Biology subjects

Fitzgerald, D.

Publications and source records attributed to Fitzgerald, D..

17 recordsLinked to original sources

bioRxiv2026

Neurodevelopment of Corticostriatal Circuits and Risk for Alcohol Use During the Transition from Adolescence to Adulthood

Adolescence and early adulthood are marked by rapid neurobehavioral development in reward and decision-making processes, coinciding with the initiation and escalation of alcohol use. While adolescent and young adulthood alcohol initiation is not atypical, adult trajectories diverge: some individuals reduce or discontinue use, whereas others escalate to more frequent or problematic patterns leading to substance use disorders. Corticostriatal circuits, including the ventral striatum (nucleus accumbens; NAcc) and dorsal striatum (caudate and putamen), support reward processing, goal-directed behavior, and habit formation, and are thought to contribute to distinct stages of alcohol use. Yet, how the normative maturation of these circuits relates to alcohol initiation and the transition to habitual consumption remains unclear. We used data from the National Consortium on Alcohol and NeuroDevelopment in Adolescence and Adulthood (NCANDA-A) cohort (822 participants, baseline ages 12 - 22 years old, 1 - 9 visits per participant, 4,356 total visits), a large multisite longitudinal neuroimaging sample spanning adolescence to young adulthood. We observed that rsfMRI functional connectivity (FC) patterns varied systematically across striatal subdivisions: NAcc FC followed an inverted U-shaped trajectory, peaking during adolescence; while caudate and putamen FC showed monotonic decreases with age. Adolescent peak NAcc connectivity was associated with alcohol use initiation, while a lack of normative decrease in putamen connectivity was linked to more frequent alcohol use in adulthood. Together, results suggest that the maturation of reward processing circuitry may support alcohol initiation, while a lack of habit system specialization may contribute to continued alcohol use, with potential implications for the timing of interventions aimed at limiting at-risk drinking.

neuroscience↗

bioRxiv2025

Developmental variation in dopamine neurobiology, neurocognitive functioning, and impulsivity shape substance use trajectories in youth

Theoretical neurodevelopmental models implicate increases in dopamine (DA) function and limitations in neurocognitive control in risk-taking behavior, including substance use, during the transition from adolescence to adulthood. However, developmental relationships between DA, neurocognitive control, and the emergence of substance use are poorly understood. Here, we tested the role of basal ganglia tissue iron, reflecting DA neurophysiology, as well as impulsivity and inhibitory control in longitudinal developmental trajectories of substance use. We leveraged the National Consortium on Alcohol and NeuroDevelopment in Adolescence and Adulthood (NCANDA-A) cohort, a large, multisite longitudinal neuroimaging study of 807 participants (baseline ages 12 - 22 years old, 50% female, 1 - 9 annual visits per participant, 6164 sessions total). Substance use, inhibitory control, and tissue iron increased non-linearly during adolescence into young adulthood, concurrent with decreases in impulsivity. Non-linear Growth Mixture Models identified four common trajectories of substance use: low (no- or low levels of use across visits; 30% of participants), youth peak (peak use in adolescence/young adulthood followed by declines; 26%), adolescent increasing (early, steep linear increases in use from adolescence into adulthood; 17%), and adult increasing (low use in adolescence, followed by late linear increases into adulthood; 26%). We show that increased substance use was associated with a phenotype of high impulsivity, low inhibitory control, and low basal ganglia tissue iron, particularly in early adolescence in individuals who displayed youth peak patterns in substance use trajectories. These findings highlight that early developmental differences in DA-related neurobiology and associated impulsivity and cognitive control shape distinct trajectories of adolescent substance use, underscoring adolescence as a critical window for the early identification and implementation of neurodevelopmentally sensitive interventions for substance use disorders.

neuroscience↗

bioRxiv2025

Human iPSC derived alveolar macrophages reveal macrophage subtype specific functions of itaconate in M. tuberculosis host defense

