Biology subjects

Reed, C.

Publications and source records attributed to Reed, C..

10 recordsLinked to original sources

bioRxiv2025

Chronic Ethanol Drinking Alters Medial Prefrontal Cortex and Nucleus Accumbens Astrocyte Translatome and Extracellular Matrix Glycosaminoglycans

Alcohol Use Disorder is a leading preventable cause of morbidity and mortality, yet knowledge of mechanisms driving ethanol-related neuroplasticity remains incomplete. While research has traditionally focused on neuronal signaling, emerging evidence implicates astrocytes in addiction-related adaptations. Here, we investigated the astrocyte-specific molecular consequences of chronic ethanol consumption in the prefrontal cortex and nucleus accumbens, two brain regions critical for executive control and reward processing. Using Translating Ribosome Affinity Purification RNA-seq and bulk RNA-seq in Aldh1l1-EGFP/Rpl10a mice, expressing an EGFP tag on astrocyte ribosomes, we identified hundreds of differentially translated astrocytic genes following chronic continuous two-bottle choice ethanol drinking. Sex-specific analyses revealed greater astrocytic changes in the female PFC and male NAc. Pathway enrichment highlighted extracellular matrix remodeling, synaptic signaling, mitochondrial function, and immune-related pathways. Analyses of individual drinking levels further demonstrated distinct correlations between ethanol intake and astrocytic translation. The major components of the brain extracellular matrix are chondroitin sulfate proteoglycans, produced primarily by astrocytes and covalently bound to chondroitin sulfate glycosaminoglycan chains. Complementary mass spectrometry/liquid chromatography analyses of chondroitin sulfate, heparan sulfate, and hyaluronic acid glycosaminoglycan disaccharides revealed ethanol-induced alterations in chondroitin sulfate glycosaminoglycan sulfation patterns, with additional baseline differences identified between selectively bred high- and low-ethanol preference lines. Together, these findings indicate that astrocytes undergo profound sex- and region-specific adaptations to chronic ethanol, implicating extracellular matrix and glycosaminoglycan remodeling as key risk-factors for and mediators of chronic ethanol-related neuroplasticity.

neuroscience↗

bioRxiv2025

Genomic and Behavioral Signatures of Selection for Ethanol Preference from the Heterogeneous Stock Collaborative Cross Mice: The Central Nucleus of the Amygdala

Alcohol use disorder (AUD) is a complex disease with heritability of [~]0.5, indicating genetic and non-genetic factors contribute to risk. Identifying gene expression networks contributing to risk using post-mortem human brain tissue has the limitation of conflating risk for AUD with consequences of alcohol use. We leveraged mice selectively bred for differential ethanol preference from a highly genetically diverse population to overcome this limitation. Ethanol intake was highly correlated with preference, high-preferring (HP) mice consumed more sweet-but not bitter-tasting solutions compared to low-preferring (LP) mice, and the lines did not differ in rate of ethanol elimination. Adult, ethanol-naive HP and LP mice contributed tissue from the central nucleus of the amygdala (CeA), a region critical to ethanol preference and intake. Single-nuclei and bulk RNA sequencing data were used to identify cell types and transcriptome changes related to selective breeding for differential risk for ethanol preference. Single nuclei analysis identified populations of inhibitory ([~]48% of cells) and excitatory ([~]23%) neurons, and non-neuronal ([~]29%) cells, but no differences in cell-type composition or gene expression were identified between the lines. Bulk CeA analysis identified differences between the lines for: (1) gene expression (2996 genes), (2) expression variability (426 genes), and (3) wiring (407 significant gene-gene correlations). Overall, lower variance was found in the HP line. Reduced gene-gene correlation, also found in HP mice, suggested that selection for high preference induced changes in transcriptional regulation resulting in reduced connectivity, specific to gene networks enriched in markers for inhibitory neurons expressing Isl1 and Tac1.

genomics↗

bioRxiv2023

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria

The wobble bases of tRNAs that decode split codons are often heavily modified. In Bacteria tRNAGlu, Gln, Asp contain a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the installation of this modification. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the Radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathways intermediates in regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. The occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in nature. ImportanceThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in Bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile Radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

microbiology↗

bioRxiv2023

A transposase-derived gene required for human brain development

DNA transposable elements and transposase-derived genes are present in most living organisms, including vertebrates, but their function is largely unknown. PiggyBac Transposable Element Derived 5 (PGBD5) is an evolutionarily conserved vertebrate DNA transposase-derived gene with retained nuclease activity in human cells. Vertebrate brain development is known to be associated with prominent neuronal cell death and DNA breaks, but their causes and functions are not well understood. Here, we show that PGBD5 contributes to normal brain development in mice and humans, where its deficiency causes disorder of intellectual disability, movement, and seizures. In mice, Pgbd5 is required for the developmental induction of post-mitotic DNA breaks and recurrent somatic genome rearrangements. In the brain cortex, loss of Pgbd5 leads to aberrant differentiation and gene expression of distinct neuronal populations, including specific types of glutamatergic neurons, which explains the features of PGBD5 deficiency in humans. Thus, PGBD5 might be a transposase-derived enzyme required for brain development in mammals. One-Sentence SummaryPiggyBac Transposable Element Derived 5 (PGBD5) is required for brain development in humans and mice through genetic and epigenetic mechanisms.

neuroscience↗

bioRxiv2023

Control of working memory maintenance by theta-gamma phase amplitude coupling of human hippocampal neurons

