Biology subjects

Stephens, C.

Publications and source records attributed to Stephens, C..

12 recordsLinked to original sources

bioRxiv2026

Multimodal Human Scalp Atlas Defines Cell Landscape and Lineage Architecture In Situ

Human scalp hair has an extraordinary ability to grow continuously for years while maintaining structural and functional integrity. However, the cell states and lineage organization that enable this capacity and how they are disrupted in inflammatory hair loss disorders remain poorly defined in humans. Here we establish a high-resolution, multimodal atlas of human scalp by integrating deep-coverage spatial transcriptomics with single-cell RNA-seq and multiomics data. This reference resolves spatially organized epithelial and mesenchymal states and links in situ transcriptional programs to chromatin accessibility dynamics and lineage trajectories at single-cell resolution, revealing human-specific principles of tissue organization and previously unrecognized features of hair follicle architecture and lineage progression. We validate key aspects of matrix cell organization and cell activities using live imaging, connecting molecularly defined cell states to dynamic cell behaviors and lineage progression in the matrix. Leveraging the atlas as a spatial reference, we project patient scRNA-seq profiles from alopecia areata and lichen planopilaris onto defined cell compartments, resolving disease-specific perturbations in fibroblasts, epithelial and immune populations. This comparison delineates distinct cellular programs associated with non-scarring versus scarring hair loss and highlights compartment- and state-specific pathways with diagnostic and therapeutic potential. Together, this work provides a foundational resource for human hair biology and establishes a generalizable framework for spatially resolved, multimodal interrogation of tissue organization and disease in complex human tissues.

Developmental Biology↗

bioRxiv2026

High-purity stem cell-derived β-cells recapitulate key transcriptional and functional features of human islets

Human pluripotent stem cell-derived islets (SC-islets) offer an excellent medium for human pancreatic disease modelling and mechanistic studies into diabetes. While substantial progress has been made in differentiation protocols, their implementation in different laboratories result in variable {beta}-cell proportions with contaminant non-endocrine and proliferative cell types. To date, no facility-level implementation exists for producing SC-islets that can be shipped and benchmarked across multiple sites. Here, we describe the scalable optimisation, standardization, and facility-level implementation of an established human stem cell differentiation strategy that consistently results in a high proportion of {beta}-cells, with up to 75% of cells co-expressing C-peptide and the pancreatic endocrine marker, ISL1. Functionally, SC-islets exhibit glucose-responsive calcium influx and insulin secretion, recapitulating key physiological {beta}-cell functions. Single-cell transcriptomic profiling reveals a simplified endocrine landscape dominated by {beta}-cells, with a striking transcriptional similarity to human primary {beta}-cells (Pearsons r2[~]0.9). We observe smaller fractions of - and enterochromaffin-like cells with very low levels of poly-hormonal or proliferating cell types (<3%). Taken together, we provide a well-defined, reproducible and accessible in vitro SC-islet platform benchmarked for functionality at multiple recipient sites.

cell biology↗

bioRxiv2026

Student-Driven Microbiome Exploration: A Low-Cost 16S rRNA Sequencing Curriculum for Undergraduate Biology Education

Microbiome science is increasingly important in modern biology education because microbial communities influence human health, ecosystems, and environmental processes. However, undergraduate microbiome instruction is often limited by the high cost and technical complexity of sequencing-based workflows, restricting opportunities for authentic student-driven research. To address this challenge, we developed a low-cost, inquiry-based curriculum that enables undergraduate students to conduct complete microbiome studies using 16S rRNA gene sequencing. The module integrates project design, environmental sample collection, microbial cell processing, PCR amplification, sequencing, and bioinformatic analysis using open-source tools such as QIIME 2. Cost-reduction strategies included centrifugation-based cell collection and a surfactant-assisted direct PCR workflow that eliminated the need for commercial DNA extraction kits. Students designed independent research projects investigating microbial communities in local environments, including campus water sources and gym equipment surfaces. Assessment data from post-course surveys, knowledge checks, and student research products demonstrated strong learning gains in microbiome concepts, molecular biology techniques, scientific communication, and computational analysis. Students reported high confidence in PCR, experimental design, and microbiome interpretation, while also identifying bioinformatics as the most challenging yet rewarding component of the curriculum. All participants expressed increased interest in future research in microbiology or bioinformatics. Overall, this curriculum provides an accessible, scalable framework for integrating next-generation sequencing into undergraduate education while promoting inquiry-driven learning, student ownership, and engagement in authentic scientific research.

microbiology↗

bioRxiv2026

A broad-host-range Rhizobium rhizogenes strain enables transient expression across diverse crops and establishes functional assays in faba bean

Agrobacterium-mediated transient expression has revolutionized plant research, enabling numerous landmark discoveries across diverse areas of plant biology. Yet this powerful approach remains largely confined to solanaceous species, leaving most economically important crop families without a comparable rapid assay platform. Here, we show that an engineered Rhizobium rhizogenes strain, AS109, mediates efficient transient expression across diverse dicot species spanning multiple taxonomic families, consistently outperforming commonly used laboratory agrobacterial strains. Leveraging the broad host range of AS109, we establish a suite of functional assays in faba bean (Vicia faba), including protein localisation, RNA interference-mediated gene silencing, cell-surface elicitor recognition screens, nucleotide-binding leucine-rich repeat receptor (NLR) activation, and infection cell biology at the host-pathogen interface. We further demonstrate that both singleton NLRs and sensor-helper NLR pairs from Solanaceae retain effector recognition and cell death activity when transferred into faba bean, establishing a rapid platform for evaluating cross-family transferability of disease-resistance genes. AS109 thus provides an accessible and versatile chassis for functional genomics in non-model crops, bridging the widening gap between hypothesis generation and experimental validation across diverse plant species.

plant biology↗

bioRxiv2025

A fungal root endophyte functionally complements host immunity and mitigates natural immune variation in Arabidopsis

Beneficial root-associated microbes can enhance plant resilience by complementing aspects of host immunity. The fungal root endophyte Serendipita indica (Si) is known to promote plant growth and confer broad stress tolerance. To assess how natural host genetic variation influences Si-mediated protection, we screened 47 Arabidopsis thaliana accessions for susceptibility to the fungal pathogen Bipolaris sorokiniana (Bs) with and without Si colonization. All accessions benefited from Si, indicating that endophyte-mediated disease mitigation occurs broadly across diverse host genotypes. A focused comparison of two genetically and geographically proximate Swedish accessions, T510 and T530, which displayed the most divergent protection scores, revealed substantial differences in Bs susceptibility. Transcriptome profiling under bi- and tripartite colonization showed conserved defense responses in both accessions. Bs infection downregulated growth- and development-related genes, consistent with a growth-immunity trade-off, with T530 exhibiting higher Bs colonization and a stronger transcriptional response than T510. Co-colonization with Si effectively suppressed pathogen growth and disease symptoms in both accessions. Comparative genomic and transcriptomic analyses identified four immune receptor genes, including the TIR-NLR ISI, present in T510 but absent in T530. An isi T-DNA insertion mutant phenocopied the heightened Bs susceptibility of T530, confirming that ISI contributes to root immunity, while Si-mediated protection remained intact despite increased pathogen susceptibility. Together, these findings demonstrate that fungal endophytes can mitigate the functional consequences of natural immune variation and enhance the resilience of genetically diverse plant populations. HighlightsO_LIS. indica confers broad protection against B. sorokiniana largely independent of host genotype or pathogen susceptibility. C_LIO_LIThe TIR-NLR immune receptor ISI contributes to root immunity but is not essential for S. indica-mediated protection. C_LIO_LIBeneficial endophytes can mitigate natural immune variation effects and support overall plant health. C_LI

ecology↗

bioRxiv2024

Genomes of two invasive Adelges species (hemlock woolly adelgid and pineapple gall adelgid) enable characterization of nicotinic acetylcholine receptors

Two invasive hemipteran adelgids are associated with widespread damage to several North American conifer species. Adelges tsugae, hemlock woolly adelgid, was introduced from Japan and reproduces parthenogenetically in North America, where it has rapidly decimated Tsuga canadensis and Tsuga caroliniana (the eastern and Carolina hemlocks, respectively). Adelges abietis, eastern spruce gall adelgid, introduced from Europe, forms distinctive pineapple-shaped galls on several native spruce species. While not considered a major forest pest, it weakens trees and increases susceptibility to additional stressors. Broad-spectrum insecticides that are often used to control adelgid populations can have off-target impacts on beneficial insects. Whole genome sequencing was performed on both species to aid in development of targeted solutions that may minimize ecological impact. Adelges abietis was sequenced using Illumina Linked-Read technology from 30 pooled individuals, with Hi-C scaffolding performed using data from a single individual collected from the same host plant. Adelges tsugae used Oxford Nanopore long-read sequencing from pooled nymphs. The assembled A. tsugae and A. abietis genomes, pooled from several parthenogenetic females, are 220.75 Mbp and 253.16 Mbp, respectively. Each consists of eight autosomal chromosomes, as well as two sex chromosomes (X1/X2), supporting the XX-XO sex determination system. The genomes are over 96% complete based on BUSCO assessment. Genome annotation identified 11,424 and 12,060 protein-coding genes in A. tsugae and A. abietis, respectively. Comparative analysis of proteins across 29 hemipteran species and 14 arthropod outgroups identified 31,666 putative gene families. Gene family evolution analysis with CAFE revealed lineage-specific expansions in immune-related aminopeptidases (ERAP1) and juvenile hormone binding proteins (JHBP), contractions in juvenile hormone acid methyltransferases (JHAMT), and conservation of nicotinic acetylcholine receptors (nAChR). These genes were explored as candidate families towards a long-term objective of developing adelgid-selective insecticides. Structural comparisons of proteins across seven focal species (Adelges tsugae, Adelges abietis, Adelges cooleyi, Rhopalosiphum maidis, Apis mellifera, Danaus plexippus, and Drosophila melanogaster) revealed high conservation of nAChR and ERAP1, while JHAMT exhibited species-specific structural divergence. The potential of JHAMT as a lineage-specific target for pest control was explored through virtual drug and pesticide screening.

