Biology subjects

Abate, A. R.

Publications and source records attributed to Abate, A. R..

17 recordsLinked to original sources

bioRxiv2026

Spatially resolved transcriptome-metabolome integration reveals region-specific glial lipid dysregulation associated with Alzheimer's pathology

Glial cells maintain the brains lipid and energy balance, and their breakdown is increasingly recognized as a causal contributor to Alzheimers disease (AD). While this concept is established, no approach has directly shown how glial homeostatic failure manifests across brain regions and microenvironments or how it links local pathology, such as plaques, to global metabolic imbalance. To address this gap, we developed iMIST, an integrated platform that combines MALDI-based metabolite imaging, histology, and spatial transcriptomics within a single tissue section to align molecular and anatomical information. Using a mouse model of late-onset AD that recapitulates both amyloid deposition and metabolic vulnerability, iMIST revealed that glial lipid dysregulation is widespread but spatially specialized. In gray matter, plaque-associated microglia were associated with upregulated glycerophospholipid-remodeling in cortico-thalamic areas indicating metabolic stress around local pathology. In contrast, white matter tracts rich in lipid-producing oligodendrocytes show plaque-independent deficits in galactosylceramide metabolism reflecting their high myelin demand. Both processes intensify with age, transforming adaptive glial responses into persistent metabolic dysfunction. Together, these findings demonstrate the spatial interplay between global glial metabolic imbalance and local microenvironmental stressors associated with AD pathology. By integrating transcriptomic and metabolomic information in situ, iMIST provides a framework for uncovering how regional glial vulnerability shapes the pathogenesis of neurodegenerative diseases.

neuroscience↗

bioRxiv2025

The Immunophenotype and Proviral Landscape of HIV-infected CD4 T Cells During Antiretroviral Therapy

In individuals on effective antiretroviral therapy, integrated HIV proviruses persist within CD4 T cells, forming a viral reservoir that rebounds if treatment is stopped. Identifying and targeting these rare, infected cells is critical for advancing therapies, but methods to study reservoir cells are limited and their unique properties remain largely unknown. We applied DAb-seq, a high-throughput method that combines single-cell DNA and surface protein sequencing, to profile over five hundred and twenty thousand CD4 T cells from the blood of six individuals on ART. Infected cells were unequally distributed in T cell subsets, and differential protein expression between infected and uninfected cells revealed significant heterogeneity across cell subsets. Attempts to identify surface markers that differentiate infected from uninfected cells found antigens that mirrored the enrichment of HIV in central memory subsets. However, while central memory T cells harbored the majority of HIV, cells with intact provirus were enriched relative to their defective counterparts in Naive and Regulatory T cell subsets, suggesting that they differentially maintain intact proviruses. In summary, we developed DAb-seq as an open-source platform for linking the proviral landscape to diverse cellular phenotypes, revealing heterogeneity in surface protein expression and provirus maintenance across infected subsets.

genomics↗

bioRxiv2025

High-Throughput Antibody Neutralization Screening in Massively Parallel Droplet Arrays

Neutralizing antibodies provide rapid immune defense against infectious diseases, but are difficult to discover at scale because neutralization assays require live reporter cells and soluble monoclonal antibodies. Here we report Droplet Reporter Cell Testing for Neutralization (DrReCT-Neutralization) to screen antibody gene libraries for their ability to neutralize viral infections. We established the necessary engineered cell lines and validated the DrReCT screening platform using synthetic oligoclonal libraries, followed by an example discovery campaign that demonstrated scalable functional antibody data collection against viral diseases.

immunology↗

bioRxiv2024

PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well- characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long- term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

bioengineering↗

bioRxiv2024

Multiplatform molecular profiling and functional genomic screens identify prognostic signatures and mechanisms underlying MEK inhibitor response in somatic NF1 mutant glioblastoma

