Biology subjects

Sheppard, S.

Publications and source records attributed to Sheppard, S..

16 recordsLinked to original sources

bioRxiv2026

In vivo screening reveals new regulators of Natural Killer cell development and functional response to acute cytomegalovirus infection

Natural killer (NK) cells are critical innate immune effectors in antiviral and antitumour responses. However, the factors governing NK cell development and function remain incompletely understood. To address this, we adopted an in vivo screening approach, generating a panel of mice deficient in genes differentially expressed across NK cell maturation stages or following viral infection. We performed a combinational screen comprising baseline immunophenotyping and mouse cytomegalovirus (MCMV) challenge. We identified novel regulators of NK cell development, including transcriptional regulators and proteins with putative trafficking functions. MCMV challenge studies additionally identified proteins that impacted antiviral immunity independently of NK cell phenotypes, including a novel regulator of NK cell degranulation, Synaptotagmin-like protein 3. Identified genes exhibited reduced tolerance to loss-of-function in large scale human sequencing studies and evidence for reduced NK cell numbers in a cohort of immunodeficiency patients. Together, these findings provide a resource of NK-expressed genes important for NK cell maturation and function, with relevance to human health. SummaryPring et al. conduct an in vivo screen in mice identifying novel regulators of natural killer (NK) cell development and/or antiviral functionality. Large-scale human sequencing and human immunodeficiency patients revealed evidence for the relevance of identified genes in human health.

immunology↗

bioRxiv2025

Single-cell neural network classifiers reveal that PM21 NK cell expansion is dependent on B cell signaling

In the field drug development ML/AI methods are being applied to improve drug production speed, costs, and reliability. In allogenic NK cell therapy production, one of the biggest challenges is the inherent variability in the donors that provide the starting material for NK cell expansion. In this study we performed PM21-mediated NK cell expansion on 26 donors, and in parallel performed single-cell transcriptomics on the same donor sample prior to expansion. Canonical differential expression analysis and cell state abundance did not highlight any significant difference between donors with high and low NK cell expansion yield. Instead, training neural networks classifiers for high-yield donors enabled identifying several highly predictive models with perfect cross-validation recall. Further investigation of the most predictive models unveiled a previously unknown role for B cell in supportive NK cell expansion. Overall, this study represents a blueprint for combining deep phenotyping and machine learning methods to unveil novel biology and improve the quality and speed of delivery of cell therapeutics to patients.

systems biology↗

bioRxiv2025

Non-catalytic and catalytic TREHALOSE-6-PHOSPHATE SYNTHASES interact with RAMOSA3 to control maize development.

Trehalose-6-phosphate (Tre6P) is the intermediate in the two-step pathway of trehalose biosynthesis mediated by Tre6P-synthases (TPSs) and Tre6P-phosphatases (TPPs). Plants harbor small families of TPS and TPP genes, however most plant TPSs lack enzymatic activity, suggesting they have regulatory functions. The classical mutant ramosa3 (ra3) increases inflorescence branching in maize, and RA3 encodes a catalytic TPP. We found that RA3 interacts with maize ZmTPS1, a non-catalytic TPS. Mutants in ZmTPS1 and its close paralog ZmTPS12 enhance ra3 phenotypes, suggesting their physical interaction is biologically significant. ZmTPS1 also interacts with the two catalytically active maize TPSs, ZmTPS11 and ZmTPS14, however zmtps11;zmtps14 double mutants fail to complete embryogenesis, suggesting that they are essential, as in arabidopsis. Interestingly, the non-catalytic ZmTPS1 protein stimulated the coupled activity of RA3 and ZmTPS14, suggesting that RA3, ZmTPS1, and ZmTPS14 form a complex, and we confirmed this by expressing and purifying the three proteins and by Alphafold predictions. Our results suggest that non-catalytic TPSs form a complex with catalytic TPSs and TPPs to stimulate catalytic activity and regulate plant development.

plant biology↗

bioRxiv2025

Zinc-finger proteins with a co-opted capsid domain anchor nucleosomes over transposon sequences

