Moldenhauer, T. | bioRxiv Science

bioRxiv2023

Differentiation of vaginal cells from epidermal cells using morphological and autofluorescence properties: Implications for sexual assault casework involving digital penetration

Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. There is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across >80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving digital penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue ([≥] 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types.

cell biology↗

bioRxiv2017

Naturally-segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster

Identifying the causative sequence polymorphisms underlying complex trait variation is a key goal of evolutionary and biomedical genetics. By knowing the precise molecular events that confer phenotypic changes we can gain insight into the pathways underlying complex traits and the evolutionary forces acting on variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative nucleotide-level polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource, and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNAi of this gene reduced resistance. We discovered a naturally-segregating 22-bp frameshift deletion in Ugt86Dd, and overexpression of the insertion-containing allele in a range of tissues enhanced resistance. Accounting for the InDel event during mapping largely eliminates the QTL, implying the InDel explains the bulk of the effect associated with the mapped locus. Finally, we edited a relatively resistant genetic background to generate lesions in Ugt86Dd that recapitulate the naturally-occurring putative loss-of-function allele, and succeeded in radically reducing resistance. The putatively causative coding InDel in Ugt86Dd can be a launchpad for future mechanistic exploration of xenobiotic detoxification.\n\nARTICLE SUMMARYResolving the mutations that contribute to among-individual trait variation is a major goal of biomedical and evolutionary genetics. In general however, genetic mapping experiments do not allow immediate resolution of the underlying causative variants. Previous mapping work revealed several loci contributing to nicotine resistance in Drosophila melanogaster. We employed a battery of functional tests to demonstrate that the detoxification gene Ugt86Dd has a major phenotypic effect, and that a segregating frameshift mutation is likely causative. Editing the gene to introduce a premature stop codon led to a significant reduction in resistance, validating its role in xenobiotic detoxification.

genetics↗