Biology subjects

Holt, R. A.

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

11 recordsLinked to original sources

bioRxiv2026

Vitamin D deficiency alters prostate epithelial differentiation and increases prostate cancer aggressiveness in ex vivo and in vivo models

Here, we examined the consequences of biologically relevant vitamin D deficiency, a known risk factor for aggressive prostate cancer, using ex vivo and in vivo models. Phenotypic and single-cell RNA sequencing of mouse prostate organoids showed that vitamin D deficiency stunted luminal cell differentiation more than androgen deficiency, which is a known driver of prostate development. Mice fed a vitamin D-deficient diet showed significantly altered expression of androgen-responsive genes in their prostate luminal cells, as determined by single-cell RNA sequencing. MDA-PCa-2b human prostate cancer cells, when maintained for 6 months in 1,25-dihydroxyvitamin D, lost the ability to form xenografts, despite normal proliferation in vitro. RNA sequencing showed that these cells also had disruptions in androgen signaling and multiple cancer-related pathways. This study offers new insights and validation of vitamin Ds role in both benign and malignant prostate biology, underscoring its essential hormonal functions and supporting strategies for vitamin D supplementation to reduce prostate cancer risk in vulnerable populations. STATEMENT OF SIGNIFICANCEVitamin D is an essential hormone, however, the non-calcemic consequences of vitamin D deficiency remain poorly defined, despite its high prevalence in the population. This study demonstrates significant biological consequences of vitamin D deficiency on prostate cells at biologically relevant levels in multiple systems.

cancer biology↗

bioRxiv2025

A De Novo Algorithm for Allele Reconstruction from Oxford Nanopore Amplicon Reads, with Application to CYP2D6

The Oxford Nanopore Technologies sequencing platform offers a path towards bedside genomics, producing long reads that can completely cover a gene of interest, and thus detect any known or novel variant the gene contains. However, the analysis of these long reads to identify actionable genotypes remains challenging and typically requires customization depending on the target gene. Here, we describe a generic algorithm to accurately reconstruct allele sequences derived from long-reads of genomic-amplicon origin. Rather than calling variants directly from these long-reads, our method takes a "sequence-first" approach, performing an unbiased reconstruction of the underlying amplicon sequences to generate high-confidence reconstructed allele sequences. This is done without user input of the expected target gene, allowing for any source amplicon to be reconstructed. These high-confidence reconstructed allele sequences are then compared to the genomic reference sequence of the gene to infer the specific diplotype present in the sample. This approach is agnostic towards the number of genes and alleles present and readily detects novel variants. We demonstrate our approach using three independent data sets for CYP2D6, a diverse and complex gene with over 175 known alleles of clinical significance affecting drug dosing. We show how our approach can accurately recover validated CYP2D6 diplotypes from 20 Coriell samples sequenced using different primer sets, on different Oxford Nanopore Technologies flow cell versions, and to different depths. This includes inferring occurrences of copy number variation from relative abundances of each allele, a critical factor for ascribing functional effects to a diplotype. Further, we demonstrate our approachs utility for other genomic regions, including HLA.

bioinformatics↗

bioRxiv2022

Behavioral dissection of hunger states in Drosophila

Hunger is a motivational drive that promotes feeding, and it can be generated by the physiological need to consume nutrients as well as the hedonic properties of food. Brain circuits and mechanisms that regulate feeding have been described, but which of these contribute to the generation of motive forces that drive feeding is unclear. Here, we describe our first efforts at behaviorally and neuronally distinguishing hedonic from homeostatic hunger states in Drosophila melanogaster and propose that this system can be used as a model to dissect the molecular mechanisms that underlie feeding motivation. We visually identify and quantify behaviors exhibited by hungry flies and find that increased feeding duration is a behavioral signature of hedonic feeding motivation. Using a genetically-encoded marker of neuronal activity, we find that the mushroom body (MB) lobes are activated by hedonic food environments, and we use optogenetic inhibition to implicate a dopaminergic neuron cluster (PAM) to /{beta} MB circuit in hedonic feeding motivation. The identification of discrete hunger states in flies and the development of behavioral assays to measure them offers a framework to begin dissecting the molecular and circuit mechanisms that generate motivational states in the brain.

animal behavior and cognition↗

bioRxiv2022

Encoding of hunger by the neuronal epigenome slows aging in Drosophila

Hunger is, by necessity, an ancient motivational drive, yet the molecular nature of homeostatic pressures of this sort and how they modulate health and physiology are largely unknown. Here we show that the molecular encoding of hunger slows aging in Drosophila. We identify the branched-chain amino acids (BCAAs) as dietary hunger signals that extend lifespan despite increasing food intake when reduced, and in parallel show that optogenetic activation of a subset of hunger-promoting neurons is sufficient to recapitulate these effects. We find that remodeling of the neuronal histone acetylome is associated with dietary BCAA reduction, and that this requires BCAA metabolism in specific subsets of neurons. Preventing the histone acetylome from being molded by dietary BCAAs abrogates both increased feeding and extended lifespan. However, the mechanisms that promote feeding and modulate aging downstream of alterations in histone acetylation occur through spatially and temporally distinct responses; differential usage of the histone variant H3.3A in the brain is an acute response to hunger that promotes increased feeding without modulating lifespan, while a prolonged experience of hunger may slow aging by promoting a beneficial decrease of a set-point around which hunger levels are regulated. Identification of a molecular basis for the encoding of hunger and demonstration of its sufficiency in extending lifespan reveals that motivational states alone are deterministic drivers of aging and behavior.

physiology↗

bioRxiv2022

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies' MinION device

BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications. ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 % to 0.53 %, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100 % accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

bioinformatics↗

bioRxiv2021

Modulation of the host cell transcriptome and epigenome by Fusobacterium nucleatum

Fusobacterium nucleatum (Fn) is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal and other cancer types. Fn can adhere to and invade host cells in a manner that varies across Fn strains and host cell phenotypes. Here we performed pairwise co-cultures between three Fn strains and two immortalized primary host cell types (colonic epithelial cells and vascular endothelial cells) followed by RNA-seq and ChIP-seq to investigate transcriptional and epigenetic host cell responses. We observed that Fn-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Further, we identified genes strongly upregulated specifically in conditions of host cell invasion, including overexpression of both EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon Fn exposure. To further explore this relationship, we used the ChIP-seq data to annotate chromatin states genome-wide. We found significant chromatin remodeling following Fn exposure in conditions of host cell invasion, with substantial increases in the frequency of states corresponding to active enhancers as well as low signal or quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to Fn exposure, and extensive host cell epigenomic changes associated with Fn host cell invasion. These results extend our understanding of Fn as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of Fn.

genomics↗

bioRxiv2020

Invasion by Fusobacterium nucleatum Causes Upregulation of dll4 and klf4 Expression in Caco-2 Cells

Fusobacterium nucleatum is an emerging microbe of importance in the pathogenesis of colorectal cancer. Strains of this enigmatic bacterial species vary in their capacity to invade human epithelial cells, a virulence determinant which has important implications in disease. Here, we infected human colorectal epithelial (Caco-2) cells in vitro with a known, highly invasive strain of F. nucleatum isolated from a Crohns Disease patient, as well as a further invasive isolate of F. nucleatum derived from a colorectal cancer tumour. We used transcriptional profiling to determine the human genes upregulated during the invasion process compared to exposure to a non-invasive E.coli control strain. Infection with F. nucleatum strains resulted in the upregulation of several host genes, including two associated with tumorigenesis: dll4 and klf4.

cancer biology↗

bioRxiv2020

Recombinant T cell receptors specific for HLA-A*02:01-restricted neoepitopes containing KRAS codon 12 hotspot mutations

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify {beta} T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (<2% tetramer positive). We amplified and sequenced the full-length TCR alpha and beta chains from each of the three T cell clones and determined that these three TCRs comprised distinct combinations of two different TCR alpha chains and two distinct TCR beta chains. We resynthesized these TCR alpha and beta chain nucleotide sequences and reconstituted the original pairs in healthy donor CD8+ T cells by lentiviral transduction, substituting the human {beta} TCR constant gene segments with murine {beta} TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-{gamma} ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

immunology↗

bioRxiv2020

Selective B cell depletion upon infusion of replication-incompetent anti-CD19 CAR lentivirus

Anti-CD19 CAR-T therapy for B cell malignancies has shown clinical success, but a major limitation is the logistical complexity and high cost of manufacturing autologous cell products. If engineered for improved safety, direct infusion of viral gene transfer vectors to initiate in vivo CAR-T transduction, expansion and anti-tumor activity could provide an alternative, universal approach. To explore this approach we administered approximately 20 million replication-incompetent VSV-G lentiviral particles carrying an anti-CD19CAR-2A-GFP transgene comprising either an FMC63 (human) or 1D3 (murine) anti-CD19 binding domain, or a GFP-only control transgene, to wild-type C57BL/6 mice by tail vein infusion. The dynamics of immune cell subsets isolated from peripheral blood were monitored at weekly intervals. We saw emergence of a persistent CAR-transduced CD3+ T cell population beginning week 3-4 that reached a maximum of 13.5 +/-0.58% (mean +/-SD) and 7.8 +/-0.76% of the peripheral blood CD3+ T cell population in mice infused with ID3-CAR or FMC63-CAR lentivector, respectively, followed by a rapid decline in each case of the B cell content of peripheral blood. Complete B cell aplasia was apparent by week 5 and was sustained until the end of the protocol (week 8). No significant CAR positive populations were observed within other immune cell subsets or other tissues. These results indicate that direct IV infusion of conventional VSV-G pseudotyped lentiviral particles carrying a CD19 CAR transgene can transduce T cells that then fully ablate endogenous B cells in wild type mice.

immunology↗

bioRxiv2017

The interface of malignant and immunologic clonal dynamics in high-grade serous ovarian cancer

High-grade serous ovarian cancer exhibits extensive intratumoral heterogeneity coupled with widespread intraperitoneal disease. Despite this, metastatic spread of tumor clones is non-random, implying the existence of local microenvironmental factors that shape tumor progression. We interrogated the molecular interface between tumor-infiltrating lymphocytes (TIL) and cancer cells in 143 samples from 21 patients using whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T- and B-cell receptor sequencing. We identify 3 immunologic response categories, which frequently co-exist within individual patients. Furthermore, epithelial CD8+ TIL were inversely associated with malignant cell diversity, evidenced by subclonal neoepitope elimination and spatial tracking between tumor and T-cell clones. Intersecting mutational signatures and immune analysis showed that foldback inversion genomic aberrations lead to worse outcomes even in the presence of cytotoxic TIL (n=433). Thus, regional variation in immune contexture mirrors the pattern of intraperitoneal malignant spread, provoking new perspectives for treatment of this challenging disease.

cancer biology↗

bioRxiv2017

It’s okay to be green: Draft genome of the North American Bullfrog (Rana [Lithobates] catesbeiana)

Frogs play important ecological roles as sentinels, insect control and food sources. Several species are important model organisms for scientific research to study embryogenesis, development, immune function, and endocrine signaling. The globally-distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Consequently, the extensive Xenopus genomic resources are of limited utility for Ranids and related frog species. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines.\n\nHerein, we report on the first genome sequence of a Ranid species, an adult male North American bullfrog (Rana [Lithobates] catesbeiana). We assembled high-depth Illumina reads (66-fold coverage), into a 5.8 Gbp (NG50 = 57.7 kbp) draft genome using ABySS v1.9.0. The assembly was scaffolded with LINKS and RAILS using pseudo-long-reads from targeted denovo assembler Kollector and Illumina Synthetic Long-Reads, as well as reads from long fragment (MPET) libraries. We predicted over 22,000 protein-coding genes using the MAKER2 pipeline and identified the genomic loci of 6,227 candidate long noncoding RNAs (IncRNAs) from a composite reference bullfrog transcriptome. Mitochondrial sequence analysis supported Lithobates as a subgenus of Rana. RNA-Seq experiments identified ~6,000 thyroid hormone- responsive transcripts in the back skin of premetamorphic tadpoles; the majority of which regulate DNA/RNA processing. Moreover, 1/6th of differentially-expressed transcripts were putative lncRNAs. Our draft bullfrog genome will serve as a useful resource for the amphibian research community.

genomics↗