Biology subjects

Hopkins, J.

Publications and source records attributed to Hopkins, J..

12 recordsLinked to original sources

bioRxiv2026

A combined program of induced stemness and differentiation in response to interferon gamma drives acute myeloid leukaemia growth

How inflammation shapes acute myeloid leukaemia (AML) has come under scrutiny, as it may explain the disease resistance to immunotherapy approaches. IFNg has emerged as a key cytokine with paradoxical roles in suppressing and supporting AML growth, and the fundamental question of how leukemic stem cells (LSCs) respond to IFNg and whether IFNg signaling influences LSC quiescence and their capacity to regenerate disease over the long term remains unanswered. Here, we study primary human AML cells and murine models and combine bioinformatics analyses and functional assays to show that AML hierarchical heterogeneity is responsive to IFNg challenge. We uncover that IFNg triggers parallel stemness and differentiation programs; HSC/MPP-like cells enter deeper stemness, associated with quiescence and high leukaemia regeneration potential, while the surviving pool of progenitor-like cells divides faster but produces progeny that is quickly lost. Finally, with murine models we show that exposure to inflammation in vivo results in only transient impairment of leukaemia propagating capacity. This mechanism is driven by a previously unrecognised intraclonal fate bifurcation, relevant for the development of more effective therapeutic approaches.

cancer biology↗

bioRxiv2024

Formation of an Enduring Ensemble of Accumbens Neurons Leads to Prepotent Seeking for Cocaine Over Natural Reward Cues

Neuronal activity in the nucleus accumbens core (NAcore) is necessary for reward-seeking behaviors. We hypothesized that the differential encoding of natural and drug rewards in the NAcore contributes to substance use disorder. We leveraged single-cell calcium imaging of dopamine D1- and D2-receptor-expressing medium spiny neurons (MSNs) in the NAcore of mice to examine differences between sucrose and cocaine rewarded (self-administration) and unrewarded (abstinent and cue-induced) seeking. Activity was time-locked to nose-poking for reward, clustered, and compared between sucrose and cocaine. Only in cocaine-trained mice were excited D1-MSNs securely stable, capable of decoding nose-poking in all rewarded and unrewarded sessions and correlated with the intensity of nose-poking for unrewarded seeking. Furthermore, D1-MSNs formed a stable ensemble predictive of seeking behavior after extended cocaine, but not sucrose abstinence. The excited D1-MSN ensemble uniquely drives cue-induced cocaine seeking and may contribute to why drug seeking is prepotent over natural reward seeking in cocaine use disorder.

neuroscience↗

bioRxiv2023

Genomic structures and regulation patterns at HPV integration sites in cervical cancer

Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.

genomics↗

bioRxiv2022

Structure and mechanism of LARGE1 matriglycan polymerase

Matriglycan is a linear polysaccharide of alternating xylose and glucuronate that binds extracellular matrix proteins and acts as a receptor for Lassa fever virus. LARGE1 synthesizes matriglycan on dystroglycan and mutations in LARGE1 cause muscular dystrophy with abnormal brain development. However, the mechanism of matriglycan polymerization by LARGE1 is unknown. Here, we report the cryo-EM structure of LARGE1. We show that LARGE1 functions as a dimer to polymerize matriglycan by alternating activities between the xylose transferase domain on one protomer and the glucuronate transferase domain on the other protomer. Biochemical analyses using a recombinant Golgi form of dystroglycan reveal that LARGE1 polymerizes matriglycan processively. Our results provide mechanistic insights into LARGE1 function and may facilitate novel therapeutic strategies for treating neuromuscular disorders or arenaviral infections. One-Sentence SummaryDimeric LARGE1 processively polymerizes matriglycan on dystroglycan using orthogonal active sites on alternate protomers.

biochemistry↗

bioRxiv2021

OPTN limits herpes stromal keratitis severity and demyelination through negative regulation of IL-17 and hyperinflammatory T-cell response

