Biology subjects

Walsh, D. J.

Publications and source records attributed to Walsh, D. J..

6 recordsLinked to original sources

bioRxiv2026

Fluorescent non-canonical amino acid as a site-specific conformational probe of prion formation

The pathogenic conversion of the cellular prion protein (PrPC) into the {beta}-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive non-canonical amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and non-infectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new and broadly applicable strategy for studying conformational dynamics in amyloid-forming proteins.

biochemistry↗

bioRxiv2026

Embarrassingly_FASTA: Enabling Recomputable, Population-Scale Pangenomics by Reducing Commercial Genome Processing Costs from $100 to less than $1

Computational preprocessing has become the dominant bottleneck in genomics, frequently exceeding sequencing costs and constraining population-scale analysis, even as large repositories grow from tens of petabytes toward exabyte-scale storage to support World Genome Models. Legacy CPU-based workflows require many hours to days per 30x human genome, driving many repositories to distribute aligned or derived intermediates such as BAM and VCF files rather than raw FASTQ data. These intermediates embed reference- and model-dependent assumptions that limit reproducibility and impede reanalysis as reference genomes, including pangenomes, continue to evolve. Although recent work has established that GPUs can dramatically accelerate genomic pipelines, enabling large-cohort processing to shrink from years to days given sufficient parallelism, such workflows remain cost-prohibitive. Here, we introduce Embarrassingly_FASTA, a GPU-accelerated preprocessing pipeline built on NVIDIA Parabricks that fundamentally changes the economics of genomic data management. By rendering intermediate files transient rather than archival, Embarrassingly_FASTA enables retention of raw FASTQ data and reliable use of highly discounted ephemeral cloud infrastructure such as spot instances, reducing compute spend from [~]$17/genome (CPU on-demand) to <$1/genome (GPU spot), and commercial secondary-analysis pricing from [~]$120/genome to compute spend under $1/genome. We demonstrate the impact of this efficiency using a simulated large-cohort pangenome build-up (using variant-union accumulation as a proxy for diversity growth) in Caenorhabditis elegans and humans, highlighting the long tail of unsampled human genetic diversity. Beyond GPU kernels, Embarrassingly_FASTA contributes a transient-intermediate lifecycle and spot-friendly orchestration that makes FASTQ retention and routine recomputation economically viable. Embarrassingly_FASTA thus provides enabling infrastructure for recomputable, population-scale pangenomics and next-generation genomic models. Non-Expert DescriptionReading a persons complete DNA sequence has become fast and inexpensive, but turning that raw data into something scientists can analyze is now one of the biggest obstacles in modern genetics. Today, processing a single genome can take many hours or even days, which makes it difficult and expensive to study large populations or reanalyze data when better methods become available. As a result, many databases store only partially processed DNA instead of the original data, limiting future discoveries. In this work, we present a new system that dramatically speeds up this processing step using graphics processing units (GPUs), the same type of hardware used in modern artificial intelligence. With our approach, a human genome can be processed in about 35 minutes instead of more than 15 hours, and at a fraction of the cost. This makes it practical to keep the original DNA data and reprocess it whenever new tools or reference genomes become available, rather than being locked into outdated results. We also show that this speed and affordability allow researchers to explore genetic diversity at an unprecedented scale. By analyzing both human genomes and those of a small worm species commonly used in research, we demonstrate how new genetic variations continue to emerge as more individuals are studied, especially in humans, where much diversity remains unexplored. Overall, our work helps remove a major barrier to studying DNA at population scale and lays the foundation for future genetic models that could better explain disease, evolution, and human health. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/703356v2_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@11b8fc3org.highwire.dtl.DTLVardef@7b8b60org.highwire.dtl.DTLVardef@fb5a3borg.highwire.dtl.DTLVardef@1e11276_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

IspE Kinase as an Anti-infective Target: Role of a Hydrophobic Pocket in Inhibitor Binding

Enzymes of the methylerythritol phosphate (MEP) pathway are potential targets for antimicrobial drug discovery. Here we focus on 4-diphosphocytidyl-2-C-methyl-D-erythritol (IspE) kinase from the MEP pathway. We use biochemical and structural biology methods to investigate homologs from the pathogenic microorganisms; Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. We determined the X-ray structures of IspE-inhibitor complexes and studied selected inhibitors binding modes targeting the substrate pocket. The experimental results indicate the need for distinct inhibitor strategies due to structural differences among IspE homologs, particularly for A. baumannii IspE, which displays unique inhibitory profile due to a tighter hydrophobic subpocket in the substrate binding site. This study enhances our understanding of the MEP enzymes and sets the stage for structure-based drug design of selective inhibitors to combat pathogenic microorganisms.

biophysics↗

bioRxiv2023

Convergent generation of atypical prions in knock-in mouse models of genetic prion disease

Most cases of human prion disease arise due to spontaneous misfolding of wild-type or mutant prion protein. Though recapitulating spontaneous prion conversion in animal models has proven challenging, transgenic mice expressing the misfolding-prone bank vole prion protein (BVPrP) recreate certain key aspects of sporadic and genetic prion disease. However, it remains unclear whether spontaneous prion generation can occur in the absence of protein over-expression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knock-in mice expressing physiological levels of either wild-type or mutant BVPrP with isoleucine at codon 109. While mice expressing wild-type BVPrP remained free from neurological disease, a subset of knock-in mice expressing BVPrP with mutations that cause either fatal familial insomnia (D178N) or familial Creutzfeldt-Jakob disease (E200K) developed progressive neurological illness. Brains from spontaneously ill knock-in mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant prion protein. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP knock-in mice transmitted disease to mice expressing wild-type BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical both pre- and post-transmission, suggesting that both mutations guide the formation of a highly similar atypical prion strain. These findings imply that knock-in mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action. Therefore, these mice represent useful tools for studying the early stages of genetic prion diseases.

neuroscience↗

bioRxiv2023

Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice as they age.

One-carbon metabolism is a complex network of metabolic reactions that are essential for cellular function including DNA synthesis. Vitamin B12 and folate are micronutrients that are utilized in this pathway and their deficiency can result in the perturbation of one-carbon metabolism and subsequent perturbations in DNA replication and repair. This effect has been well characterized in nuclear DNA but to date, mitochondrial DNA (mtDNA) has not been investigated extensively. Mitochondrial variants have been associated with several inherited and age-related disease states; therefore, the study of factors that impact heteroplasmy are important for advancing our understanding of the mitochondrial genomes impact on human health. Heteroplasmy studies require robust and efficient mitochondrial DNA enrichment to carry out in-depth mtDNA sequencing. Many of the current methods for mtDNA enrichment can introduce biases and false positive results. Here we use a method that overcomes these limitations and have applied it to assess mitochondrial heteroplasmy in mouse models of altered one-carbon metabolism. Vitamin B12 deficiency was found to cause increased levels of mitochondrial DNA heteroplasmy across all tissues that were investigated. Folic acid supplementation also contributed to elevated mitochondrial DNA heteroplasmy across all mouse tissues investigated. Heteroplasmy analysis of human data from the Framingham Heart Study suggested a potential sex-specific effect of folate and vitamin B12 status on mitochondrial heteroplasmy. This is a novel relationship that may have broader consequences for our understanding of one-carbon metabolism, mitochondrial related disease and the influence of nutrients on DNA mutation rates. Significance StatementUsing a sensitive method for mitochondrial heteroplasmy analysis, we show that both vitamin B12 and folic acid can impact mitochondrial DNA mutation. This effect requires further investigation of the potential impact on humans.

molecular biology↗

bioRxiv2022

Mito-SiPE: A sequence-independent, PCR-free mitochondrial DNA enrichment method for ultra-deep sequencing that minimises amplification and alignment artifacts.

BackgroundDeep sequencing is often used to measure somatic variation in the mitochondrial genome. Selective enrichment methods, such as PCR amplification or probe hybridization/capture are commonly used. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs); elements that can introduce artefacts into analyses such as an assessment of mitochondrial heteroplasmy. ResultsHere, we demonstrate a method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome by selectively purifying the intact organelle itself using differential centrifugation and alkaline lysis. We applied this approach to seven different mouse tissues. Isolation of mitochondria yields a preparation of highly enriched mtDNA. We compared this method to the commonly used PCR-based method. Mito-SiPE avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mtDNA enrichment. DiscussionWe have described a modified version of a long-established protocol for purifying mtDNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low-frequency heteroplasmy without introducing PCR biases or NUMT contamination that are falsely identified as heteroplasmy when long-range PCR is used.

genomics↗