Biology subjects

Shepherd, C.

Publications and source records attributed to Shepherd, C..

8 recordsLinked to original sources

bioRxiv2026

Modular Scaffold Crystals for Programmable Installation and Structural Observation of DNA-Binding Proteins

Inducing biomacromolecules to self-assemble into diffraction-quality crystals remains a major challenge, typically overcome by brute-force experimental screening. Inspired by Seemans vision of DNA-junction-based scaffolds organizing guest biomacromolecules, we developed a protein-DNA co-crystal combining modular DNA programmability with robust protein-lattice diffraction. Our engineered co-crystals are composed of stacked double-stranded DNA scaffolded by protein columns, surrounding solvent channels designed to enable guest protein diffusion. DNA strut variation allows positionally-controlled installation of diverse DNA binding guest proteins. Experimentally, we simply grow scaffold crystals under standardized conditions, ligate the scaffold, and soak guest proteins. Decoupling crystal growth from guest installation will enable high-throughput structure determination of diverse DNA-binding proteins and protein-macromolecule conjugates. Sub-nanometer position and orientation control of guest macromolecules will also enable functional applications beyond structural biology.

bioengineering↗

bioRxiv2026

Coxsackievirus B Escapes Antiviral CD8⁺ T Cells but Triggers Robust CD4⁺ Memory Responses

Coxsackieviruses B (CVBs) are plausible triggers of the pancreatic islet autoimmunity leading to type 1 diabetes. Islet autoantibody seroconversion correlates with persistent CVB infections in the gut and pancreas, suggesting defective antiviral control and the need to define immune mechanisms at the intestinal entry site. We investigated how CVB3 modulates antigen presentation, the viral immunopeptidome of enterocytes and antigen-presenting cells, and downstream T-cell immunity. CVB3-infected enterocytes escaped immune recognition by downregulating HLA Class I and viral peptide presentation, impairing CD8+ T-cell responses in vitro. In CVB-seropositive individuals, circulating CVB-reactive CD8+ T cells were stalled in naive-like and exhausted effector/memory states. In contrast, CVB3 induced HLA class II upregulation, promoting robust CD4 T-cell activation. Circulating CVB3-reactive CD4 T cells fully differentiated into polyfunctional T helper memory. These findings indicate that CVB3 antiviral control is predominantly CD4 T-cell-mediated and provide a rationale for mucosal vaccination strategies and immune monitoring tools to follow infection or vaccination. TeaserCoxsackievirus evades CD8+ T-cell immunity in the gut, leaving CD4+ T cells as the main line of antiviral defense.

immunology↗

bioRxiv2025

Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites

Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.

molecular biology↗

bioRxiv2022

The demand for wildlife not protected by the CITES multilateral treaty

The international wildlife trade presents severe conservation and environmental security risks. However, no international regulatory framework exists to monitor the trade of species not listed in the appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). We explored the composition and dynamics of internationally regulated versus non-regulated trade, focussing on importations of wild-caught terrestrial vertebrates entering the United States of America (US) from 2009-2018. The prominence of the US in global wildlife imports and its detailed data collection conventions allows a unique opportunity to formally assess this substantial but often overlooked and understudied component of the legal wildlife trade. We found 3.6 times the number of unlisted species in US imports compared with CITES-listed species (1,366 versus 378). CITES-listed species were more likely to face reported conservation threats relative to the unlisted species (71.7% vs 27.5%). Yet, we found 376 unlisted species facing conversation threats, 297 species with unknown population trends and 139 species without an evaluation by the IUCN Red List of Threatened Species. Unlisted species appeared novelly in imports at 5.5 times higher rates relative to CITES-listed species, where unlisted reptiles saw the largest rate of entry, averaging 53 unique species appearing in imports for the first time per year. Overall trade volumes were substantially larger for unlisted imports with approximately 11 times the number of animals relative to CITES-listed imports, however, import volumes were similar when compared at a species-by-species level. We found that the countries that were top exporters for CITES-listed shipments were mostly different from exporters of unlisted species. In highlighting the vulnerabilities of the wild-caught unlisted vertebrate trade entering the US and in the face of increasing global demand, we recommend governments adapt policies to monitor the trade of all wildlife.