Herpes stromal keratitis (HSK) is a result of the inflammatory sequelae following primary and recurrent Herpes simplex virus type-1 (HSV-1) infections. This pathology is known to be mediated by immunopathogenic T cell responses against viral antigens, however most individuals infected with HSV-1 never exhibit signs of this immunopathology. Recent studies have identified the host restriction factor, optineurin (OPTN), as an inhibitor of viral spread in the central nervous system, protecting hosts from viral encephalopathy. In an HSV-1 corneal infection mouse model on OPTN knockout mice, we assess the contribution of OPTN to ameliorating the clinical manifestations of HSK. We identify that OPTN protects the host from loss of ocular and whisker sensitivity and opacification of the cornea. scRNA-seq of the trigeminal ganglion (TG) reveals that transcription changes to the peripheral neurons and immune cell populations drive the expression of Il-17A in CD4 and CD8 T cells, as well as increased infiltration of T cells into the TG. This leads to demyelination and the observed HSK pathology.

immunology↗

bioRxiv2021

Chemogenetic Inhibition of Corticostriatal Circuits Reduces Cued Reinstatement of Methamphetamine Seeking

Methamphetamine (meth) causes enduring changes within the medial prefrontal cortex (mPFC) and the nucleus accumbens (NA). Projections from the mPFC to the NA have a distinct dorsal-ventral distribution, with the prelimbic (PL) mPFC projecting to the NAcore, and the infralimbic (IL) mPFC projecting to the NAshell. Inhibition of these circuits has opposing effects on cocaine relapse. Inhibition of PL-NAcore reduces cued reinstatement of cocaine seeking and IL-NAshell inhibition reinstates cocaine seeking. Meth, however, exhibits a different profile, as pharmacological inhibition of either the PL or IL decrease cued reinstatement of meth-seeking. The potentially opposing roles of the PL-NAcore and IL-NAshell projections remain to be explored in the context of cued meth seeking. Here we used an intersectional viral vector approach that employs a retrograde delivery of Cre from the NA and Cre-dependent expression of DREADD in the mPFC, in both male and female rats to inhibit or activate these parallel pathways. Inhibition of the PL-NAcore circuit reduced cued reinstatement of meth seeking under short and long-access meth self-administration and after withdrawal with and without extinction. Inhibition of the IL-NAshell also decreased meth cued reinstatement. Activation of the parallel circuits was without an effect. These studies show that inhibition of the PL-NAcore or the IL-NAshell circuits can inhibit reinstated meth seeking. Thus, the neural circuitry mediating cued reinstatement of meth seeking is similar to cocaine in the dorsal, but not ventral, mPFC-NA circuit.

neuroscience↗

bioRxiv2020

Dynamic regulation of hierarchical heterogeneity in Acute Myeloid Leukemia serves as a tumor immunoevasion mechanism

Acute Myeloid Leukemia, a hematological malignancy with poor clinical outcome, is composed of hierarchically heterogeneous cells. We examine the contribution of this heterogeneity to disease progression in the context of anti-tumor immune responses and investigate whether these responses regulate the balance between stemness and differentiation in AML. Combining phenotypic analysis with proliferation dynamics and fate-mapping of AML cells in a murine AML model, we demonstrate the presence of a terminally differentiated, chemoresistant population expressing high levels of PDL1. We show that PDL1 upregulation in AML cells, following exposure to IFN{gamma} from activated T cells, is coupled with AML differentiation and the dynamic balance between proliferation, versus differentiation and immunosuppression, facilitates disease progression in the presence of immune responses. This microenvironment-responsive hierarchical heterogeneity in AML may be key in facilitating disease growth at the population level at multiple stages of disease, including following bone marrow transplantation and immunotherapy.