Retaining information in working memory (WM) is a demanding process that relies on cognitive control to protect memoranda-specific persistent activity from interference. How cognitive control regulates WM storage, however, remains unknown. We hypothesized that interactions of frontal control and hippocampal persistent activity are coordinated by theta-gamma phase amplitude coupling (TG-PAC). We recorded single neurons in the human medial temporal and frontal lobe while patients maintained multiple items in WM. In the hippocampus, TG-PAC was indicative of WM load and quality. We identified cells that selectively spiked during nonlinear interactions of theta phase and gamma amplitude. These PAC neurons were more strongly coordinated with frontal theta activity when cognitive control demand was high, and they introduced information-enhancing and behaviorally relevant noise correlations with persistently active neurons in the hippocampus. We show that TG-PAC integrates cognitive control and WM storage to improve the fidelity of WM representations and facilitate behavior.

neuroscience↗

bioRxiv2021

INO-4800 DNA Vaccine Induces Neutralizing Antibodies and T cell Activity Against Global SARS-CoV-2 Variants

Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with broadened host specificity, pathogenicity, and immune evasion to vaccine induced immunity. Here we compared humoral and cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFN{gamma} T cell responses were fully maintained against all variants tested.

immunology↗

bioRxiv2019

Genomic interrogation of the burden and transmission of multidrug-resistant pathogens within and across hospital networks

BackgroundMultidrug-resistant organisms (MDROs) disproportionately affect hospitalized patients due to the combination of comorbidities, frequent antimicrobial use, and in-hospital MDRO transmission. Identification of MDRO transmission by hospital microbiology laboratories is difficult due to limitations of existing typing methods.\n\nMethodsWe conducted a prospective multicenter genomics implementation study (8 hospitals, 2800 beds) from 24th April to 18th June 2017 in Melbourne, Australia. Clinical and screening isolates from hospital inpatients were collected for six MDROs (vanA VRE, MRSA, ESBL E. coli [ESBL-Ec] and Klebsiella pneumoniae [ESBL-Kp], and carbapenem-resistant Pseudomonas aeruginosa [CRPa] and Acinetobacter baumannii [CRAb]), sequenced (Illumina NextSeq) and analyzed using open-source tools. MDRO transmission was assessed by genomics (core SNP phylogeny, grouped by species and ST) and compared to epidemiologic data.\n\nResults408 isolates were collected from 358 patients; 47.5% were screening isolates. ESBL-Ec was most common (52.5%), then MRSA (21.6%), vanA VRE (15.7%) and ESBL-Kp (7.6%).\n\nWe define the transmission rate for each MDRO by genomics and epidemiology; 31.6% of all study patients had potential genomic links to other study isolates; 86% of these were confirmed by epidemiologic links (probable or possible transmission). The highest transmission rates occurred with vanA VRE (88.4% of patients).\n\nConclusionsCombining genomics with high-quality epidemiologic data gives substantial insights into the burden and distribution of critical MDROs in hospitals, including in-hospital transmission. By defining transmission rates by genomics, we hope to enable comparisons over time and between sites, and introduce this as a new outcome measure to assess the efficacy of infection control interventions.

genomics↗

bioRxiv2017

Therapeutic targeting of PGBD5-induced DNA repair dependency in pediatric solid tumors

Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have now found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma and Ewing sarcoma, express an active DNA transposase PGBD5 that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we found that mouse and human cells deficient in non-homologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity-deficient mutant, was sufficient to induce hypersensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment, and underwent apoptosis in both dividing and G1 phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against the majority of neuroblastoma, medulloblastoma, Ewing sarcoma and rhabdoid tumor cells tested, while sparing non-transformed human and mouse embryonic fibroblasts in vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient mice in vivo. This effect was potentiated by combined treatment with cisplatin, including significant anti-tumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.

cancer biology↗

bioRxiv2017

Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors

Genomic rearrangements are a hallmark of childhood solid tumors, but their mutational causes remain poorly understood. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as an enzymatically active human DNA transposase expressed in the majority of rhabdoid tumors, a lethal childhood cancer. Using assembly-based whole-genome DNA sequencing, we observed previously unknown somatic genomic rearrangements in primary human rhabdoid tumors. These rearrangements were characterized by deletions and inversions involving PGBD5-specific signal (PSS) sequences at their breakpoints, with some recurrently targeting tumor suppressor genes, leading to their inactivation. PGBD5 was found to be physically associated with human genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. We found that ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote penetrant cell transformation in vitro and in immunodeficient mice in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain, as well as end-joining DNA repair, and induced distinct structural rearrangements, involving PSS-associated breakpoints, similar to those found in primary human rhabdoid tumors. This defines PGBD5 as an oncogenic mutator and provides a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

cancer biology↗

bioRxiv2017

MEF2C phosphorylation is required for chemotherapy resistance in acute myeloid leukemia

HIGHLIGHTSO_LIMEF2C S222 phosphorylation is a specific marker of chemotherapy resistance in diagnostic AML patient specimens.\nC_LIO_LIMEF2C S222 phosphorylation is dispensable for normal hematopoiesis in mice, as established using genome editing in vivo, but is required for MLL-AF9 induced leukemogenesis.\nC_LIO_LIMARK kinases specifically phosphorylate MEF2C S222, potentiating its transcriptional activity.\nC_LIO_LIChemical inhibition of MARK-induced MEF2C phosphorylation overcomes chemotherapy resistance of and exhibits selectivity toxicity against MEF2C-activated human AML cells.\nC_LI\n\nSUMMARYIn acute myeloid leukemia, chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9. MEF2C phosphorylation was required for leukemia stem cell maintenance, and induced by MARK kinases in cells. Treatment with the selective MARK inhibitor MRT199665 caused apoptosis of MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.

cancer biology↗