Transposable elements (TEs) are frequently co-opted as cis-regulatory sequences that govern multiple aspects of host biology. The regulatory activity of these domesticated sequences are controlled by host factors, notably KRAB domain-containing zinc-finger proteins (KZFPs) in tetrapods. Here, we report that SCAN domain-containing zinc-finger proteins (SZFPs), which originally arose through capture of a retroviral capsid domain by a KZFP gene, have expanded and diversified their DNA recognition specificity to bind distinct TE subfamilies. We further demonstrate that SZFPs anchor nucleosomes at their target sites, and that their depletion leads to global shifts of nucleosomes away from underlying TE-derived sequences, occasionally accompanied by a gain of enhancer-associated chromatin states. Thus, SZFPs represent a novel layer of chromatin regulation centered on rapidly evolving TE-derived regulatory sequences.

genetics↗

bioRxiv2024

Tracing Endometriosis: Coupling deeply phenotyped, single-cell based Endometrial Differences and AI for disease pathology and prediction

Endometriosis, affecting 1 in 9 women, presents treatment and diagnostic challenges. To address these issues, we generated the biggest single-cell atlas of endometrial tissue to date, comprising 466,371 cells from 35 endometriosis and 25 non-endometriosis patients without exogenous hormonal treatment. Detailed analysis reveals significant gene expression changes and altered receptor-ligand interactions present in the endometrium of endometriosis patients, including increased inflammation, adhesion, proliferation, cell survival, and angiogenesis in various cell types. These alterations may enhance endometriosis lesion formation and offer novel therapeutic targets. Using ScaiVision, we developed neural network models predicting endometriosis of varying disease severity (median AUC = 0.83), including an 11-gene signature-based model (median AUC = 0.83) for hypothesis-generation without external validation. In conclusion, our findings illuminate numerous pathway and ligand-receptor changes in the endometrium of endometriosis patients, offering insights into pathophysiology, targets for novel treatments, and diagnostic models for enhanced outcomes in endometriosis management.

molecular biology↗

bioRxiv2024

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments

Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping 3D genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here we reveal the essential role of ZNF274, a SCAN-bearing member of the Kruppel-associated box (KRAB)-containing zinc finger proteins (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods - encompassing, among others, protocadherin and KZFP-encoding genes - with loss of repressive chromatin marks, altered 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NADs organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.

molecular biology↗

bioRxiv2023

Contrasting genes conferring short and long-term biofilm adaptation in Listeria

Listeria monocytogenes is an opportunistic food-borne bacterium that is capable of infecting humans with high rates of hospitalisation and mortality. Natural populations are genotypically and phenotypically variable, with some lineages being responsible for most human infections. The success of L. monocytogenes is linked to its capacity to persist on food and in the environment. Biofilms are an important feature that allow these bacteria to persist and infect humans, therefore, understanding the genetic basis of biofilm formation is key to understanding transmission. We sought to investigate the biofilm forming ability of L. monocytogenes by identifying genetic variation that underlies biofilm formation in natural populations using genome-wide association studies. Changes in gene expression of specific strains during biofilm formation were then investigated using RNAseq. Genetic variation associated with enhanced biofilm formation was identified in 273 genes by GWAS and differential expression in 220 genes by RNAseq. Statistical analyses show that number of overlapping genes flagged by either type of experiment is less than expected by random sampling. This is consistent with an evolutionary scenario where rapid adaptation is driven by variation in gene expression of pioneer genes, and this is followed by slower adaptation driven by nucleotide changes within the core genome. Impact statementListeria monocytogenes is a problematic food-borne bacterium that can cause severe illness and even death in humans. Some strains are known to be more common in disease and biofilms are crucial for survival in the environment and transmission to humans. To unravel the genetic basis of biofilm formation, we undertook a study employing genome-wide association studies (GWAS) and gene transcription profiling. We identified 273 genes associated with robust biofilm formation through GWAS and discovered differential expression in 220 genes through RNAseq. Statistical analysis revealed fewer overlapping genes than expected by chance, supporting an evolutionary scenario where initial adaptation relies on gene expression variation, followed by slower adaptation through genetic changes within the core genome. Data summaryShort read genome data are available from the NCBI (National Center for Biotechnology Information) SRA (Sequence Read Archive), associated with BioProject PRJNA971143 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA971143). Assembled genomes and supplementary material are available from FigShare: doi: 10.6084/m9.figshare.23148029. RNA sequence data and differential gene expression profiles have been deposited in the NCBI Gene Expression Omnibus.

microbiology↗

bioRxiv2023

Deconvolution of ex-vivo drug screening data and bulk tissue expression predicts the abundance and viability of cancer cell subpopulations

Ex-vivo drug sensitivity screening allows the prediction of cancer treatment effectiveness in a personalized fashion. However, it only provides a readout on mixtures of cells, potentially occulting important information on clinically relevant cell subtypes. To address this shortcoming, we developed a machinelearning framework to decompose drug sensitivity recorded at the bulk level into cell subtype-specific drug sensitivity. We first determined that our method could decipher the cellular composition of bulk samples with top-ranking accuracy across five cancer types compared to state-of-the-art bulk deconvolution methods. We emphasize its effectiveness in the realm of Acute Myeloid Leukemia, where it appears to offer the most precise estimation of leukemic stem cell fractions across three test datasets and three patient cohorts. We then optimized an algorithm capable of estimating cell subtype- and single-cell-specific drug sensitivity, which we evaluated by performing in-vitro drug studies and in-depth simulations. We then applied our deconvolution strategy to the beatAML cohort dataset, currently the most extensive database of ex-vivo drug screening data. We developed a drug sensitivity profile tailored to specific cell subtypes, focusing on four therapeutic compounds predicted to target leukemic stem cells: the previously known midostaurin and A-674563, as well as SNS-032 and foretinib, which have not been previously linked to leukemic stem cells. Our work provides an attractive new computational tool for drug development and precision medicine.

bioinformatics↗

bioRxiv2022

Statistical learning quantifies transposable element-mediated cis-regulation

BackgroundTransposable elements (TEs) have colonized the genomes of most metazoans, and many TE-embedded sequences function as cis-regulatory elements (CREs) for genes involved in a wide range of biological processes from early embryogenesis to innate immune responses. Because of their repetitive nature, TEs have the potential to form CRE platforms enabling the coordinated and genome-wide regulation of protein-coding genes by only a handful of trans-acting transcription factors (TFs). ResultsHere, we directly test this hypothesis through mathematical modeling and demonstrate that differences in expression at protein-coding genes alone are sufficient to estimate the magnitude and significance of TE-contributed cis-regulatory activities, even in contexts where TE-derived transcription fails to do so. We leverage hundreds of overexpression experiments and estimate that, overall, gene expression is influenced by TE-embedded CREs situated within approximately 200kb of promoters. Focusing on the cis-regulatory potential of TEs within the gene regulatory network of human embryonic stem cells, we find that pluripotency-specific and evolutionarily young TE subfamilies can be reactivated by TFs involved in post-implantation embryogenesis. Finally, we show that TE subfamilies can be split into truly regulatorily active versus inactive fractions based on additional information such as matched epigenomic data, observing that TF binding may better predict TE cis-regulatory activity than differences in histone marks. ConclusionOur results suggest that TE-embedded CREs contribute to gene regulation during and beyond gastrulation. On a methodological level, we provide a statistical tool that infers TE-dependent cis-regulation from RNA-seq data alone, thus facilitating the study of TEs in the next-generation sequencing era.

genomics↗

bioRxiv2022

Transposable elements contribute to the spatiotemporal microRNA landscape in human brain development