Mycobacterium tuberculosis (Mtb) must survive within multiple macrophage populations during infection, including alveolar macrophages (AM) and recruited inflammatory macrophages. In mice, itaconate, produced in macrophages by ACOD1 mediated decarboxylation of aconitate, has direct antimicrobial activity, modulates inflammatory cytokines, and is required for resistance to M. tuberculosis (Mtb) infection. The role of itaconate in human macrophages is less clear and whether itaconate mediates distinct effects in macrophage subtypes is unknown. Here, we investigated the role of itaconate in human iPSC-derived macrophages, either induced by GM-CSF to resemble alveolar macrophages (AM-Like cells), or treated with M-CSF to generate control macrophages (MCDM cells). Both types of human macrophages produce substantially less itaconate than mouse macrophages and AM-Ls produced 4-fold less itaconate than MCDMs. Surprisingly, ACOD1 deficient AM-L macrophages, but not MCDM macrophages, were permissive for Mtb growth. Moreover, itaconate functioned to dampen the Mtb induced inflammatory response in MCDMs, but not AM-L macrophages, affecting both the Type I IFN and TNF pathways. These results indicate that itaconate is involved in human macrophage responses to TB, with distinct roles in different macrophage subsets. These results also show that genetically tractable hiPSC-derived macrophages are a robust and versatile model to dissect cellular host pathogen interactions.

immunology↗

bioRxiv2024

Functional phenotyping of genomic variants using multiomic scDNA-scRNA-seq

Genomic variation ranging from single nucleotide polymorphisms to structural variants can impact gene function and expression, contributing to disease mechanisms such as cancer progression. The systematic study of this variation is hindered by inefficient precision editing tools making it challenging to confidently link genotype and gene expression in pooled screens. Additionally, assessing heterogenous variants in primary tumor samples at scale is difficult with current single-cell technologies. We developed droplet-based multiomic targeted scDNA-scRNAseq (SDR-seq) to precisely link genotypes with gene expression profiles in high-throughput. SDR-seq simultaneously assesses up to 480 RNA and gDNA targets with high coverage and sensitivity across thousands of cells. Using SDR-seq, we associate coding and non-coding variants with distinct gene expression profiles in human iPSCs. Furthermore, we demonstrate that in primary B-cell lymphoma samples, cells with a higher mutational burden exhibit elevated B-cell receptor signaling and tumorigenic gene expression. SDR-seq has broad potential for gaining functional insights into regulatory mechanisms encoded by genetic variants at diverse loci, advancing our ability to study gene expression regulation and its implications for disease.

molecular biology↗

bioRxiv2023

A single-cell multi-omic and spatial atlas of nodal B-cell lymphomas reveals B-cell maturation drives intratumor heterogeneity

Intratumor heterogeneity underpins cancer pathogenesis and evolution, although it is typically considered independent from the differentiation processes that drive physiological cell-type diversity. As cancer types and subtypes arise from different cell types, we investigated whether cellular differentiation influences intratumor heterogeneity. Nodal B-cell non-Hodgkin lymphomas are a diverse set of cancers originating from different stages of B-cell maturation. Through single-cell transcriptome and surface epitope profiling (CITE-Seq) of diffuse large B-cell, mantle cell, follicular, and marginal zone lymphomas in addition to reactive lymph nodes from 51 patients, we found multiple B-cell maturation states within tumors. Intratumor maturation states emerged from the same clone, revealing divergent differentiation from a shared cell of origin. Maturation state composition varied across subtypes and tumors, which encompassed mixed cell-of-origin diagnostic subtypes. Through highly multiplexed immunohistochemistry (CODEX) of samples from 19 of these patients, we found that intratumor maturation states inhabited distinct spatial niches, displaying cellular interactions and regulatory networks typical of their maturation states while harboring different genetic variants. By deconvoluting intratumor maturation states from a microarray dataset of 507 patients, we identified risk groups within diagnoses with striking differences in survival, including IgM memory-enriched germinal center B-cell (M = 1.9 vs >10 years, p = 0.00039) and activated B-cell (M = 2.4 vs 9.6 years, p = 0.016) diffuse large B-cell lymphoma, and dark zone-enriched follicular lymphoma (M = 8.6 vs 13 years; p = 0.0019). Our findings reveal cellular differentiation remains plastic in B-cell lymphomas, driving tumor variation, evolution, and response. Key PointsO_LICellular differentiation remains plastic in B-cell lymphomas, driving tumor variation, evolution, and response. C_LIO_LIIntratumor maturation states occupy unique immune niches, harbor distinct genetic variants, and are tied to different survival outcomes. C_LI

cancer biology↗

bioRxiv2023

Genome-wide mapping of the Escherichia coli PhoB regulon reveals many transcriptionally inert, intragenic binding sites

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the Escherichia coli transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved. Many intragenic PhoB sites are located in regions bound by H-NS, likely due to shared sequence preferences of PhoB and H-NS. However, these PhoB binding sites are not associated with transcription regulation even in the absence of H-NS. We propose that for many transcription factors, including PhoB, binding sites not associated with promoter sequences are transcriptionally inert, and hence are tolerated as genomic "noise". IMPORTANCERecent studies have revealed large numbers of transcription factor binding sites within the genes of bacteria. The function, if any, of the vast majority of these binding sites has not been investigated. Here, we map the binding of the transcription factor PhoB across the Escherichia coli genome, revealing that the majority of PhoB binding sites are within genes. We show that PhoB binding sites within genes are not associated with regulation of the overlapping genes. Indeed, our data suggest that bacteria tolerate the presence of large numbers of non-regulatory, intragenic binding sites for transcription factors, and that these binding sites are not under selective pressure.

microbiology↗

bioRxiv2022

Multimodal and spatially resolved profiling identifies distinct patterns of T-cell infiltration in nodal B-cell lymphoma entities

T-cell-engaging immunotherapies have improved the treatment of nodal B-cell lymphoma, but responses vary highly. Future improvements of such therapies require better understanding of the variety of lymphoma-infiltrating T-cells. We employed single-cell RNA and T-cell receptor sequencing alongside quantification of surface proteins, flow cytometry and multiplexed immunofluorescence on 101 lymph nodes from healthy controls, and patients with diffuse large B-cell, mantle cell, follicular, or marginal zone lymphoma. This multimodal resource revealed entity-specific quantitative and spatial aberrations of the T-cell microenvironment. Clonal PD1+ TCF7- but not PD1+ TCF7+ cytotoxic T-cells converged into terminally exhausted T-cells, the proportions of which were variable across entities and linked to inferior prognosis. In follicular and marginal zone lymphoma, we observed expansion of follicular helper and IKZF3+ regulatory T-cells, which were clonally related and inversely associated with tumor grading. Overall, we portray lymphoma-infiltrating T-cells with unprecedented comprehensiveness and decipher both beneficial and adverse dimensions of T-cell response.

cancer biology↗

bioRxiv2022

The Free Fatty Acid-Binding Pocket is a Conserved Hallmark in Pathogenic β-Coronavirus Spike Proteins from SARS-CoV to Omicron

As COVID-19 persists, severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor-binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA-binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic {beta}-coronaviruses ({beta}-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2 and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation while the open, infectious conformation is LA-free. Electron tomography of SARS-CoV-2 infected cells reveals that LA-treatment inhibits viral replication, resulting in fewer, deformed virions. Our results establish FFA-binding as a hallmark of pathogenic {beta}-CoV infection and replication, highlighting potential antiviral strategies. One-Sentence SummaryFree fatty acid-binding is conserved in pathogenic {beta}-coronavirus S proteins and suppresses viral infection and replication.

