Biology subjects

Wilson, F.

Publications and source records attributed to Wilson, F..

6 recordsLinked to original sources

bioRxiv2025

Identification of Functional Genetic Components Modulating Toxicity Response to PFOS using Genome-wide CRISPR Screens in HepG2/C3A cells

Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 {micro}M) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.

pharmacology and toxicology↗

bioRxiv2025

Neonatal Mouse Ovary Culture: An In Vitro Model for Studying Primordial Follicle Regulation

In mammalian females, primordial follicles form during fetal ovarian development and serve as the only source to sustain adult ovarian function. Mechanisms underlying how primordial follicles assemble, maintain dormancy, activate for follicular development, and undergo cell death are important for understanding ovarian physiology and pathological conditions. Here, we demonstrate a protocol of culturing postnatal mouse ovaries on membrane inserts - an approach allowing culture, pharmaceutical treatment, and live-imaging of intact ovaries for up to 10 days depending on the developmental stage of the ovary. The change of culture conditions can be done by transferring inserts containing cultured ovaries between wells on a plate, avoiding physical interference with tissues during culture. In this experiment, we use postnatal day 5 (P5) CD1 mouse ovary culture as an example. P5 ovaries were isolated and placed on a 12 mm insert in a 24-well plate. Each ovary was separated within a droplet of DMEM/F12 medium supplemented with 10% FBS, 3 mg/ml BSA, 10 mIU/ml FSH, and Gibco Antibiotic-Antimycotic, and gently stabilized to the membrane insert. The medium was changed every two days, and the culture was maintained for five days. Following the culture, ovaries were fixed in 4% paraformaldehyde for two hours and processed for whole-mount antibody staining. Primordial follicles were visualized using confocal microscopy with anti-DDX4 antibody, allowing for the analysis of oocyte number and morphology. We showed that the number of primordial follicles in each ovary was significantly affected by whether tissues were properly placed on the membrane insert. Difference in the number of ovaries on each insert may contribute to non-biological variations and should be avoided.

developmental biology↗

bioRxiv2024

Primary oocytes with cellular senescence features are involved in ovarian aging in mice

In mammalian females, quiescent primordial follicles serve as the ovarian reserve and sustain normal ovarian function and egg production via folliculogenesis. The loss of primordial follicles causes ovarian aging. Cellular senescence, characterized by cell cycle arrest and production of the senescence-associated secretory phenotype (SASP), is associated with tissue aging. In the present study, we report that some quiescent primary oocytes in primordial follicles become senescent in adult mouse ovaries. The senescent primary oocytes share senescence markers characterized in senescent somatic cells. The senescent primary oocytes were observed in young adult mouse ovaries, remained at approximately 15% of the total primary oocytes during ovarian aging from 6 months to 12 months, and accumulated in aged ovaries. Administration of a senolytic drug ABT263 to 3-month-old mice reduced the percentage of senescent primary oocytes and the transcription of the SASP cytokines in the ovary. In addition, led to increased numbers of primordial and total follicles and a higher rate of oocyte maturation and female fertility. Our study provides experimental evidence that primary oocytes, a germline cell type that is arrested in meiosis, become senescent in adult mouse ovaries and that senescent cell clearance reduced primordial follicle loss and mitigated ovarian aging phenotypes.

cell biology↗

bioRxiv2018

CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry

Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement. We demonstrate use of CRISPR/Cas methodology in diploid Fragaria vesca ssp. vesca Hawaii 4 and octoploid F. x ananassa Calypso by targeting the visible endogenous marker gene PDS (phytoene desaturase). Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of either the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% ( Hawaii 4) and 50% ( Calypso) putative transgenic shoot lines exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 55% ( Calypso) and 75% ( Hawaii 4) included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in Calypso and clearly demonstrate that CRISPR/Cas9 can be used to edit single copy genes at high frequency within the genome of the diploid and the same target in octoploid strawberry.

molecular biology↗

bioRxiv2018

Identification of powdery mildew resistance QTL in Fragaria x ananassa

The obligate biotrophic fungus Podosphaera aphanis is the causative agent of powdery mildew on cultivated strawberry (Fragaria x ananassa). Genotypes from two bi-parental mapping populations Emily x Fenella and Redgauntlet x Hapil were phenotyped for powdery mildew disease severity in a series of field trials. Here we report multiple QTL associated with resistance to powdery mildew, identified in ten phenotyping events conducted across different years and locations. Seven QTL show a level of stable resistance across multiple phenotyping events however many other QTL were represented in a single phenotyping event and therefore must be considered transient. One of the identified QTL was closely linked to an associated resistance gene across the wider germplasm. Furthermore, a preliminary association analysis identified a novel conserved locus for further investigation. Our data suggests that resistance is highly complex and that multiple additive sources of quantitative resistance to powdery mildew exist across strawberry germplasm. Implementation of the reported markers in marker-assisted breeding or genomic selection would lead to improved powdery mildew resistant strawberry cultivars, particularly where the studied parents, progeny and close pedigree material are included in breeding germplasm.\n\nKey MessagePowdery mildew resistance in two strawberry mapping populations is controlled by both stable and transient novel QTL of moderate effect. Some transferability of QTL across wider germplasm was observed.

pathology↗

bioRxiv2017

Pangenomic analysis reveals pathogen-specific regions and novel effector candidates in Fusarium oxysporum f.sp. cepae

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage.\n\nGenome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core.\n\nRNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

pathology↗