NF1 is recurrently mutated in glioblastoma yet the molecular landscape and efficacy of targeted therapies remain unclear. Here, we combine bulk and single cell genomics of human somatic NF1 mutant, IDH-wildtype glioblastomas with functional genomic analysis of cell lines and mouse intracranial tumor models to identify molecular subgroups within NF1 mutant glioblastomas and mechanisms underlying MEK inhibitor response. Targeted DNA sequencing showed homozygous deletion of the cell cycle regulator CDKN2A/B is a poor prognostic marker in somatic NF1 mutant, but not NF1 wildtype, tumors. DNA methylation array profiling revealed three epigenetic groups highlighted by distinct clinical features, co-mutation patterns, and reference methylation classifier identities. Genome-wide CRISPRi screens in glioblastoma cells revealed cell cycle regulators are conserved mediators of cell growth while response to the MEK inhibitor selumetinib converges on Ras/RAF/MEK pathway activation. Repression of the RAF regulator SHOC2 sensitizes glioblastomas to selumetinib in vitro and in vivo in mouse intracranial glioblastoma models. Single cell RNA-sequencing of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib reveals distinct responses between mesenchymal-like (MES-like) and non MES-like subpopulations suggesting non-MES like cells are intrinsically resistant to MEK inhibition. Finally, single nuclear RNA-sequencing (snRNA-seq) of human NF1 mutant, CDKN2A/B deleted glioblastomas reveals MES-like tumor cells are associated with selumetinib sensitivity signatures while non MES-like cells exhibit increased cell cycle progression and lack selumetinib sensitivity, further supporting the notion that MEK inhibition specifically targets MES-like tumor cell subpopulations. Taken together, our data underscores the heterogeneity between and within somatic NF1 mutant glioblastomas and delineates mechanisms of MEK inhibitor response across distinct tumor subpopulations, guiding the development of future therapeutic strategies that may synergize with MEK inhibition for NF1 mutant tumors. The tumor suppressor NF1 is mutated in 15% of glioblastomas,1-3 the most common malignant brain tumor with poor outcomes and few effective treatments.4 NF1 is a GTPase activating protein (GAP) that negatively regulates Ras, and thus, NF1 loss leads to induction of Ras/RAF/MEK/ERK signaling, driving tumorigenesis and comprising a targetable molecular cascade.5,6 Genomic analysis of glioblastoma demonstrates NF1 mutation is associated with a mesenchymal-like (MES-like) transcriptomic tumor cell subpopulation and altered tumor microenvironment.7,8 More broadly, DNA methylation analysis reveals multiple epigenetic subgroups with overlapping relationships to transcriptomic subtype and DNA alterations, underscoring the complex relationship between genetic drivers and molecular signatures.9 While the updated 2021 Central Nervous System WHO tumor classification incorporates an ever increasing amount of molecular criteria for diffuse astrocytic tumors,10 the existence and clinical significance of molecular subgroups within somatic NF1 mutant, IDH-wildtype glioblastomas based on genetic co-mutations, epigenetic profile, or transcriptomic signatures remain unclear. Preclinical data support the utility of MEK inhibition in NF1 mutant gliomas,11,12 and the MEK inhibitor selumetinib is FDA approved for tumors arising