cancer biology↗

bioRxiv2020

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we follow heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

cell biology↗

bioRxiv2020

Cancer-specific associations of driver genes with immunotherapy outcome

Genomic features such as microsatellite instability (MSI) and tumor mutation burden (TMB) are predictive of immune checkpoint inhibitor (ICI) response. However, they do not account for the functional effects of specific driver gene mutations, which may alter the immune microenvironment and influence immunotherapy outcomes. By analyzing a multi-cancer cohort of 1,525 ICI-treated patients, we identified 12 driver genes in 6 cancer types associated with treatment outcomes, including genes involved in oncogenic signaling pathways (NOTCH, WNT, FGFR) and chromatin remodeling. Mutations of PIK3CA, PBRM1, SMARCA4, and KMT2D were associated with worse outcomes across multiple cancer types. In comparison, genes showing cancer-specific associations--such as KEAP1, BRAF, and RNF43--harbored distinct variant types and variants, some of which were individually associated with outcomes. In colorectal cancer, a common RNF43 indel was a putative neoantigen associated with higher immune infiltration and favorable ICI outcomes. Finally, we showed that selected mutations were associated with PD-L1 status and could further stratify patient outcomes beyond MSI or TMB, highlighting their potential as biomarkers for immunotherapy.

genomics↗

bioRxiv2019

The spread of chloramphenicol-resistant Neisseria meningitidis in Southeast Asia

Invasive disease caused by Neisseria meningitidis is a significant health concern globally, but our knowledge of the prevailing serogroups, antimicrobial susceptibility patterns, and genetics of N. meningitidis in Southeast Asia is limited. Chloramphenicol resistance in N. meningitidis has rarely been reported, but was first described in isolates from Vietnam in 1998. Using whole-genome sequencing of meningococcal isolates from 18 patients collected between 2007 and 2018 from diagnostic microbiology laboratories in Cambodia, Thailand and the Lao Peoples Democratic Republic (Laos), of which eight were non-susceptible to chloramphenicol, we report the spread of this chloramphenicol-resistant lineage of N. meningitidis across Southeast Asia. Strains resistant to penicillin, tetracycline, and ciprofloxacin were also observed, including a chloramphenicol-resistant strain from the previously-described lineage which has acquired penicillin and ciprofloxacin resistance, and most isolates were of serogroup B. This study suggests that chloramphenicol-resistant N. meningitidis is more widespread than previously thought.

microbiology↗

bioRxiv2019

Dissociation of solid tumour tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase associated stress responses

BackgroundSingle-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6{degrees}C) protease and collagenase (37{degrees}C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines.\n\nResultsWe observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically \"dying\" cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. From the contrast between tissue protease dissociation at 37{degrees}C or 6{degrees}C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37{degrees}C), which are minimized by dissociation with a cold active protease (6{degrees}C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods.\n\nConclusionsThe method and conditions of tumour dissociation influence cell yield and transcriptome state and are both tissue and cell type dependent. Interpretation of stress pathway expression differences in cancer single cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

genomics↗

bioRxiv2018

Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus-2 Release from Cells

Herpes simplex virus-2 (HSV-2) can productively infect many different cell types of human and non-human origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L in HSV-2 release from cells. In vaginal epithelial cells and other cell lines tested, HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-{kappa}B nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface, and thus facilitate virus release from cells.\n\nImportanceGenital infections by HSV-2 represent one of the most common sexually transmitted viral infections. The virus causes painful lesions, and sores around the genitals or rectum. Intermittent release of the virus from infected tissues during sexual activities is the most common cause of transmission. At the molecular level, cell surface heparan sulfate (HS) is known to provide attachment sites for HSV-2. While the removal of HS during HSV-1 release has been shown, not much is known about the host factors and their regulators that contribute to HSV-2 release from natural target cell types. Here we suggest a role for the host enzyme heparanase in HSV-2 release. Our work reveals that in addition to the regulation of transcription by NF-{kappa}B, HPSE is also regulated post-translationally by cathepsin L and that inhibition of heparanase activity directly affects HSV-2 release. We provide unique insights into the host mechanisms controlling HSV-2 egress and spread.

microbiology↗