Transposable elements (TEs) contribute to the evolution of gene regulatory networks and are dynamically expressed throughout human brain development and disease. One gene regulatory mechanism influenced by TEs is the miRNA system of post-transcriptional control. miRNA sequences frequently overlap TE loci and this miRNA expression landscape is crucial for control of gene expression in adult brain and different cellular contexts. Despite this, a thorough investigation of the spatiotemporal expression of TE-embedded miRNAs in human brain development is lacking. Here, we identify a spatiotemporally dynamic TE-embedded miRNA expression landscape between childhood and adolescent stages of human brain development. These miRNAs sometimes arise from two apposed TEs of the same subfamily, such as for L2 or MIR elements, but in the majority of cases stem from solo TEs. They give rise to in silico predicted high-confidence pre-miRNA hairpin structures, likely represent functional miRNAs and have predicted genic targets associated with neurogenesis. TE-embedded miRNA expression is distinct in the cerebellum when compared to other brain regions, as has previously been described for gene and TE expression. Furthermore, we detect expression of previously non-annotated TE-embedded miRNAs throughout human brain development, suggestive of a previously undetected miRNA control network. Together, as with non-TE-embedded miRNAs, TE-embedded sequences give rise to spatiotemporally dynamic miRNA expression networks, the implications of which for human brain development constitute extensive avenues of future experimental research. To facilitate interactive exploration of these spatiotemporal miRNA expression dynamics, we provide the "Brain miRTExplorer" web application freely accessible for the community.

genomics↗

bioRxiv2021

Repeated out-of-Africa expansions of Helicobacter pylori driven by replacement of deleterious mutations

Helicobacter pylori lives in the human stomach and has a population structure which resembles that of its host. However, H. pylori from Europe and the Middle East trace a substantially higher fraction of ancestry from modern African populations than the humans that carry them. Here, we used a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations compared to either non-segregating or synonymous sites, implying selection to remove them. We used simulations to show that demographic bottlenecks can lead to long-term segregation of deleterious mutations, despite high rates of homologous recombination, but that population fitness can be restored by migration of small numbers of bacteria from non-bottlenecked populations, leading to mosaic patterns of ancestry like that seen for H. pylori. We conclude that H. pylori have been able to spread repeatedly from Africa by outcompeting strains that carried deleterious mutations accumulated during the original out-of-Africa bottleneck.

evolutionary biology↗

bioRxiv2021

Digital 3D Brain MRI Arterial Territories Atlas

The locus and extent of brain damage in the event of vascular insult can be quantitatively established quickly and easily with vascular atlases. Although highly anticipated by clinicians and clinical researchers, no digital MRI arterial atlas is readily available for automated data analyses. We created a digital arterial territory atlas based on lesion distributions in 1,298 patients with acute stroke. The lesions were manually traced in the diffusion-weighted MRIs, binary stroke masks were mapped to a common space, probability maps of lesions were generated and the boundaries for each arterial territory was defined based on the ratio between probabilistic maps. The atlas contains the definition of four major supra- and infra-tentorial arterial territories: Anterior, Middle, Posterior Cerebral Arteries and Vertebro-Basilar, and sub-territories (thalamoperforating, lenticulostriate, basilar and cerebellar arterial territories), in two hierarchical levels. This study provides the first publicly-available, digital, 3D deformable atlas of arterial brain territories, which may serve as a valuable resource for large-scale, reproducible processing and analysis of brain MRIs of patients with stroke and other conditions.

neuroscience↗

bioRxiv2020

Transposable elements and their KZFP controllers are drivers of transcriptional innovation in the developing human brain

Transposable elements (TEs) constitute 50% of the human genome and many have been co-opted throughout human evolution due to gain of advantageous regulatory functions controlling gene expression networks. Several lines of evidence suggest these networks can be fine-tuned by the largest family of TE controllers, the KRAB-containing zinc finger proteins (KZFPs). One tissue permissive for TE transcriptional activation (termed transposcription) is the adult human brain, however comprehensive studies on the extent of this process and its potential contribution to human brain development are lacking. In order to elucidate the spatiotemporal transposcriptome of the developing human brain, we have analysed two independent RNA-seq datasets encompassing 16 distinct brain regions from eight weeks post-conception into adulthood. We reveal an anti-correlated, KZFP:TE transcriptional profile defining the late prenatal to early postnatal transition, and the spatiotemporal and cell type specific activation of TE-derived alternative promoters driving the expression of neurogenesis-associated genes. We also demonstrate experimentally that a co-opted antisense L2 element drives temporal protein re-localisation away from the endoplasmic reticulum, suggestive of novel TE dependent protein function in primate evolution. This work highlights the widespread dynamic nature of the spatiotemporal KZFP:TE transcriptome and its potential importance throughout neurotypical human brain development.

