the nuclear mechanophenotype. Mechanistic studies in human induced pluripotent stem cells further reveal that these phenotypical changes and the associated active fluctuations of the nuclear envelope arise from growth factor signaling-controlled changes in chromatin mechanics and cytoskeletal confinement. These collective mechano-osmotic changes trigger global transcriptional repression and a condensation-prone environment that primes chromatin for a cell fate transition by attenuating repression of differentiation genes. However, while this mechano-osmotic chromatin priming has the potential to accelerate fate transitions and differentiation, sustained biochemical signals are required for robust induction of specific lineages. Our findings uncover a critical mechanochemical feedback mechanism that integrates nuclear mechanics, shape and volume with biochemical signaling and chromatin state to control cell fate transition dynamics.

cell biology↗

bioRxiv2024

TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7-/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

microbiology↗

bioRxiv2024

Sex differences in brain-behavior relationships in the first two years of life

BackgroundEvidence for sex differences in cognition in childhood is established, but less is known about the underlying neural mechanisms for these differences. Recent findings suggest the existence of brain-behavior relationship heterogeneities during infancy; however, it remains unclear whether sex underlies these heterogeneities during this critical period when sex-related behavioral differences arise. MethodsA sample of 316 infants was included with resting-state functional magnetic resonance imaging scans at neonate (3 weeks), 1, and 2 years of age. We used multiple linear regression to test interactions between sex and resting-state functional connectivity on behavioral scores of working memory, inhibitory self-control, intelligence, and anxiety collected at 4 years of age. ResultsWe found six age-specific, intra-hemispheric connections showing significant and robust sex differences in functional connectivity-behavior relationships. All connections are either with the prefrontal cortex or the temporal pole, which has direct anatomical pathways to the prefrontal cortex. Sex differences in functional connectivity only emerge when associated with behavior, and not in functional connectivity alone. Furthermore, at neonate and 2 years of age, these age-specific connections displayed greater connectivity in males and lower connectivity in females in association with better behavioral scores. ConclusionsTaken together, we critically capture robust and conserved brain mechanisms that are distinct to sex and are defined by their relationship to behavioral outcomes. Our results establish brain-behavior mechanisms as an important feature in the search for sex differences during development. Plain language summaryEarly childhood differences exist in mental processes and behavior between males and females. The brain-basis for these sex differences may arise in infancy. Indeed, small brain differences in infancy may contribute to major changes in cognitive ability throughout childhood. However, few studies have examined sex differences in brain functionality in infancy and their relationship to future behaviors in early childhood. In this study, we aimed to study this relationship by using sex differences in brain functional measures in neonate, 1, and 2-year-olds and 4-year behavioral outcomes. We identified six functional connections with robust brain-behavior sex differences. These connections were unique to frontal brain regions. Also, these connections were not specific to the brain and were only evident when associated with future behavior. In brief, our analysis shows distinct age-specific brain-behavior relationships in males and females in early childhood. This is helpful for a better understanding of brain-based prediction of behavior and informed intervention of future disorders and disabilities characterized by a sex bias. HighlightsO_LIMultiple linear regression was used to test the interaction between sex and early childhood resting-state functional connectivity on future behavioral scores C_LIO_LISix age-specific, intra-hemispheric functional connections displayed sex differences C_LIO_LIMost connections exist within prefrontal regions (with one connection in the temporal pole) C_LIO_LIFunctional connections are specific to brain-behavior relationships and not in brain connectivity alone C_LIO_LISex differences in brain-behavior relationships are robust at smaller sample sizes C_LI

neuroscience↗

bioRxiv2023

Boosting Live Malaria Vaccine with Cytomegalovirus Vector Can Prolong Immunity through Innate and Adaptive Mechanisms