ecology↗

bioRxiv2018

Heritability and genetic variance of dementia with Lewy bodies

Recent large-scale genetic studies have allowed for the first glimpse of the effects of common genetic variability in dementia with Lewy bodies (DLB), identifying risk variants with appreciable effect sizes. However, it is currently well established that a substantial portion of the genetic heritable component of complex traits is not captured by genome-wide significant SNPs. To overcome this issue, we have estimated the proportion of phenotypic variance explained by genetic variability (SNP heritability) in DLB using a method that is unbiased by allele frequency or linkage disequilibrium properties of the underlying variants. This shows that the heritability of DLB is nearly twice as high as previous estimates based on common variants only (31% vs 59.9%). We also determine the amount of phenotypic variance in DLB that can be explained by recent polygenic risk scores from either Parkinsons disease (PD) or Alzheimers disease (AD), and show that, despite being highly significant, they explain a low amount of variance. Additionally, to identify pleiotropic events that might improve our understanding of the disease, we performed genetic correlation analyses of DLB with over 200 diseases and biomedically relevant traits. Our data shows that DLB has a positive correlation with education phenotypes, which is opposite to what occurs in AD. Overall, our data suggests that novel genetic risk factors for DLB should be identified by larger GWAS and these are likely to be independent from known AD and PD risk variants.

neuroscience↗

bioRxiv2018

Functional Testing of Thousands of Osteoarthritis-Associated Variants for Regulatory Activity

To date, genome-wide association studies have implicated at least 35 loci in osteoarthritis, but due to linkage disequilibrium, we have yet to pinpoint the specific variants that underlie these associations, nor the mechanisms by which they contribute to disease risk. Here we functionally tested 1,605 single nucleotide variants associated with osteoarthritis for regulatory activity using a massively parallel reporter assay. We identified six single nucleotide polymorphisms (SNPs) with differential regulatory activity between the major and minor alleles. We show that our most significant hit, rs4730222, drives increased expression of an alternative isoform of HBP1 in a heterozygote chondrosarcoma cell line, a CRISPR-edited osteosarcoma cell line, and in chondrocytes derived from osteoarthritis patients.

genomics↗

bioRxiv2018

Hookworm-derived small molecule extracts suppress pathology in a mouse model of colitis and inhibit secretion of key inflammatory cytokines in primary human leukocytes

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Here we use a metabolomics approach to characterize the non-protein small molecule complement of hookworms. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of somatic tissue extracts revealed the presence of 52 polar metabolites and 22 non-polar components including short chain fatty acids (SCFA). Several of these small metabolites, notably the SCFA, have been shown to have anti-inflammatory properties in various diseases, including IBD. Using a murine model of colitis and human peripheral blood mononuclear cells, we demonstrate that somatic tissue extracts of the hookworm Ancylostoma caninum contain small molecules with anti-inflammatory activities. Of the five extracts tested, two of them significantly protected mice against T cell-mediated immunopathology and weight loss in a chemically-induced colitis model. Moreover, one of the anti-colitic extracts suppressed ex vivo production of inflammatory cytokines from primary human leukocytes. While the origin of the SCFA (parasite or host microbiota-derived) present in the hookworm somatic tissue extracts cannot be ascertained from this study, it is possible that A. caninum may be actively promoting an anti-inflammatory host microbiome by facilitating immune crosstalk through SCFA production.

microbiology↗

bioRxiv2017

Revisiting The Ancylostoma caninum Secretome Provides New Information On Hookworm-Host Interactions

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anaemia and other complications. To avoid the hosts immune response the parasite releases excretory/secretory products (ESPs), a complex mixture of glycans, lipids and proteins that represent the major host-parasite interface. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalysed the dog hookworm, Ancylostoma caninum, ESPs (AcES). We identified 315 proteins present in the AcES, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (90 of the 315 proteins), and the most abundant constituents of AcES are homologues of the tissue inhibitors of metalloproteases (TIMP) family. We identified putative vaccine candidates and proteins that could have immunomodulatory effects for treating inflammatory diseases. This study provides novel information about the proteins involved in host-hookworm interactions, and constitutes a comprehensive dataset for the development of vaccines and the discovery of new immunoregulatory biologics.

microbiology↗