Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA.

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.

Detection of offender DNA following skin-to-skin contact with a victim.

Forensic medical examiners are frequently asked to examine persons who claim to have been assaulted. If the suspect is unknown and there has been contact between his or her skin and the alleged victim, there is an expectation that DNA can be collected from the victim's skin. In this way, the retrieved suspect DNA from the skin of the victim can be used to support the proposition that places the suspect at the scene. This study investigated the transfer and persistence of offender DNA on a victim following a mock physical assault and further transfer of the offender DNA onto clothing worn over the assaulted area. Mock assault scenarios were conducted with the offender using medium pressure without friction and heavy pressure with friction on the wrist and upper arm of the victim. Samples were taken at either 0 h, 3 h or 24 h post assault, with 18 assault scenarios conducted at each time point. Samples from the victim's skin where the assault had taken place and, where applicable, clothing worn over the assaulted area were collected using the double swabbing method. Offender DNA was observed on the victim's skin at 0 h in the majority of samples with a higher transfer rate observed where heavy pressure and friction was used. The presence of the offender profile was detected in the samples collected at 3 h and 24 h, with 25% and 12% of samples respectively producing a LR in support of Hp. Transfer of offender DNA to clothing worn over the assaulted area was demonstrated, with 19% of samples producing a LR in support of Hp. Samples taken from clothing were complex mixtures, with 23% of samples producing four or more person mixtures. Indirect transfer of other DNA was also observed, with background DNA on the offender's skin observed in the clothing swabs of the victim on a number of occasions. Our data suggests that sampling from clothing worn over the assaulted area may be an additional or better avenue for the recovery of offender DNA post assault where there has been significant time between assault and sampling.

Toenails as an alternative source material for the extraction of DNA from decomposed human remains.

The DNA identification of decomposed human remains for coronial investigations at the Victorian Institute of Forensic Medicine routinely requires the retrieval and processing of a bone sample obtained from the deceased. Bone is a difficult sample type to work with as it requires surgical removal from the deceased, refrigerated storage, and additional processing steps prior to DNA analysis in comparison to other samples types such as buccal swabs or blood stains. In an attempt to overcome the issues posed by bone, a DNA extraction method utilising toenails as an alternate source material was optimised and trialled. Two DNA extraction methods were optimised for digestion of toenail material, with the method utilising the QIAGEN DNA Investigator Kit selected for a casework trial. Single source DNA profiles, matching those of the conventional samples taken, were obtained for toenail samples collected from 28 of 30 coronial cases available for this study. Of these, 26 toenail samples produced full profiles. Although the overall DNA profile quality from the toenails was less than that of the conventional sample, the profiles from toenails met the reporting requirements for identification. Based on the results obtained, the Victorian Institute of Forensic Medicine will be implementing toenails as the primary sample type for collection from decomposed remains when blood is not a suitable sample type.

Genomic organization and expression analysis for hcstk, a serine/threonine protein kinase gene of Haemonchus contortus, and comparison with Caenorhabditis elegans par-1.

The organization and expression of a putative serine/threonine kinase gene (designated hcstk), proposed to relate to a conserved eukaryotic signal transduction pathway, was characterized for the socio-economically important pathogen Haemonchus contortus (Nematoda). The entire hcstk gene is approximately 26.7 kb in size, has 26 exons and is inferred to produce multiple isoforms via alternative splicing in its N-terminal header and spacer domains. Comparison of hcstk with its Caenorhabditis elegans homologue, par-1, revealed major differences in genomic organization, exon number and inferred mRNA processing. The expression of hcstk transcripts was highest in the first- and late-fourth-stage larvae of the parasite compared with other developmental stages, somewhat distinct from par-1 in C. elegans. In spite of a substantial amino acid sequence identity in the functional domains between the predicted proteins HcSTK and PAR-1, overall, the findings suggest a unique functional role for each molecule.

HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus.

A putative serine/threonine protein kinase (HcSTK) from the parasitic nematode Haemonchus contortus was characterised at the mRNA and amino acid levels. HcSTK displays a high level of identity (85-93% in the catalytic domain) with proteins of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily, which represent signal transduction molecules involved in establishing and maintaining polarity in proliferating and differentiating cells. The transcript of hcstk is expressed in different developmental stages (second-, third-, fourth-stage larvae and adults) and various organs (muscle, intestine and reproductive) of H. contortus. In addition, there are several isoforms which appear to relate to a single gene. The expression profile of hcstk is similar to that of Caenorhabditis elegans PAR-1, and the level of sequence identity among members of the PAR-1/MARK STK subfamily, representing a range of species of vertebrates (e.g. humans and rodents), invertebrates (e.g. insects and C. elegans) and yeast, suggests that HcSTK may be involved in a conserved signal transduction pathway.