Vaccines to persistent parasite infections have been challenging, and current iterations lack long-term protection. Cytomegalovirus (CMV) chronic vaccine vectors drive protection against SIV, tuberculosis and liver-stage malaria correlated with antigen-specific CD8 T cells with a Tem phenotype. This phenotype is likely driven by a combination of antigen-specific and innate adjuvanting effects of the vector, though these mechanisms are less well understood. Sterilizing immunity from live Plasmodium chabaudi vaccination lasts less than 200 days. While P. chabaudi-specific antibody levels remain stable after vaccination, the decay of parasite-specific T cells correlates with loss of challenge protection. Therefore, we enlisted murine CMV as a booster strategy to prolong T cell responses against malaria. To study induced T cell responses, we included P. chabaudi MSP-1 epitope B5 (MCMV-B5). We found that MCMV vector alone significantly protected against a challenge P. chabaudi infection 40-60 days later, and that MCMV-B5 was able to make B5-specific Teff, in addition to previously-reported Tem, that survive to the challenge timepoint. Used as a booster, MCMV-B5 prolonged protection from heterologous infection beyond day 200, and increased B5 TCR Tg T cell numbers, including both a highly-differentiated Tem phenotype and Teff, both previously reported to protect. B5 epitope expression was responsible for maintenance of Th1 and Tfh B5 T cells. In addition, the MCMV vector had adjuvant properties, contributing non-specifically through prolonged stimulation of IFN-{gamma}. In vivo neutralization of IFN-{gamma}, but not IL-12 and IL-18, late in the course of MCMV, led to loss of the adjuvant effect. Mechanistically, sustained IFN-{gamma} from MCMV increased CD8+ dendritic cell numbers, and led to increased IL-12 production upon Plasmodium challenge. In addition, neutralization of IFN-{gamma} before challenge reduced the polyclonal Teff response to challenge. Our findings suggest that, as protective epitopes are defined, an MCMV vectored booster can prolong protection through the innate effects of IFN-{gamma}. Authors summaryMalaria represents a challenging target for vaccination. This is in part because of the requirement for CD4 T cell immunity in addition to the standard B cell responses current vaccines induce. However, human malaria vaccine approaches thus far have limited longevity of protection due to decay of T cell responses. This includes the most advanced malaria vaccine, a virus-like particle expressing one recombinant liver-stage antigen (RTS,S), and liver-stage parasites attenuated by radiation (PfSPZ), as well as live vaccination with drug-treatment. Our work seeks to prolong this protection using MCMV, a promising vaccine vector known to promote CD8 T cell responses. We observed that boosting the live malaria vaccine with MCMV including a Plasmodium antigen led to longer protection from P. chabaudi parasitemia, and can be used to promote maintenance of antigen-specific CD4 T cells. In investigating the mechanisms of the MCMV booster, we found that the cytokine IFN-{gamma} is required for prolonged protection and enhances priming of the innate immune system for prolonged protection from malaria. Our research informs both the quest for a longer-lived malaria vaccine and that to understand mechanisms of protection from persistent infection.

immunology↗

bioRxiv2022

Effects of Low-level Persistent Infection on maintenance of immunity by CD4 T cell subsets and Th1 cytokines

CD4 T cells are required, along with antibodies, for complete protection from blood-stage infection with Plasmodium spp., which cause malaria. Without continuous exposure, as on emigration of people from endemic areas, protection from malaria decays. As in other persistent infections, low-level P. chabaudi protects the host from re-infection at two months post-infection, a phenomenon termed premunition. Premunition is correlated with T cell responses, rather than antibody levels. We previously showed that while both effector T cells (Teff) and memory T cells (Tmem) are present after infection, Teff protect better than Tmem. Here we studied T cell kinetics post-infection by labelling dividing Ifng+ T cells with BrdU in infected Ifng-reporter mice. A large drop in specific T cell numbers and Ifng+ cells upon clearance of parasite suggest a mechanism for decay of protection. Although protection decays, CD4 Tmem persist, including a highly-differentiated CD27- Effector Memory (Tem) subset that maintains some Ifng expression. In addition, pre-treatment of chronically-infected animals with neutralizing antibody to IFN-{gamma}, or clodronate liposomes before re-infection decrease premonition supporting a role for Th1-type immunity to re-infection. A pulse/chase experiment comparing chronically infected to treated animals showed that recently divided Ifng+ T cells, particularly IFN-{gamma}+TNF+IL-2- T cells, are promoted by persistent infection. These data suggest that low-level persistent infection reduces CD4+ Tmem survival and multi-functional Teff but promotes IFN-{gamma}+TNF+IL-2- Late Effector Memory and Terminally Differentiated Effector T cells and prolongs immunity.

immunology↗

bioRxiv2020

Structural basis of the human Scribble-Vangl2 association in health and disease

Scribble is a critical cell polarity regulator that has been shown to work as either an oncogene or tumor suppressor in a context dependent manner, and also impacts cell migration, tissue architecture and immunity. Mutations in Scribble lead to neural tube defects in mice and humans, which has been attributed to a loss of interaction with the planar cell polarity regulator Vangl2. We show that the Scribble PDZ domains 1, 2 and 3 are able to interact with the C-terminal PDZ binding motif of Vangl2 and have now determined crystal structures of these Scribble PDZ domains bound to the Vangl2 peptide. Mapping of mammalian neural tube defect mutations reveal that mutations located distal to the canonical PDZ domain ligand binding groove can not only ablate binding to Vangl2 but also disrupt binding to multiple other signaling regulators. Our findings suggest that PDZ-associated neural tube defect mutations in Scribble may not simply act in a Vangl2 dependent manner but as broad-spectrum loss of function mutants by disrupting the global Scribble-mediated interaction network.

cell biology↗

bioRxiv2019

RAS Internal Tandem Duplication Disrupts GAP-binding to Activate Oncogenic Signaling

Molecular testing of oncogenic RAS mutations in colorectal cancer (CRC) have led to increased identification of mutations in patients with CRC. NRAS-mutated CRC has not been well characterized because it is less common (<6%) than KRAS mutations. Here, we report a novel 10 amino acid internal tandem duplication (ITD) in NRAS, which disrupts the switch II domain, in a patient with widely disseminated CRC. Hotspot next generation sequencing of a brain metastasis identified the NRAS ITD and a TP53 missense mutation (p.P275F). Whole exome sequencing of the primary tumor and two metastatic lesions (lung and brain) confirmed that the NRAS ITD and TP53 mutation were conserved between the primary tumor and both metastatic tumors, and identified an additional pathogenic mutation in CSMD1 (a tumor suppressor gene). Structural biology and biochemical analyses demonstrated that the NRAS ITD prevented binding to GAP protein, leading to sustained RAS activation, increased interaction with RAF, and downstream MAPK activation. Additionally, we provide the first crystal structure of the RAS ITD. In conclusion, these studies indicate that the NRAS ITD was the probable primary driver mutation of this aggressive CRC. Identical or biologically similar ITDs in NRAS and KRAS may be rare drivers of CRC and other aggressive malignancies.

molecular biology↗

bioRxiv2019

Increased Th1 bias in memory T cells corresponds with protection from reinfection in Plasmodium infection, and is regulated by T cell-intrinsic STAT3

Hybrid Th1/Tfh cells (IFN-{gamma}+IL-21+CXCR5+) predominate in response to persistent infections; however, molecular regulation of their function is poorly defined. In infection with Plasmodium spp, an IFN-{gamma}+ T helper-1 (Th1) response controls initial parasitemia, while antibody and IL-21+CXCR5+ T follicular helper (Tfh) function effect final clearance. Here, we found that CD4-intrinsic Bcl6, Blimp-1 and STAT3 all regulate T-bet expression, which controls IFN-{gamma} expression. While Bcl6 and Blimp-1 regulate the level of CXCR5, only T-bet and STAT3 affected the functional bias of the Th1/Tfh phenotype. Infected mice with STAT3-deficient T cells produced less antibody, and more IFN-{gamma}+IL-21-CXCR5lo T cells, significantly increasing protection from re-infection. Conversely, reduced Th1 bias in re-infected T-bet KO was reflected in prolonged secondary parasitemia. In summary, each feature of hybrid Th1/Tfh population in Plasmodium infection is uniquely regulated and the cytokine bias of memory T cells can be modified to enhance the effectiveness of the response.

immunology↗

bioRxiv2019

Harnessing patient-specific response dynamics to optimize evolutionary therapies for metastatic clear cell renal cell carcinoma - Learning to adapt

Renal cell carcinoma (RCC) is one of the ten most common and lethal cancers in the United States. Tumor heterogeneity and development of resistance to treatment suggest that patient-specific evolutionary therapies may hold the key to better patients prognosis. Mathematical models are a powerful tool to help develop such strategies; however, they depend on reliable biomarker information. In this paper, we present a dynamic model of tumor-immune interactions, as well as the treatment effect on tumor cells and the tumor-immune environment. We hypothesize that the neutrophil-to-lymphocyte ratio (NLR) is a powerful biomarker that can be used to predict an individual patients response to treatment. Using randomly sampled virtual patients, we show that the model recapitulates patient outcomes from clinical trials in RCC. Finally, we use in silico patient data to recreate realistic tumor behaviors and simulate various treatment strategies to find optimal treatments for each virtual patient.

cancer biology↗