Biology subjects

Carter, E.

Publications and source records attributed to Carter, E..

9 recordsLinked to original sources

bioRxiv2024

Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches.

Aggregating immune cells within perivascular niches (PVN) can regulate tissue immunity in infection, autoimmunity and cancer. How cells are assembled at PVNs and the activation signals imparted within remain unclear. Here, we integrate dynamic time-resolved in vivo imaging with a novel spatially-resolved platform for microanatomical interrogation of transcriptome, immune phenotype and inflammatory mediators in skin PVNs. We uncover a complex positive-feedback loop within CXCL10+ PVNs that regulates myeloid and Th1 cell positioning for exchange of critical signals for Th1 activation. Th1 cells spend [~]24h in the PVN, receiving initial peripheral activation signals, before redeploying to the inflamed dermal parenchyma. Niche-enriched, CCR2-dependent myeloid cells were critical for Th1 IFN{gamma}-production. In turn, PVN instructional signals enabled Th1s to orchestrate PVN assembly by CXCR2-dependent intra-tissue myeloid cell aggregation. The results reveal a critical tissue organizing role for Th1s, gained rapidly on tissue entry, that could be exploited to boost regional immunity. HIGHLIGHTSO_LIPerivascular niche (PVN): myeloid hubs in inflamed mouse and healthy human skin C_LIO_LITh1 cells enter, get activated, and leave the PVN within first 24h of tissue entry C_LIO_LIAntigen-specific signals in the PVN promote the tissue organizing functions of Th1s C_LIO_LITh1 cells assemble the PVN via CXCR2-dependent myeloid cell aggregation C_LI

immunology↗

bioRxiv2024

A Water Relaxation Atlas for Age and Region specific Metabolite Concentration Correction

Metabolite concentration estimates from Magnetic Resonance Spectroscopy (MRS) are typically quantified using water referencing, correcting for relaxation-time differences between metabolites and water. One common approach is to correct the water reference signal for differential relaxation within three tissue compartments (gray matter, white matter, and cerebrospinal fluid) using fixed literature values. However, water relaxation times (T1 and T2) vary between brain locations, in pathology, and with age. MRS studies, even those measuring metabolite levels across the lifespan, often ignore these effects, because of a lack of reference data. The purpose of this study is to develop a water relaxometry atlas and to integrate location- and age-appropriate relaxation values into the MRS analysis workflow. 101 volunteers (51 men, 50 women; [~]10 male and 10 female participants per decade (from the 20s to 60s), were recruited. T1-weighted MPRAGE images ((1-mm)3 isotropic resolution) were acquired. Whole-brain water T1 and T2 measurements were made with DESPOT ((1.4 mm)3 isotropic resolution). T1 and T2 maps were registered to the JHU MNI-SS/EVE atlas using affine and LDDMM transformation. The atlass 268 parcels were reduced to 130 by combining homologous parcels. Mean T1 and T2 values were calculated for each parcel in each subject. Linear models of T1 and T2 as functions of age were computed, using age - 30 as the predictor. Reference atlases of "age-30-intercept" and age-slope for T1 and T2 were generated. The atlas-based workflow was integrated into Osprey, which co-registers MRS voxels to the atlas and calculates location- and age-appropriate water relaxation parameters for quantification. The water relaxation aging atlas revealed significant regional and tissue differences in water relaxation behavior across adulthood. Using location- and subject-appropriate reference values in the MRS analysis workflow removes a current methodological limitation and is expected to reduce quantification biases associated with water-referenced tissue correction, especially for studies of aging.

neuroscience↗

bioRxiv2024

Id1, Spp1 and Pak3 are biomarkers of Smad4 and TGF-β1 dependency in conditional intestinal adenoma, organoids and colorectal cancer.

TGF-{beta} ligand activation suppresses cell growth yet can paradoxically and potently promote cancer invasion and metastasis depending on downstream pathway mutational context. Here, we evaluated the basis of this observation in conditional murine intestinal adenoma models with and without loss of Mothers against decapentaplegic homolog 4 (Smad4), with the aim of identifying TGF-{beta}-BMP-SMAD4 pathway dependent gene expression biomarkers for translational application. Conditional Lgr5-CreERT2 activation in Apcfl/flSmad4fl/flresulted in adenoma formation with recombined homozygote floxed alleles (Apc{Delta}/{Delta}Smad4{Delta}/{Delta}). The adenoma phenotype was discordant, with a reduced small intestinal adenoma burden yet development of large non-metastatic caecal adenoma with nuclear localisation of phospho-Smad2/3. Derived Apc{Delta}/{Delta}Smad4{Delta}/{Delta} adenoma organoids resisted TGF-{beta}1 dose dependent growth arrest and cell death (IC50 534pM) compared to Apc{Delta}/{Delta}Smad4+/+ (IC 24pM). TGF-{beta}1 (390pM) modified adenoma mRNA expression (bulk RNA-Seq) most significantly for Id1low and Spp1high in Apc{Delta}/{Delta}Smad4{Delta}/{Delta}. Single cell RNAseq of caecal adenoma identified expansion of Lgr5low, Pak3high and Id1low progenitor populations in Apc{Delta}/{Delta}Smad4{Delta}/{Delta}. Of the 76 Smad4 and TGF-{beta}1 dependent genes identified in adenoma organoids, 7 human equivalent genes were also significantly differentially expressed in colorectal cancer, including ID1low, SPP1high and PAK3high that also correlated with poorer survival (TCGA cohorts). Murine conditional models identified Smad4 loss of function mRNA expression biomarkers that require further evaluation as functional classifiers of colorectal cancer subtypes.

cancer biology↗

bioRxiv2023

Insight into the emerging insect to human pathogen Photorhabdus revealing geographic differences in immune cell tropism.

Photorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide, however actual clinical infections have so far only been identified in North America, Australia and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells, based on the geographical site of isolation. In this study we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. We present findings from the clinical isolates from Australia (Kingscliff) and North America (ATCC43949), and soil borne nematode isolates from Thailand (PB68) and Northern Europe non-clinical P. asymbiotica genospecies HIT and JUN. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells. Infection assays were done using both cultured cell lines (THP-1, CHO and HEK cells) and also primary immune cells, Peripheral Blood Mononuclear cells (PBMCs) isolated from human blood. Here for the first time, we show how P. asymbiotica, selectively infects certain immune cells while avoiding others, and that infectivity varies depending on growth temperature. We also show that the infected immune cells vary depending on the geographical location a strain is isolated from, and that the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture.

microbiology↗

bioRxiv2022

Extracellular ATP drives pancreatic cancer cell invasion via purinergic receptor-integrin interactions

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet clinical need. Given the elevated ATP levels seen in PDAC, the purinergic axis represents an attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, it plays essential roles in fibrosis, inflammation response and immune function. We have analysed the PDAC purinome using publicly available databases to discern which members may impact patient survival. We identified P2RY2 to be the purinergic gene with the strongest association to hypoxia, the highest cancer cell specific expression and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification or pharmacological strategies we identify the mechanism of this ATP-driven invasion to require direct protein-protein interactions between P2Y2 and V integrins. Using DNA-PAINT super-resolution fluorescence microscopy, we found that P2Y2 regulates the amount and distribution of integrin V in the plasma membrane. This work highlights a novel GPCR-integrin interaction in cancer invasion and its potential for therapeutic targeting.

cancer biology↗

bioRxiv2022

Low HER2 enables dedifferentiation and transformation of normal breast epithelial cells via chromatin opening

Overexpression of the human epidermal growth factor 2 (HER2) protein in breast cancer patients is a predictor of poor prognosis and resistance to therapies. Despite significant advances in the development of targeted therapies and improvements in the 5-year survival rate of metastatic HER2-positive breast cancer patients, a better understanding of the disease at an early stage is needed to prevent its progression. Here, we used an inducible breast cancer transformation system that allows investigation of early molecular changes at high temporal resolution. HER2 overexpression to similar levels as those observed in a subtype of HER2 positive breast cancer patients induced transformation of MCF10A cells and resulted in gross morphological changes, increased anchorage-independent growth of cells, and altered transcriptional programme of genes associated with oncogenic transformation. Global phosphoproteomic analysis during the first few hours of HER2 induction predominantly detected an increase in protein phosphorylation. Intriguingly, this correlated with a wave of chromatin opening, as measured by ATAC-seq on acini isolated from 3D cell culture. We observed that HER2 overexpression leads to reprogramming of many distal regulatory regions and promotes reprogramming-associated heterogeneity. We found that a subset of cells acquired a dedifferentiated breast stem-like phenotype, making them likely candidates for malignant transformation. Our data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance and increased chromatin accessibility, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not displaying a stem-like phenotype. Our data provide a discovery platform for signalling to chromatin pathways in HER2-driven cancers, offering an opportunity for biomarker discovery and identification of novel drug targets.

cancer biology↗

bioRxiv2021

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype, and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A, and recorded up-regulated ISG expression and interferon alpha protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy. SummaryDominant-negative mutations in ATAD3A, a ubiquitously expressed mitochondrial protein, cause mitochondrial DNA-dependent up-regulation of type I interferon signaling in the context of neurological disease and autoimmunity, thereby defining a novel type I interferonopathy.

immunology↗

bioRxiv2020

Inferring brain-wide interactions using data-constrained recurrent neural network models

Behavior arises from the coordinated activity of numerous anatomically and functionally distinct brain regions. Modern experimental tools allow unprecedented access to large neural populations spanning many interacting regions brain-wide. Yet, understanding such large-scale datasets necessitates both scalable computational models to extract meaningful features of inter-region communication and principled theories to interpret those features. Here, we introduce Current-Based Decomposition (CURBD), an approach for inferring brain-wide interactions using data-constrained recurrent neural network models that directly reproduce experimentally-obtained neural data. CURBD leverages the functional interactions inferred by such models to reveal directional currents between multiple brain regions. We first show that CURBD accurately isolates inter-region currents in simulated networks with known dynamics. We then apply CURBD to multi-region neural recordings obtained from mice during running, macaques during Pavlovian conditioning, and humans during memory retrieval to demonstrate the widespread applicability of CURBD to untangle brain-wide interactions underlying behavior from a variety of neural datasets.

neuroscience↗

bioRxiv2019

Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, we introduced Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis performed on the soluble ectodomain BG505 SOSIP Env trimers, cell-surface expressed BG505 full-length trimers and single-molecule Forster Resonance Energy Transfer (smFRET) performed on the full-length virion-bound Env confirmed that these mutations prevented CD4-induced transitions of the HIV-1 Env. Structural analysis by single-particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins revealed rearrangements in the gp120 topological layer contacts with gp41. Specifically, a conserved tryptophan at position 571 (W571) was displaced from its typical pocket at the interface of gp120 topological layers 1 and 2 by lysine 567, disrupting key gp120-gp41 contacts and rendering the Env insensitive to CD4 binding. Vector based analysis of closed Env SOSIP structures revealed the newly designed trimers exhibited a quaternary structure distinct from that typical of SOSIPs and residing near a cluster of Env trimers bound to vaccine-induced fusion peptide-directed antibodies (vFP Mabs). These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.

biochemistry↗