Post mortem sampling of the bladder for the identification of victims of fire related deaths.

In a coronial setting a deceased person must be formally identified. It is difficult to identify a deceased person when their physical features are disrupted and identification by visual means cannot occur. In the absence of visual identification, the confirmation of identity of a deceased person relies on the scientific comparison of information obtained post mortem with ante mortem information. The ante mortem information may include dental and medical records, fingerprints, and DNA profiling. For cases involving incinerated remains, this traditionally requires the collection of blood, muscle or bone samples from the deceased (depending on the severity of the burns) for DNA analysis and subsequent comparison to a reference sample for kinship determination. Following on from work conducted during the DVI response to a plane crash in Papua New Guinea in 2011, a study has been performed examining the viability of utilising material obtained from bladder swabs in deaths associated with fires. Twenty-eight cases were analysed during 2012 with deaths occurring in motor vehicle and aviation accidents, as well as house fires, homicides and from self-immolation. Bladder and conventional (blood, muscle or bone) samples were subjected to DNA analysis and compared. Our findings demonstrate that the bladder samples all gave DNA of sufficient quality for DNA profiling. This easily obtained sample (when available) can be now recommended in the scientific identification process of fire affected deceased persons.

Haemonchus contortus: evaluation of two signal sequence trapping systems for detection of secreted molecules.

Given that signal sequences between secreted proteins of different species can be interchanged, it is reasonable to expect that both mammalian and yeast signal sequence trapping (SST) systems would secrete Haemonchus contortus proteins with similar efficiency and quality. To determine if H. contortus cDNAs that contain a signal sequence could re-establish secretion of a reporter protein, mammalian and yeast SST vectors were designed, 10 H. contortus genes selected, and their respective cDNAs cloned into these two SST vectors. The selected molecules included genes known to code for excretory/secretory or membrane-bound proteins as potential test 'positives', and genes known to code for non-secreted proteins as test 'negatives'. While differentiation between secretion and non-secretion was evident in both systems, the results indicated greater efficiency was achieved when the mammalian system was used. Therefore, mammalian SST using COS cells would be a more useful tool to screen H. contortus cDNA libraries for potential secreted and type-1 integral membrane proteins than yeast SST.

Haemonchus contortus: prokaryotic expression and enzyme activity of recombinant HcSTK, a serine/threonine protein kinase.

Members of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily are important regulators of the cytoskeleton, and their characterization can provide insights into a number of critical processes relating to the development and survival of an organism. We previously investigated the mRNA expression for and organization of a gene (hcstk) representing HcSTK, an STK from the parasitic nematode Haemonchus contortus. In the present study, a recombinant form of HcSTK was expressed and characterized. Affinity-purified anti-HcSTK antibodies reacted with native HcSTK in protein homogenates extracted from third-stage larvae (L3) of H. contortus and were also used to immunolocalize the protein around the nuclei of ovarian and intestinal tissues of adult H. contortus. The enzyme activity of the recombinant HcSTK protein was also demonstrated. The findings show that recombinant HcSTK is a functional protein kinase, with activity directed to KXGS motifs, consistent with other members of the PAR-1/MARK STK subfamily.

Haemonchus contortus: molecular cloning, sequencing, and expression analysis of the gene coding for the small subunit of ribonucleotide reductase.

Gastro-intestinal (GI) parasites are of great agricultural importance, annually costing the livestock industry vast amounts in resources to control parasitism. One such GI parasite, Haemonchus contortus, is principally pathogenic to sheep; with the parasite's blood-feeding behaviour causing effects ranging from mild anaemia to mortality in young animals. Current means of control, which are dependent on repeated treatment with anthelmintic chemicals, have led to increasing drug resistance. Together with the growing concern over residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with the aim to develop alternate or supplementary means of control, including the development of molecular vaccines. As a first step towards the understanding of the synthesis of deoxyribonucleotides in H. contortus, and its potential application to therapeutic control of this economically important parasite, we report the cloning, sequencing, and mRNA expression analysis of the ribonucleotide reductase R2 gene.

Haemonchus contortus: molecular characterisation of a small heat shock protein.

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development. Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein