The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface – one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100%) of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x10
3 and ~1x10
5) could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence.
Bacteria from the genus Bacillus are able to transform into metabolically dormant states called (endo) spores in response to nutrient deprivation and other harsh conditions. These morphologically distinct spores are fascinating constructs, amongst the most durable cells in nature, and have attracted attention owing to their relevance in food-related illnesses and bioterrorism. Observing the course of bacterial spore formation (sporulation) spatially, temporally and mechanically, from the vegetative cell to a mature spore, is critical for a better understanding of this process. Here, we present a fast and versatile strategy for monitoring both the morphological and mechanical changes of Bacillus cereus bacteria at the nanoscale using atomic force microscopy. Through a strategy of imaging and nanomechanical mapping, we show the morphogenesis of the endospore and released mature endospore. Finally, we investigate individual spores to characterize their surface mechanically. The progression in elasticity coupled with a similarity of characteristic distributions between the incipient endospores and the formed spores show these distinct stages. Taken together, our data demonstrates the power of atomic force microscopy applied in microbiology for probing this important biological process at the single cell scale.
Forensic samples comprised of cell populations from multiple contributors often yield DNA profiles that can be extremely challenging to interpret. This frequently results in decreased statistical strength of an individual's association to the mixture and the loss of probative data. The purpose of this study was to test a front-end cell separation workflow on complex mixtures containing as many as five contributors. Our approach involved selectively labelling certain cell populations in dried whole blood mixture samples with fluorescently labeled antibody probe targeting the HLA-A*02 allele, separating the mixture using Fluorescence Activated Cell Sorting (FACS) into two fractions that are enriched in A*02 positive and A*02 negative cells, and then generating DNA profiles for each fraction. We then tested whether antibody labelling and cell sorting effectively reduced the complexity of the original cell mixture by analyzing STR profiles quantitatively using the probabilistic modeling software, TrueAllele Casework. Results showed that antibody labelling and FACS separation of target populations yielded simplified STR profiles that could be more easily interpreted using conventional procedures. Additionally, TrueAllele analysis of STR profiles from sorted cell fractions increased statistical strength for the association of most of the original contributors interpreted from the original mixtures.
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