biochemistry↗

bioRxiv2022

Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

Multiple Sclerosis (MS) is a highly heterogeneous demyelinating disease of the central nervous system (CNS) that needs for reliable biomarkers to foresee disease severity. Previous retrospective investigations in the MS model, experimental autoimmune encephalomyelitis (EAE), highlighted the important relationship between monocytic-myeloid-derived suppressor cells (M-MDSCs) and the experimented severity of the clinical course. In this work, we show for the first time cells resembling M-MDSCs associated to MS lesions, whose abundance was related to milder MS clinical courses. Moreover, Ly-6Chi cells (which are indistinguishable from circulating M-MDSCs in mice) are useful biomarkers to predict a milder severity of the EAE disease course and a lesser tissue damage extent. Finally, the abundance of M-MDSCs in blood from untreated MS patients at their first relapse was inversely correlated with EDSS at baseline and relapse recovery one-year later. In summary, our data point to M-MDSC load as a promising biomarker of patients clinical course severity. TeaserThe abundance of myeloid-derived suppressor cells is related to a milder clinical course in multiple sclerosis patients.

neuroscience↗

bioRxiv2021

Structure and mechanism of SARS-CoV-2 Spike N679-V687 deletion variant elucidate cell-type specific evolution of viral fitness

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS{Delta} variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

biochemistry↗

bioRxiv2020

Whole-genome analysis of Nigerian patients with breast cancer reveals ethnic-driven somatic evolution and distinct genomic subtypes

Black women of African ancestry experience more aggressive breast cancer with higher mortality rates than White women of European ancestry. Although inter-ethnic germline variation is known, differential somatic evolution has not been investigated in detail. Analysis of deep whole genomes of 97 breast tumors, with RNA-seq in a subset, from indigenous African patients in Nigeria in comparison to The Cancer Genome Atlas (n=76) revealed a higher rate of genomic instability and increased intra-tumoral heterogeneity as well as a unique genomic subtype defined by early clonal GATA3 mutations and a 10.5-year younger age at diagnosis. We also found evidence for non-coding mutations in two novel drivers (ZNF217 and SYPL1) and a novel INDEL signature strongly associated with African ancestry proportion. This comprehensive analysis of an understudied population underscores the need to incorporate diversity of genomes as a key parameter in fundamental research with potential to tailor clinical intervention and promote equity in precision oncology care.

genomics↗

bioRxiv2020

Unexpected free fatty acid binding pocket in the cryo-EM structure of SARS-CoV-2 spike protein

COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2. One Sentence SummaryA direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.

biochemistry↗

bioRxiv2020

Cell-specific cargo delivery using synthetic bacterial spores

SSHELs are synthetic bacterial spore-like particles wherein the spores cell surface is partially reconstituted around 1 {micro}m-diameter silica beads coated with a lipid bilayer. Via a unique cysteine engineered in one of the surface proteins, the surface of SSHELs may be covalently decorated with molecules of interest. Here, we modified SSHELs with an affibody directed against HER2, a cell surface protein overexpressed in some breast and ovarian cancer cells, and loaded them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduced tumor growth with lower toxicity in a mouse tumor xenograft model compared to free drug by specifically binding to HER2-positive cancer cells. We show that SSHELs bound to target cells are taken up and trafficked to acidic compartments, whereupon the cargo is released in a pH-dependent manner. Finally, we demonstrate that SHELLs can clear small tumor lesions in a complex tumor microenvironment in a zebrafish model of brain metastasis. We propose that SSHELs represent a versatile strategy for targeted drug delivery.

synthetic biology↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2019

STARRPeaker: Uniform processing and accurate identification of whole human STARR-seq active regions

