Comparison of immunoassay and LC-tandem mass spectrometry analyses of ractopamine in hog oral fluid.

The accurate detection of ractopamine in food animals is crucial for marketing since some entities require animals or animal carcasses to be free of ractopamine residues. Field-based ractopamine screening tests that are rapid, sensitive, and capable of high-throughput are highly desirable to ensure that inadvertent exposure to ractopamine did not occur in animals marketed as animals that have not been fed ractopamine. An immunochemically based lateral flow assay was used to analyze oral fluids from hogs never exposed to ractopamine and from hogs that were presumed positives and results were confirmed using an enhanced sensitivity LC-MSMS method. We found that an immunochemically based lateral flow system having a working range of 2.5 to 15 ng mL-1 worked well as a screening assay with 1.7% false positive results in freshly collected hog oral fluids. Using ractopamine glucuronide standards and LC-MSMS, we determined that the false positive results were not due to the presence of ractopamine glucuronide metabolites in oral fluids.

The secret hidden in dust: Assessing the potential to use biological and chemical properties of the airborne fraction of soil for provenance assignment and forensic casework.

The airborne fraction of soil (dust) is both ubiquitous in nature and contains localised biological and chemical signatures, making it a potential medium for forensic intelligence. Metabarcoding of dust can yield biological communities unique to the site of interest, similarly, geochemical analyses can uncover elements and minerals within dust that can be matched to a geographic location. Combining these analyses presents multiple lines of evidence as to the origin of dust collected from items of interest. In this work, we investigated whether bacterial and fungal communities in dust change through time and whether they are comparable to soil samples of the same site. We integrated dust metabarcoding into a framework amenable to forensic casework, (i.e., using calibrated log-likelihood ratios) to predict the origin of dust samples using models constructed from both dust samples and soil samples from the same site. Furthermore, we tested whether both metabarcoding and geochemical/mineralogical analyses could be conducted on a single swabbed sample, for situations where sampling is limited. We found both analyses could generate results from a single swabbed sample and found biological and chemical signatures unique to sites. However, we did find significant variation within sites, where this did not always correlate with time but was a random effect of sampling. This variation within sites was not greater than between sites and so did not influence site discrimination. When modelling bacterial and fungal diversity using calibrated log-likelihood ratios, we found samples were correctly predicted using dust 67% and 56% of the time and using soil 56% and 22% of the time for bacteria and fungi communities respectively. Incorrect predictions were related to within site variability, highlighting limitations to assigning dust provenance using metabarcoding of soil.

Are infrared thermography, feeding behavior, and heart rate variability measures capable of characterizing group-housed sow social hierarchies?

Group gestation housing is quickly becoming standard practice in commercial swine production. However, poor performance and welfare in group housed sows may result from the formation and maintenance of the social hierarchy within the pen. In the future, the ability to quickly characterize the social hierarchy via precision technologies could be beneficial to producers for identifying animals at risk of poor welfare outcomes. Therefore, the objective of this study was to investigate the use of infrared thermography (IRT), automated electronic sow feeding systems, and heart rate monitors as potential technologies for detecting the social hierarchy within five groups of sows. Behavioral data collection occurred for 12 h after introducing five sow groups (1-5; n = 14, 12, 15, 15, and 17, respectively) to group gestation housing to determine the social hierarchy and allocate individual sows to 1 of 4 rank quartiles (RQ 1-4). Sows within RQ1 were ranked highest while RQ4 sows were ranked lowest within the hierarchy. Infrared thermal images were taken behind the neck at the base of the ear of each sow on days 3, 15, 30, 45, 60, 75, 90, and 105 of the experiment. Two electronic sow feeders tracked feeding behavior throughout the gestation period. Heart rate monitors were worn by 10 randomly selected sows per repetition for 1 h prior to and 4 h after reintroduction to group gestation housing to collect heart rate variability (HRV). No differences were found between RQ for any IRT characteristic. Sows within RQ3 and RQ4 had the greatest number of visits to the electronic sow feeders overall (P < 0.04) but spent shorter time per visit in feeders (P < 0.05) than RQ1 and RQ2 sows. There was an interaction of RQ with hour for feed offered (P = 0.0003), with differences between RQ occurring in hour 0, 1, 2, and 8. Higher-ranked sows (RQ1 and RQ2) occupied the feeder for longer during the first hour than lower ranking sows (RQ3 and RQ4; P < 0.04), while RQ3 sows occupied the feeder longer than RQ1 sows during hour 6, 7, and 8 (P < 0.02). Heart beat interval (RR) collected prior to group housing introduction differed between RQ (P < 0.02 for all), with RQ3 sows exhibiting the lowest RR, followed by RQ4, RQ1, and RQ2. Rank quartile also affected standard deviation of RR (P = 0.0043), with RQ4 sows having the lowest, followed by RQ1, RQ3, and RQ2 sows. Overall, these results indicate that feeding behavior and HRV measures may be capable of characterizing social hierarchy in a group housing system.

Sows that are housed in groups establish a social hierarchy to gain preferential access to needed resources, such as feed. The establishment and maintenance of this social hierarchy may lead to reduced welfare and performance for certain sows. Therefore, the purpose of this study was to determine whether infrared thermography, feeding behavior, and heart rate variability measurement could identify the social hierarchy. In the future, the ability to automatically detect the social hierarchy within group housed pens using technology may give a producer the ability to mitigate any hierarchy-related welfare and performance issues on their own farm. Our results show that feeding behavior collected by an automated feeding system may be a promising tool for future social hierarchy detection. Additionally, measures related to changes in heart rate over time are capable of identifying high- and low-ranked sows when the measures are collected before sows are moved into groups. Therefore, technologies capable of measuring feeding behavior and changes in heart rate over time may be promising for future automated detection of the sow social hierarchy.

Environmental DNA as an innovative technique to identify the origins of falsified antimalarial tablets-a pilot study of the pharmabiome.

Falsified medicines are a major threat to global health. Antimalarial drugs have been particularly targeted by criminals. As DNA analysis has revolutionized forensic criminology, we hypothesized that these techniques could also be used to investigate the origins of falsified medicines. Medicines may contain diverse adventitious biological contamination, and the sealed nature of blister-packages may capture and preserve genetic signals from the manufacturing processes allowing identification of production source(s). We conducted a blinded pilot study to determine if such environmental DNA (eDNA) could be detected in eleven samples of falsified and genuine artesunate antimalarial tablets, collected in SE Asia, which could be indicative of origin. Massively Parallel Sequencing (MPS) was used to characterize microbial and eukaryote diversity. Two mitochondrial DNA analysis approaches were explored to detect the presence of human DNA. Trace eDNA from these low biomass samples demonstrated sample specific signals using two target markers. Significant differences in bacterial and eukaryote DNA community structures were observed between genuine and falsified tablets and between different packaging types of falsified artesunate. Human DNA, which was indicative of likely east Asian ancestry, was found in falsified tablets. This pilot study of the 'pharmabiome' shows the potential of environmental DNA as a powerful forensic tool to assist with the identification of the environments, and hence location and timing, of the source and manufacture of falsified medicines, establish links between seizures and complement existing tools to build a more complete picture of criminal trade routes. The finding of human DNA in tablets raises important ethical issues that need to be addressed.

A targeted capture approach to generating reference sequence databases for chloroplast gene regions.

Metabarcoding has improved the way we understand plants within our environment, from their ecology and conservation to invasive species management. The notion of identifying plant taxa within environmental samples relies on the ability to match unknown sequences to known reference libraries. Without comprehensive reference databases, species can go undetected or be incorrectly assigned, leading to false-positive and false-negative detections. To improve our ability to generate reference sequence databases, we developed a targeted capture approach using the OZBaits_CP V1.0 set, designed to capture chloroplast gene regions across the entirety of flowering plant diversity. We focused on generating a reference database for coastal temperate plant species given the lack of reference sequences for these taxa. Our approach was successful across all specimens with a target gene recovery rate of 92%, which was achieved in a single assay (i.e., samples were pooled), thus making this approach much faster and more efficient than standard barcoding. Further testing of this database highlighted 80% of all samples could be discriminated to family level across all gene regions with some genes achieving greater resolution than others-which was also dependent on the taxon of interest. Thus, we demonstrate the importance of generating reference sequences across multiple chloroplast gene regions as no single loci are sufficient to discriminate across all plant groups. The targeted capture approach outlined in this study provides a way forward to achieve this.