genomics↗

bioRxiv2022

Primary Omicron infection elicits weak antibody response but robust cellularimmunity in children

Omicron variants of SARS-CoV-2 are globally dominant and infection rates are very high in children. We determined immune responses following Omicron BA.1/2 infection in children aged 6-14 years and related this to prior and subsequent SARS-CoV-2 infection or vaccination. Primary Omicron infection elicited a weak antibody response with poor functional neutralizing antibodies. Subsequent Omicron reinfection or COVID-19 vaccination elicited increased antibody titres with broad neutralisation of Omicron subvariants. Prior pre-Omicron SARS-CoV-2 virus infection or vaccination primed for robust antibody responses following Omicron infection but these remained primarily focussed against ancestral variants. Primary Omicron infection thus elicits a weak antibody response in children which is boosted after reinfection or vaccination. Cellular responses were robust and broadly equivalent in all groups, providing protection against severe disease irrespective of SARS-CoV-2 variant. Immunological imprinting is likely to act as an important determinant of long-term humoral immunity, the future clinical importance of which is unknown.

immunology↗

bioRxiv2022

The Universal Endurance Microbiome?

Billions of microbial cells sculpt the gut ecosystem, playing essential roles in human physiology. Since endurance athletes performance is often physiology-limited, understanding the composition and interactions within these athletes gut microbiomes could lead to improved performance. Previous studies describe differences in the relative abundance of bacterial taxa when comparing athletes versus controls or athletes before and after an endurance event, suggesting the existence of an "endurance microbiome". However, there are inconsistencies among studies in which taxa correlate with extended physical exertion. Although these studies employed similar barcoding methods, variation in downstream bioinformatic analyses makes it difficult to determine whether inconsistencies are due to methodological differences or biological factors. Herein, we created a metagenomic bioinformatics workflow reanalyzing four 16S rDNA sequence datasets reflecting endurance athletes gut microbiomes, looking at alpha diversity, changes in relative abundance of gut microbiome genera, changes in pairwise correlations between bacterial genera and compared bacterial association networks. There were no significant differences in alpha diversity between any of the four treatment group comparisons. For relative abundance, there were no consistent differences in all four datasets, and only two genera were significantly different in 50% of the datasets. Although many genera showed changes in pairwise correlations in endurance microbiome samples from individual datasets, none were consistent across datasets. Collectively, these results suggest that either there is no universal endurance microbiome, or that it remains elusive even after controlling for the bioinformatic workflow and statistical analyses. Using this data, a power analysis indicates that sample sizes 150- to 800-fold larger than these published studies would be necessary to detect a 10% difference in relative abundance. Furthermore, 10- to 20-fold more samples will be needed to control for the multitude of covariates (genetic, metabolic, dietary, environmental, and pharmacological factors) that mold the gut microbiome of athletes and non-athletes alike. Im going to work so that its a pure guts race at the end, and if it is, I am the only one who can win it. - Steve Prefontaine

microbiology↗

bioRxiv2022

GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking): A methodology to rapidly leverage whole genome sequencing of bacterial isolates for clinical identification

Whole genome sequencing of clinical bacterial isolates has the potential to transform the fields of medicine and public health, with particular impact on molecular epidemiology, infection control and assessing the spread of antibiotic resistance. To realize this potential, bioinformatic software needs to be developed that meets the quality standards of a diagnostic test to allow the reporting of identification results. Our research group has developed a methodology (GAMBIT: Genomic Approximation Method for Bacterial Identification and Tracking) using k-mer based strategies for identification of bacteria based on whole genome sequence reads. GAMBIT incorporates this algorithm with a highly curated searchable database of genomes, which is a subset of the NCBI RefSeq assembly database. In this manuscript, we describe the validation of the scoring methodology, robustness to chosen parameters, establishment of confidence thresholds and the curation of the reference database. We discuss a validation set with GAMBIT deployed as a laboratory-developed test at the Alameda County Public Health Laboratory in Oakland, California. Three advancements were required to build upon existing k-mer based strategies to allow GAMBIT to possess the quality control parameters desired for its use as a diagnostic laboratory-developed test. Firstly, we innovated the data structure used to store the database of known bacterial genomes. This allowed us to store 48,224 bacterial genomes in a k-mer based database--a majority of the bacterial genomes in the NCBI RefSeq database at the time of development. Secondly, curation of the NCBI RefSeq database was required to remove ambiguous or potentially incorrectly labeled bacterial genomes to greatly increase confidence in positive matches. Lastly, we used this curated version of the NCBI RefSeq database and our scoring method to generate confidence thresholds for identification. Thus, the end-user does not rely simply on the closest match, but is informed whether that closest match exceeds a threshold for highly confident identification. This method greatly reduces or eliminates false identifications which are often detrimental in a clinical setting.

bioinformatics↗

bioRxiv2022

A single episode of CMV reactivation initiates long-term expansion and differentiation of the NK cell repertoire

NK cells play an important role in suppression of viral replication and are critical for effective control of persistent infections such as herpesviruses. Cytomegalovirus infection is associated with expansion of adaptive-memory NK cells with a characteristic CD16brightCD56dim NKG2C+ phenotype but the mechanisms by which this population is maintained remain uncertain. We studied NK cell reconstitution in patients undergoing haemopoietic stem cell transplantation and related this to CMV reactivation. NK cells expanded in the early post-transplant period but then remained stable in the absence of viral reactivation. However, a single episode of CMV reactivation led to a rapid and sustained 10-fold increase in NK cell number. NKG2C expression was increased on all NK subsets although the kinetics of expansion peaked at 6 months on immature CD56bright cells whilst continuing to rise on the mature CD56dim pool. Phenotypic maturation was observed by acquisition of CD57 and KIR expression. Transplantation from CMV-seropositive donors was associated with more rapid NK expansion and superior control of viral reactivation. Effective control of viral reactivation was seen when the peripheral NK cell count reached 20,000/ml. These data show that a single episode of CMV reactivation acts to reprogramme hemopoiesis to drive a sustained modulation and expansion of the NK cell pool and reveal further insight into long term regulation of the innate immune repertoire by infectious challenge. Author Summaryim to highlight where your work fits within a broader context; present the significance or possible implications of your work simply and objectively; and avoid the use of acronyms and complex terminology wherever possible. The goal is to make your findings accessible to a wide audience that includes both scientists and non-scientists Innate immune cells respond rapidly to infectious challenge and it has previously been thought that they were unable to learn from specific infecitons but provided only short term control. However, in recent years it has become apparent that some subsets of innate cells, termed adaptive natural killer (NK) cells, are permanently increased in people who have become infected with cytomegalovirus. The mechanism by which this is maintained is not known. We studied the kinetics of adaptive NK cell expansion in patients who had recently undergone stem cell (bone marrow) transplantation as CMV very often reactivates in this setting and allows study of how this affects the immune system. We find that one short term episode of viral replication led to a large and prolonged expansion of adaptive NK cells. Indeed, their levls increasd 11-fold over 10 months.

immunology↗

bioRxiv2020

Remarkable recent changes in genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 alleles present in historic Z. tritici isolate collections, with up to ~ 18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of alleles allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted re-sequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world between 2013-2017. However, in contrast to the data from studies of historic isolates, our study revealed a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of alleles, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence alleles tend to be eliminated in subsequent populations.

microbiology↗

bioRxiv2019

Factors affecting pathogenicity of the turfgrass dollar spot pathogen in natural and model hosts

Clarireedia sp. (formerly called Sclerotinia homoeocarpa), the fungal pathogen that causes dollar spot of turfgrasses, produces oxalic acid but the role of this toxin in Clarireedia sp. pathogenesis is unknown. In the current study, whole plant inoculation assays were used to evaluate pathogenesis of Clarireedia sp. in various model hosts and investigate the role of oxalic acid in dollar spot disease. These assays revealed that both host endogenous oxalate content and pathogen-produced oxalic acid influence the timing and magnitude of symptom development. In time-course expression analysis, oxalate oxidase and related defense-associated germin-like protein genes in creeping bentgrass showed strong up-regulation starting at 48-72 hpi, indicating that germin-like protein genes are most likely involved in defense following initial contact with the pathogen and demonstrating the importance of oxalic acid in Clarireedia sp. pathogenesis. Overall, the results of these studies suggest that oxalic acid and host endogenous oxalate content are important for pathogenesis by Clarireedia sp. and may be associated with the transition from biotrophy to necrotrophy during host infection.

plant biology↗