Biology subjects

Hesselberth, J. R.

Publications and source records attributed to Hesselberth, J. R..

At least 19 recordsLinked to original sources

bioRxiv2025

Splice-switching antisense oligonucleotides correct cryptic exon inclusion and restore SDCCAG8 protein in Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a ciliopathy often associated with progressive blindness and obesity. A patient presenting with BBS was discovered to have two mutations within 55bp of each other in intron 7 of SDCCAG8 (BBS16). One of the biallelic mutations, c.740+356C>T, causes inclusion of cryptic exon(s) containing premature termination codons, while c.740+301G>A has not been characterized. We hypothesized that antisense oligonucleotides (ASOs) complementary to the patients mutations or to the cryptic exon splice sites would correct the splicing of SDCCAG8 between exons 7 and 8 to prevent cryptic exon inclusion and restore SDCCAG8 expression. We systematically screened 20nt-long ASOs tiled across each mutation and ASOs targeting the 3' splice sites of the cryptic exons in patient-derived fibroblasts, using RT-PCR assays to assess exon 7 and 8 splicing. We identified one ASO for each mutation and a cryptic exon-targeting ASO that restored the splicing pattern to that observed in an unaffected cell line. Lead ASOs were further investigated through RT-PCR, RNA sequencing, and western blotting to confirm ASO-mediated restoration of wild-type transcript and protein. Notably, ASO 20, which targets the cryptic exon 7a/7a' splice site rather than patient-specific mutations, achieved the greatest rescue effect, increasing exon 7-8 splicing from 0% to an average of 26% and restoring SDCCAG8 protein from undetectable levels to approximately 40% of wild-type expression. This mutation-agnostic approach could benefit multiple patients with cryptic exon inclusion in this region of SDCCAG8, expanding therapeutic impact beyond traditional N-of-1 ASO strategies. These findings establish a molecular foundation for clinical development of ASO therapy for BBS caused by SDCCAG8 splicing defects.

genetics↗

bioRxiv2025

Serine recombinases are conserved genetic markers of antiphage defense systems

Renewed interest in phage therapy has highlighted a need to understand how bacteria subvert phage infection through antiphage defense systems. Traditionally, strategies to identify antiphage defense systems lack throughput or have limitations for bacterial species where antiphage defense systems are understudied. Herein, we developed a bioinformatic pipeline that uses a small serine recombinase to identify known and unknown antiphage defense systems. Using this approach to query reference genomes and metagenomes, we show that small serine recombinase genes are genetically linked to antiphage defense systems and serve as bait for finding these systems across diverse bacterial phyla. Using co-transcription predictions and statistical analysis of protein domain abundances, we experimentally validated our bioinformatic approach by discovering that KAP P-loop NTPases are fused to putative antiphage domains and reinforce prokaryotic Schlafen proteins as a new class of antiphage defense. Our work shows that small serine recombinases are a reliable genetic marker for the discovery of antiphage defenses across diverse bacterial phyla.

microbiology↗

bioRxiv2025

GPR182 is a lipoprotein receptor for dietary fat absorption

The lymphatic system plays a central role in lipid absorption, which transports chylomicrons from the small intestine to the circulation. However, the molecular mechanism by which chylomicrons get into the intestinal lymphatics is unknown. Here we demonstrated that GPR182, a receptor in lymphatic endothelial cells (LECs), mediates dietary fat absorption. GPR182 knockout mice are resistant to dietary-induced obesity. GPR182 ablation in mice leads to poor lipid absorption and thereby a delay in growth during development. GPR182 binds and endocytoses lipoproteins broadly. Mechanistically, loss of GPR182 prevents chylomicrons from entering the lacteal lumen of the small intestine. GPR182 blockage with a monoclonal antibody (mAb) protects mice from dietary induced obesity. Together, our study identifies GPR182 as a lipoprotein receptor that mediates dietary fat absorption.

physiology↗

bioRxiv2024

Nanopore sequencing of intact aminoacylated tRNAs

Transfer RNAs (tRNA) are decorated during biogenesis with a variety of modifications that modulate their stability, aminoacylation, and decoding potential during translation. The complex landscape of tRNA modification presents significant analysis challenges and to date no single approach enables the simultaneous measurement of important but disparate chemical properties of individual, mature tRNA molecules. We developed a new, integrated approach to analyze the sequence, modification, and aminoacylation state of tRNA molecules in a high throughput nanopore sequencing experiment, leveraging a chemical ligation that embeds the charged amino acid in an adapted tRNA molecule. During nanopore sequencing, the embedded amino acid generates unique distortions in ionic current and translocation speed, enabling application of machine learning approaches to classify charging status and amino acid identity. Specific applications of the method indicate it will be broadly useful for examining relationships and dependencies between tRNA sequence, modification, and aminoacylation.

molecular biology↗

bioRxiv2024

IL-27 Stablizes Myc-Mediated Transcription In Memory-Fated, Vaccine-Elicited CD8+ T Cells

Protection from pathogens relies on both humoral (antibody-mediated) and cellular (T cell-mediated) responses. While infections robustly elicit both types of immunity, currently approved vaccine adjuvants largely fail to induce T cell responses on par with that instigated by infections. Our goal was to investigate the transcriptional programming that supports the formation of CD8+ T cells elicited by subunit vaccines compared to those elicited by infections. Our data show that vaccine-elicited T cells represent a transcriptionally unique subset of activated T cells with high proliferative capacity and a memory cell fate. This relies on IL-27 signaling, which stabilizes c-Myc and thereby supports the biomass acquisition necessary for clonal expansion. Collectively, our findings reveal that subunit vaccine-elicited T cells uniquely combine aspects of both memory and effector T cell subsets, and selectively utilize IL-27 signaling to sustain the clonal expansion of cells dedicated to a memory fate. One Sentence SummaryIn contrast to infection, subunit vaccines induce a distinct population of CD8+ T cells with memory fate characteristics which maintain their proliferative capacity during the expansion phase through IL-27 receptor signaling.

immunology↗

bioRxiv2024

Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing

Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.

molecular biology↗

bioRxiv2024

Islet-antigen reactive B cells display a unique phenotype and BCR repertoire in autoantibody positive and recent-onset type 1 diabetes patients

Autoreactive B cells play an important but ill-defined role in autoimmune type 1 diabetes (T1D). To better understand their contribution, we performed single cell gene and BCR-seq analysis on pancreatic islet antigen-reactive (IAR) B cells from the peripheral blood of nondiabetic (ND), autoantibody positive prediabetic (AAB), and recent-onset T1D individuals. We found that the frequency of IAR B cells was increased in AAB and T1D. IAR B cells from these donors had altered expression of B cell signaling, pro-inflammatory, infection, and antigen processing and presentation genes. Both AAB and T1D donors demonstrated a significant increase in certain heavy and light chain V genes, and these V genes were enriched in islet-reactivity. Public clones of IAR B cells were restricted almost entirely to AAB and T1D donors. IAR B cells were clonally expanded in the autoimmune donors, particularly the AAB group. Notably, a substantial fraction of IAR B cells in AAB and T1D donors appeared to be polyreactive, which was corroborated by analysis of recombinant monoclonal antibodies. These results expand our understanding of autoreactive B cell activation during T1D and identify unique BCR repertoire changes that may serve as biomarkers for increased disease risk. One Sentence SummaryPancreatic islet antigen-reactive B cells from individuals with prediabetes and recently diagnosed with type 1 diabetes display a unique phenotype and BCR repertoire compared to non-diabetic donors.

immunology↗

bioRxiv2024

Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). Despite its clinical importance, the factors controlling transcript-specific and context-dependent variation in NMD activity remain poorly understood. Through analysis of human genetic datasets, we discovered that the amino acid preceding the PTC strongly influences NMD activity. Notably, glycine codons promote robust NMD efficiency and show striking enrichment before PTCs but depletion before normal termination codons (NTCs). This glycine-PTC enrichment is particularly pronounced in genes tolerant to loss-of-function variants, suggesting evolutionary selection or neutrality conferred by efficient elimination of truncated proteins from non-essential genes. Using biochemical assays and massively parallel reporter analysis, we demonstrated that the peptide release rate during translation termination varies substantially with the identity of the preceding amino acid and serves as the primary determinant of NMD activity. We propose a "window of opportunity" model where translation termination kinetics modulate NMD efficiency. By revealing how sequence context shapes NMD activity through translation termination dynamics, our findings provide a mechanistic framework for improved clinical interpretation of nonsense variants.

molecular biology↗

bioRxiv2023

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

Invasive lobular carcinoma of the breast (ILC) is typically estrogen receptor (ER)-positive and presents with biomarkers of anti-estrogen sensitive disease, yet patients with ILC face particularly poor long-term outcomes with increased recurrence risk, suggesting endocrine response and ER function are unique in ILC. ER is co- regulated by the DNA repair protein Mediator of DNA Damage Checkpoint 1 (MDC1) specifically in ILC cells, driving distinct ER activity. However, this novel MDC1 activity is associated with dysfunctional canonical DNA repair activity by MDC1, but without typical features of DNA repair deficiency. To understand reciprocal activities of MDC1, we profiled the MDC1 interactome and found MDC1-associated proteins in ILC cells mirror a "BRCA-like" state lacking key homologous recombination (HR) proteins, consistent with HR dysfunction but distinct from classic "BRCAness". HR dysfunction in ILC cells is supported by single-cell transcriptome and DNA repair activity analyses, with DNA repair signaling and functional data, showing dysfunctional induction and resolution of HR. In parallel, ILC tumor data are consistent with a distinct form of HR dysfunction via impaired HR resolution, lacking BRCA-like genomic scarring but showing elevated signatures of PARP inhibitor sensitivity. We demonstrate this HR dysfunction can be exploited using PARP inhibition, and found that talazoparib treatment produced a durable growth suppression both in vitro and in multiple ILC xenografts in vivo. ILC-specific ER:MDC1 activity creates a new context for ER and MDC1 function in ILC, at the cost of a DNA repair dysfunction, which may be therapeutically targetable. SignificanceILC is rarely associated with biomarkers of HR deficiency, and as such patients are rarely eligible for treatment with PARP inhibitors. Our work suggests ILC presents with a previously unappreciated form of HR dysfunction, linked to ILC-specific genomic activity of ER and MDC1, which imparts sensitivity to PARP inhibition.

cancer biology↗

bioRxiv2022

scraps: an end-to-end pipeline for measuring alternative polyadenylation at high resolution using single-cell RNA-seq

Alternative cleavage and polyadenylation (APA) contributes to the diversity of mRNA 3' ends, affecting post-transcriptional regulation by including or excluding cis-regulatory elements in mRNAs, altering their stability and translational efficiency. While APA analysis has been applied broadly in mixed populations of cells, the heterogeneity of APA among single cells has only recently begun to be explored. We developed an approach we termed scraps (Single Cell RNA PolyA Site Discovery), implemented as a user-friendly, scalable, and reproducible end-to-end workflow, to identify polyadenylation sites at near-nucleotide resolution in single cells using 10X Genomics and other TVN-primed single-cell RNA-seq (scRNA-seq) libraries. Our approach, which performs best with long (>100bp) read 1 sequencing and paired alignment to the genome, is both unbiased relative to existing methods that utilize only read 2 and recovers more sites at higher resolution, despite the reduction in read quality observed on most modern DNA sequencers following homopolymer stretches. For libraries sequenced without long read 1, we implement a fallback approach using read 2-only alignments that performs similarly to our optimal approach, but recovers far fewer polyadenylation sites per experiment. scraps also enables assessment of internal priming capture events, which we demonstrate occur commonly but at higher frequency during apoptotic 3' RNA decay. We also provide an R package, scrapR, that integrates the results of the scaps pipeline with the popular Seruat single-cell analysis package. Refinement and expanded application of these approaches will further clarify the role of APA in single cells, as well as the effects of internal priming on expression measurements in scRNA-seq libraries.

bioinformatics↗

bioRxiv2022

Direct detection of RNA repair by nanopore sequencing

Ligation by plant and fungal RNA ligases yields an internal 2'-phosphate group on each RNA ligation product. In budding yeast, this covalent mark occurs at the splice junction of two targets of ligation: intron-containing tRNAs and the messenger RNA HAC1. The repertoire of RNA molecules repaired by RNA ligation has not been explored due to a lack of unbiased approaches for identifying RNA ligation products. Here, we define several unique signals produced by 2'-phosphorylated RNAs during nanopore sequencing. A 2'-phosphate at the splice junction of HAC1 mRNA inhibits 5'[->]3' degradation, enabling detection of decay intermediates in yeast RNA repair mutants by nanopore sequencing. During direct RNA sequencing, intact 2'-phosphorylated RNAs produce diagnostic changes in nanopore current properties and base calling features, including stalls produced as the modified RNA translocates through the nanopore motor protein. These approaches enable directed studies to identify novel RNA repair events from multiple species.

molecular biology↗

bioRxiv2021

MARCO+ lymphatic endothelial cells sequester arboviruses to limit viremia and viral dissemination

While viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the dLN and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. These findings uncover a previously unrecognized arbovirus scavenging role for LECs and improve our mechanistic understanding of viremia control during arboviral infections.

immunology↗

bioRxiv2020

Inclusion of processed cell metadata improves single cell sequencing analysis reproducibility and accessibility

Single cell RNA sequencing provides an unprecedented view of cellular diversity of biological systems. Thousands of scRNA-seq datasets have been generated, providing a wealth of biological data on the diversity of cell types across different organisms, developmental stages, and disease states. But while a tremendous number of publications and datasets have been generated using this technology, we found that a minority (< 25%) of studies provide sufficient information to enable direct reuse of their data for further studies. This problem is common across journals, data repositories, and publication dates. The lack of appropriate information not only hinders exploration and knowledge transfer of reported data, but also makes reproducing the original study prohibitively difficult and/or time-consuming. Correcting this problem is not easy but we encourage investigators, reviewers, journals, and data repositories to take steps to improve their standards and ensure proper documentation of these valuable datasets.

bioinformatics↗

bioRxiv2020

Neoplastic and Immune single cell transcriptomics define subgroup-specific intra-tumoral heterogeneity of childhood medulloblastoma.

Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. To better understand cellular heterogeneity in MB we use single-cell RNA sequencing, immunohistochemistry and deconvolution of transcriptomic data to profile neoplastic and immune populations in childhood MB samples. Neoplastic cells cluster primarily according to individual sample of origin which is in part due to the effect of chromosomal copy number gains and losses. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes and are associated with clinical outcomes, including discrete photoreceptor-like cells in MB subgroups GP3 and GP4 and nodule-associated neuronally-differentiated cells in subgroup SHH. We definitively chart the spectrum of MB immune cell infiltrates, which include subgroup/subtype-associated developmentally-related neuron-pruning and antigen presenting myeloid cells. MB cellular diversity is recapitulated in genetically engineered mouse subgroup-specific models of MB. These findings provide a clearer understanding of both the neoplastic and immune cell heterogeneity in MB.

cancer biology↗

bioRxiv2020

Molecular tracking devices quantify antigen distribution and archiving in the lymph node

Live, attenuated vaccines generate humoral and cellular immune memory, increasing the duration of protective immune memory. We previously found that antigens derived from vaccination or viral infection persist within lymphatic endothelial cells (LECs) beyond the clearance of the infection, a process we termed "antigen archiving". Technical limitations of fluorescent labeling have precluded a complete picture of antigen archiving across cell types in the lymph node. We developed a "molecular tracking device" to follow the distribution, acquisition, and retention of antigen in the lymph node. We immunized mice with an antigen conjugated to a nuclease-resistant DNA tag and used single-cell mRNA sequencing to quantify its abundance in lymph node hematopoietic and non-hematopoietic cell types. At early and late time points after vaccination we found antigen acquisition by dendritic cell populations (DCs), associated expression of genes involved in DC activation and antigen processing, and antigen acquisition and archiving by LECs as well as unexpected stromal cell types. Variable antigen levels in LECs enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention. Molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigens and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

immunology↗

bioRxiv2020

Physiologic RNA Targets and Refined Sequence Specificity of Coronavirus EndoU

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone-marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for UA and CA sequences (endoYA). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggest a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. ImpactCoronavirus EndoU cleaves UA and CA sequences (endoYA) within viral (+) strand RNA to evade dsRNA-activated host responses.

microbiology↗

bioRxiv2019

clustifyr: An R package for automated single-cell RNA sequencing cluster classification

BackgroundIn single-cell RNA sequencing (scRNA-seq) analysis, assignment of likely cell types remains a time-consuming, error-prone, and biased process. Current packages for identity assignment use limited types of reference data, and often have rigid data structure requirements. As such, a more flexible tool, capable of handling multiple types of reference data and data structures, would be beneficial. FindingsTo address difficulties in cluster identity assignment, we developed the clustifyr R package. The package leverages external datasets, including gene expression profiles from scRNA-seq, bulk RNA-seq, microarray expression data, and/or signature gene lists, to assign likely cell types. We benchmark various parameters of a correlation-based approach, and also implement a variety of gene list enrichment methods. By providing tools for exploratory data analysis, we demonstrate the feasibility of a simple and effective data-driven approach for cell type assignment in scRNA-seq cell clusters. Conclusionsclustifyr is a lightweight and effective cell type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr

bioinformatics↗

bioRxiv2019

Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Methods to measure heterogeneity among cells are rapidly transforming our understanding of biology but are currently limited to molecular abundance measurements. We developed an approach to simultaneously measure biochemical activities and mRNA abundance in single cells to understand the heterogeneity of DNA repair activities across thousands of human lymphocytes, identifying known and novel cell-type-specific DNA repair phenotypes. Our approach provides a general framework for understanding functional heterogeneity among single cells.

biochemistry↗