in patients with syndromic neurofibromatosis type 1 (NF-1) harboring a germline NF1 mutation.13,14 In NF-1 associated gliomas, MEK inhibition demonstrates efficacy in a limited case series,15 and combined BRAF/MEK inhibition shows efficacy in BRAF p.V600E mutant gliomas,16 further supporting the translational potential of Ras/Raf/MEK/ERK blockade within genetically defined glioma subtypes. Nevertheless, treatment resistance to molecular monotherapy remains a challenge,17-20 and the mechanisms underlying MEK inhibitor resistance in NF1 mutant glioma are unknown. Here, we integrate targeted DNA sequencing, DNA methylation profiling, and single nuclear RNA-sequencing (snRNA-seq) of human patient somatic NF1 mutant, IDH-wildtype glioblastomas with single cell RNA-sequencing (scRNA-seq), genome-wide clustered regularly interspaced short palindromic repeats interference (CRISPRi) screens, and pharmacologic studies in cell lines and mouse intracranial glioblastoma models to define molecular subgroups and functional mediators of MEK inhibitor response. Targeted DNA sequencing of NF1 mutant, IDH-wildtype glioblastomas (n=186 tumors) revealed CDKN2A/B deletion was associated with poor outcomes in NF1 mutant, but not NF1 wildtype, glioblastomas. DNA methylation profiling (n=129 tumors) demonstrated three epigenetic subgroups with distinct clinical features, co- mutation patterns across cell cycle genes, and reference methylation classifier identities. Genome-wide CRISPRi screens in mouse SB28 and human GBM43 glioblastoma cells identified a conserved cell cycle gene network mediating cell growth, consistent with the clinical importance of additional hits affecting the cell cycle in human somatic NF1 mutant glioblastomas. Moreover, genome-wide mediators of selumetinib response converged upon two Ras pathway effectors mediating selumetinib sensitivity: BRAF and SHOC2. SHOC2 repression in glioblastoma cells significantly improved selumetinib response both in vitro and in intracranial allografts in vivo. Single cell RNA-sequencing (scRNA-seq) of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib revealed MES-like tumor cells correlated with CDKN2A retention and the CRISPRi screen selumetinib sensitivity signature, with selumetinib resistant cells displaying Ras pathway induction. In contrast, non-MES like tumor cells were CDKN2A deficient and lacked expression of the CRISPRi screen selumetinib sensitivity signature, with selumetinib resistant cells inducing a glial de-differentiation program. Finally, snRNA-seq of NF1 mutant, CDKN2A/B deleted, IDH-wildtype glioblastomas (n=9) showed non MES-like tumor cells exhibit increased cell cycle progression and were not associated with the CRISPRi screen selumetinib sensitivity signature. MES-like tumor cells within newly diagnosed, but not recurrent, tumors retained expression of the CRISPRi screen selumetinib sensitivity signature, suggesting resistance can arise both between and within specific transcriptomic glioblastoma cell tumor cell subpopulations. Taken together, our data identifies clinically important subgroups of NF1 mutant, IDH-wildtype glioblastomas and supports a model in which heterogeneity between tumors and within tumor cell subpopulations underlies MEK inhibitor response, supporting the need for additional synergistic therapeutic approaches beyond maximal Ras pathway blockade for NF1 mutant glioblastomas.