genomics↗

bioRxiv2018

Understanding the killing mechanism of action by virus-infected yeasts

Killer yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via infection by a virus. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts have been considered as beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill species off starter cultures and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action in silico. Our deterministic model explains how killer Saccharomyces cerevisiae cells distress and consequently kill the sensitive members of the species, accounting for the K1, K2 and K28 toxin mode of action at high or low concentrations. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death, and allowed us to gain novel insight on these mechanisms. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately 3 times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.

systems biology↗

bioRxiv2017

Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

A critical step towards malaria elimination will be the interruption of Plasmodium transmission from the human host to the mosquito. At the core of the transmission cycle lies Plasmodium sexual reproduction leading to zygote formation and mosquito midgut colonisation by ookinetes. Whilst in vitro ookinete culture from the murine and avian malaria parasites, Plasmodium berghei and P. gallinaceum, has greatly increased our knowledge of transmission biology; efforts to mimic the process in the human parasite P. falciparum have, to date, had only limited success. Using fluorescence microscopy and flow cytometry with antibodies specific to the male gametocyte and developing ookinetes, we sought to evaluate P. falciparum ookinete production using previously published in vitro protocols. We then compared in vitro versus in vivo ookinete production in both P. falciparum and P. berghei parasites, exploring potential barriers to complete development. Finally, we sought to test a wide range of literature-led culture conditions towards further optimisation of in vitro P. falciparum ookinete production. Despite extensive testing, our efforts to replicate published methods did not produce appreciable quantities of fully formed P. falciparum ookinetes in vitro. In parallel, however, gametocyte cultures that failed to differentiate fully in vitro successfully developed into ookinetes in vivo with an efficiency approximating that of P. berghei. Flow cytometry analysis showed that this disparity likely lies with the poor fertilization of P. falciparum gametes in vitro. Attempts to improve gametocyte fertility or define conditions more permissive to fertilisation/ookinete survival in vitro were also unsuccessful. Current in vitro conditions for P. falciparum ookinete production are not optimal for gamete fertilisation either due to the lack of parasite-species-specific mosquito factors missing from in vitro culture, or non-permissive cues contaminating culture preparations.

microbiology↗

bioRxiv2017

Epistasis mediated alleviation of the cost of antibiotic resistance for MRSA

Understanding how multi-drug resistant pathogens evolve is key to identifying means of curtailing their further emergence and dissemination. Fitness costs imposed on bacteria by resistance mechanisms are believed to hamper their dissemination in an antibiotic free environment, however, some have been reported to have little or no cost, which suggests there are few barriers preventing their global spread. One such apparently cost-free resistance mechanism acquired by the major human pathogen Staphylococcus aureus is to the clinically important antibiotic mupirocin, which is mediated by mutation of the highly-conserved and essential isoleucyl-tRNA synthethase (ileS) gene. In Genome Wide Association Studies (GWAS) on two genetically and geographically distinct MRSA lineages we have found this mutation to be associated with changes in bacterial virulence, driven through epistatic interactions with other loci. Given the potential dual effect of this mutation on both antibiotic resistance and virulence we adopted a proteomic approach and observed pleiotropic effects. This analysis revealed that the activity of the secretory apparatus of the PSM family of cytolytic toxins, the Pmt system, is affected in the mupirocin resistant mutant, which explains why it is less toxic. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production is required. Given the widespread use of this antibiotic, and that this resistance often results from a single nucleotide substitution in the ileS gene, these hidden fitness costs provide an explanation for why this resistance mechanism is not more prevalent. This work also demonstrates how population-based genomic analysis of virulence and antibiotic resistance can contribute to uncovering hidden features of the biology of microbial pathogens.

microbiology↗