BackgroundHigh-throughput reporter assays, such as self-transcribing active regulatory region sequencing (STARR-seq), allow for unbiased and quantitative assessment of enhancers at a genome-wide scale. Recent advances in STARR-seq technology have employed progressively more complex genomic libraries and increased sequencing depths, to assay larger sized regions, up to the entire human genome. These advances necessitate a reliable processing pipeline and peak-calling algorithm. ResultsMost STARR-seq studies have relied on chromatin immunoprecipitation sequencing (ChIP-seq) processing pipelines. However, there are key differences in STARR-seq versus ChIP-seq. First, STARR-seq uses transcribed RNA to measure the activity of an enhancer, making an accurate determination of the basal transcription rate important. Second, STARR-seq coverage is highly non-uniform, overdispersed, and often confounded by sequencing biases, such as GC content and mappability. Lastly, here, we observed a clear correlation between RNA thermodynamic stability and STARR-seq readout, suggesting that STARR-seq may be sensitive to RNA secondary structure and stability. Considering these findings, we developed a negative-binomial regression framework for uniformly processing STARR-seq data, called STARRPeaker. In support of this, we generated whole-genome STARR-seq data from the HepG2 and K562 human cell lines and applied STARRPeaker to call enhancers. ConclusionsWe show STARRPeaker can unbiasedly detect active enhancers from both captured and whole-genome STARR-seq data. Specifically, we report [~]33,000 and [~]20,000 candidate enhancers from HepG2 and K562, respectively. Moreover, we show that STARRPeaker outperforms other peak callers in terms of identifying known enhancers with fewer false positives. Overall, we demonstrate an optimized processing framework for STARR-seq experiments can identify putative enhancers while addressing potential confounders.

bioinformatics↗

bioRxiv2019

The microbiota regulates inflammatory responses to toxin-induced CNS demyelination but has minimal impact on remyelination

The microbiota is now recognised as a key influence on the host immune response in the central nervous system (CNS). As such, there has been some progress towards therapies that modulate the microbiota with the aim of limiting immune-mediated demyelination, as occurs in multiple sclerosis. However, remyelination - the regeneration of myelin sheaths - also depends upon an immune response, and the effects that such interventions might have on remyelination have not yet been explored. Here, we show that the inflammatory response during CNS remyelination in mice is modulated by antibiotic or probiotic treatment, as well as in germ-free mice. We also explore the effect of these changes on oligodendrocyte progenitor cell differentiation, which is inhibited by antibiotics but unaffected by our other interventions. These results reveal that high combined doses of oral antibiotics negatively influence remyelination and further our understanding of how mammalian regeneration relates to the microbiota.

neuroscience↗

bioRxiv2017

The evolutionary impact of Intragenic FliA Promoters in Proteobacteria

Recent work has revealed that large numbers of promoters in bacteria are located inside genes. In contrast, almost all studies of transcription have focused on promoters upstream of genes. Bacterial promoters are recognized by Sigma factors that associate with initiating RNA polymerase. In Escherichia coli, one Sigma factor recognizes the majority of promoters, and six \"alternative\" Sigma factors recognize specific subsets of promoters. One of these alternative Sigma factors, FliA ({sigma}28), recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, reveal that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription is a conserved phenomenon, but that the specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved, and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence protein evolution in bacteria, and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.\n\nAUTHOR SUMMARYRecent genome-scale studies of bacterial transcription have revealed thousands of promoters inside genes. In a few cases, these promoters have been shown to transcribe functional RNAs. However, it is unclear whether most intragenic promoters have important biological function. Similarly, there are likely to be thousands of intragenic binding sites for transcription factors, but very few have been functionally characterized. Moreover, it is unclear what impact intragenic promoters and transcription factor binding sites have on evolution of the overlapping genes. In this study, we focus on FliA, a broadly conserved Sigma factor that is responsible for initiating transcription of many flagellar genes. We previously showed that FliA directs RNA polymerase to ~50 genomic sites in Escherichia coli. In our current study, we show that while most intragenic FliA promoters are actively transcribed, very few are conserved in other species. This suggests that most FliA promoters are not functional. Nonetheless, one intragenic FliA promoter is highly conserved, and we show that this promoter constrains evolution of the overlapping protein-coding gene. Given the enormous number of regulatory DNA sites within genes, we propose that the evolution of many genes is constrained by these elements.

genetics↗