genomics↗

bioRxiv2023

Reciprocal inflammatory signals establish profibrotic cross-feeding metabolism

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung remodeling and collagen deposition that leads to respiratory failure. Myeloid cells are abundant in IPF lung and in murine lung fibrosis, but their functional effects are incompletely understood. Using mouse and human lung models, we show that ornithine produced by myeloid cells expressing Arg1 serves as a substrate for proline and collagen synthesis by lung fibroblasts. The predominant Arg1-expressing myeloid cells in mouse lung were macrophages, but in IPF lung, high-dimensional imaging revealed ARG1 to be expressed mainly in neutrophils. Arg1 inhibition suppressed both ornithine levels and collagen expression in cultured, precision-cut IPF lung slices and in murine lung fibrosis. These results were confirmed in macrophage-specific Arg1 KO mice. Furthermore, we find that this pathway is regulated by cell-to-cell crosstalk, starting with purinergic signaling: Fibroblast eATP receptor P2rx4 was necessary for fibroblast IL-6 expression, which in turn was necessary for Arg1 expression by myeloid cells. Taken together, our findings define an immune-mesenchymal circuit that governs profibrotic metabolism in lung fibrosis.

immunology↗

bioRxiv2023

Microbiome single cell atlases generated with a commercial instrument

Single cell sequencing is useful for resolving complex systems into their composite cell types and computationally mining them for unique features that are masked in pooled sequencing. However, while commercial instruments have made single cell analysis widespread for mammalian cells, analogous tools for microbes are limited. Here, we present EASi-seq (Easily Accessible Single microbe sequencing). By adapting the single cell workflow of the commercial Mission Bio Tapestri instrument, this method allows for efficient sequencing of individual microbes genomes. EASi-seq allows thousands of microbes to be sequenced per run and, as we show, can generate detailed atlases of human and environmental microbiomes. The ability to capture large shotgun genome datasets from thousands of single microbes provides new opportunities in discovering and analyzing species subpopulations. To facilitate this, we develop a companion bioinformatic pipeline that clusters genome by sequence similarity, improving whole genome assembly, strain identification, taxonomic classification, and gene annotation. In addition, we demonstrate integration of metagenomic contigs with the EASi-seq datasets to reduce capture bias and increase coverage. Overall, EASi-seq enables high quality single cell genomic data for microbiome samples using an accessible workflow that can be run on a commercially available platform.

bioengineering↗

bioRxiv2021

Age-dependent Effect Imposed by Rotenone Exposure

BackgroundThe increasing awareness that environmental exposure may lead to sporadic neurological disorders has implicated rotenone to the etiology of some neurodegenerative diseases. However, the risk associated with rotenone toxicity remains controversial as a limited amount of research has studied its effects on brain health. ObjectivesThis work assessed the risk of rotenone exposure to mice of different ages, gender, and duration by examining in vivo effects on brains. MethodsUsing a mouse model, the impact of rotenone exposure was determined by analyzing the cellular phenotype in the murine brain. ResultsOur results highlight the neurological susceptibility to long-term rotenone exposure in younger ages. For such, younger mice exhibit seizures and convulsions, resulting in shorter lifespan. At the cellular level, rotenone exposure specifically alters the migrating neuroblast populations in the dentate gyrus and causes disorganized pyramidal neurons in the CA3 within the hippocampus. Our findings, albeit the absence of transgenerational inheritance, demonstrated age-related outcomes from rotenone exposure. DiscussionWe demonstrated that rotenone exposure specifically influences the population of neuroblasts and pyramidal neurons residing in the hippocampus, a brain region important for learning/memory and associated with convulsive seizure. Our understanding of how exactly rotenone affected region-specific neuronal cells and the molecular mechanism behind exposure risk is still limited. From the perspective of public health, our in vivo study highlights age-related susceptibility to rotenone toxicity. Future investigations in environmental epidemiology should determine whether age and duration of exposure to rotenone in human subjects pertains to the development of seizures or other neurological abnormalities over time.

neuroscience↗

bioRxiv2021

Mapping enzyme catalysis with metabolomic biosensing

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

bioengineering↗

bioRxiv2020

Modular barcode beads for microfluidic single cell genomics

Barcode beads allow efficient nucleic acid tagging in single cell genomics. Current barcode designs, however, are fabricated with a particular application in mind. Repurposing to novel targets, or altering to add additional targets as information is obtained is possible but the result is suboptimal. Here, we describe a modular framework that simplifies generation of multifunctional beads and allows their easy extension to new targets. One Sentence SummaryWe describe an optimized design for barcoding beads which are useful for single cell RNA and DNA sequencing.

cell biology↗

bioRxiv2020

Sequencing ultra-rare targets with compound nucleic acid cytometry

Targeted sequencing enables sensitive and cost-effective analysis by focusing resources on molecules of interest. Existing methods, however, are limited in enrichment power and target capture length. Here, we present a novel method that uses compound nucleic acid cytometry to achieve million-fold enrichments of molecules >10 kbp in length using minimal prior target information. We demonstrate the approach by sequencing HIV proviruses in infected individuals. Our method is useful for rare target sequencing in research and clinical applications, including for identifying cancer-associated mutations or sequencing viruses infecting cells.

genomics↗

bioRxiv2020

Joint profiling of proteins and DNA in single cells reveals extensive proteogenomic decoupling in leukemia

Studies of acute myeloid leukemia rely on DNA sequencing and immunophenotyping by flow cytometry as primary tools for disease characterization. However, leukemia tumor heterogeneity complicates integration of DNA variants and immunophenotypes from separate measurements. Here we introduce DAb-seq, a novel technology for simultaneous capture of DNA genotype and cell surface phenotype from single cells at high throughput, enabling direct profiling of proteogenomic states in tens of thousands of cells. To demonstrate the approach, we analyze the disease of three patients with leukemia over multiple treatment timepoints and disease recurrences. We observe complex genotype-phenotype dynamics that illustrate the subtlety of the disease process and the degree of incongruity between blast cell genotype and phenotype in different clinical scenarios. Our results highlight the importance of combined single-cell DNA and protein measurements to fully characterize the heterogeneity of leukemia.

cancer biology↗

bioRxiv2019

Linked optical and gene expression profiling of single cells at high throughput

Single cell RNA sequencing has emerged as a powerful tool for characterizing cells, but not all phenotypes of interest can be observed through gene expression alone. Linking sequencing with optical analysis has provided insight into the molecular basis behind cellular function, but current approaches have limited throughput. Here, we present a high throughput platform for linked optical and gene expression profiling of single cells. We demonstrate accurate fluorescence and gene expression measurements from thousands of cells in a single experiment and use the platform to characterize DNA and RNA changes in Jurkat cells through the cell cycle. In addition to its scalability, our integration of microfluidics and array-based molecular biology holds promise for comprehensive multi-omics profiling of single cells.

bioengineering↗

bioRxiv2018

Particle-templated emulsification for microfluidics-free digital biology

The compartmentalization of reactions in monodispersed droplets is valuable for applications across biology. However, the requirement of microfluidics to partition the sample into monodispersed droplets is a significant barrier that impedes implementation. Here, we introduce particle-templated emulsification, a method to encapsulate samples in monodispersed emulsions without microfluidics. By vortexing a mixture of hydrogel particles and sample solution, we encapsulate the sample in monodispersed emulsions that are useful for most droplet applications. We illustrate the method with ddPCR and single cell culture. The ability to encapsulate samples in monodispersed droplets without microfluidics should facilitate the implementation of compartmentalized reactions in biology.

bioengineering↗

bioRxiv2018

Robotic automation of droplet microfluidics

Droplet microfluidics enables new reactions, assays, and analytic capabilities, but often requires complex workflows involving numerous steps of macro- and micro-fluidic processing. We demonstrate robotically-automated droplet microfluidics, an approach to automate workflows with commercial fluid-handling robots. These workflows can be performed without human intervention, increasing reliability and convenience.

bioengineering↗

bioRxiv2017

Combined aptamer and transcriptome sequencing of single cells

The transcriptome and proteome encode distinct information that is important for characterizing heterogeneous biological systems. We demonstrate a method to simultaneously characterize the transcriptomes and proteomes of single cells at high throughput using aptamer probes and droplet-based single cell sequencing. With our method, we differentiate distinct cell types based on aptamer surface binding and gene expression patterns. Aptamers provide advantages over antibodies for single cell protein characterization, including rapid, in vitro, and high-purity generation via SELEX, and the ability to amplify and detect them with PCR and sequencing.

cell biology↗

bioRxiv2017

Printed Droplet Microfluidics for on demand dispensing of picoliter droplets and cells

Although the elementary unit of biology is the cell, high throughput methods for the microscale manipulation of cells and reagents are limited. The existing options are either slow, lack single cell specificity, or utilize fluid volumes out of scale with those of cells. Here, we present Printed Droplet Microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single cell printer, enabling high throughput generation of intricate arrays of droplets, cells, and microparticles. Printed Droplet Microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

bioengineering↗