Rare genera differentiate urban green space soil bacterial communities in three cities across the world.

Vegetation complexity is potentially important for urban green space designs aimed at fostering microbial biodiversity to benefit human health. Exposure to urban microbial biodiversity may influence human health outcomes via immune training and regulation. In this context, improving human exposure to microbiota via biodiversity-centric urban green space designs is an underused opportunity. There is currently little knowledge on the association between vegetation complexity (i.e. diversity and structure) and soil microbiota of urban green spaces. Here, we investigated the association between vegetation complexity and soil bacteria in urban green spaces in Bournemouth, UK; Haikou, China; and the City of Playford, Australia by sequencing the 16S rRNA V4 gene region of soil samples and assessing bacterial diversity. We characterized these green spaces as having 'low' or 'high' vegetation complexity and explored whether these two broad categories contained similar bacterial community compositions and diversity around the world. Within cities, we observed significantly different alpha and beta diversities between vegetation complexities; however, these results varied between cities. Rare genera (<1% relative abundance individually, on average 35% relative abundance when pooled) were most likely to be significantly different in sequence abundance between vegetation complexities and therefore explained much of the differences in microbial communities observed. Overall, general associations exist between soil bacterial communities and vegetation complexity, although these are not consistent between cities. Therefore, more in-depth work is required to be done locally to derive practical actions to assist the conservation and restoration of microbial communities in urban areas.

Evaluation of rapid and standard tandem mass spectrometric methods to analyse veterinary drugs and their metabolites in antemortem bodily fluids from food animals.

Antemortem bodily fluids can serve as an indicator of veterinary medicine exposure prior to food animal slaughter. A multi-residue, rapid screen electrospray ionisation mass spectrometric (RS-ESI-MS) method was developed to analyse 10 veterinary drugs or metabolites (clenbuterol, erythromycin, flunixin, 5-hydroxyflunixin, meloxicam, ractopamine, ractopamine-glucuronide, salbutamol, tylosin, and zilpaterol) in hog oral fluid and bovine urine. Simple acetonitrile extraction with salting-out was employed to remove the analytes from matrices in less than 30 minutes. Instrumental analysis time was < 1 min/injection. Regression coefficients of matrix-matched calibration curves ranged 0.9743-0.9999 across all compounds with limits of detection ranging from 0.46-108 ng mL-1 for cattle urine and 0.19-64.4 ng mL-1 for hog oral fluid across all analytes. Except for ractopamine-glucuronide, analyte recoveries ranged from 92.7-106% for oral fluid and urine fortified at 30, 100, and 300 ng mL-1, with inter-day variations of < 25%. Ractopamine-glucuronide recovery was 93.3% for oral fluid fortified at 300 ng mL-1. The RS-ESI-MS method accurately identified ractopamine and/or ractopamine-glucuronide in incurred cattle urine with results correlating well with traditional LC-MS/MS and HPLC fluorescence methods. As far as we are aware, this is the first report of the direct quantification of ractopamine-glucuronide from biological matrices without lengthy hydrolysis and cleanup steps.

GPR39 localization in the aging human brain and correlation of expression and polymorphism with vascular cognitive impairment.

INTRODUCTION: The pathogenesis of vascular cognitive impairment (VCI) is not fully understood. GPR39, an orphan G-protein coupled receptor, is implicated in neurological disorders but its role in VCI is unknown. METHODS: We performed GPR39 immunohistochemical analysis in post mortem brain samples from mild cognitive impairment (MCI) and control subjects. DNA was analyzed for GPR39 single nucleotide polymorphisms (SNPs), and correlated with white matter hyperintensity (WMH) burden on pre mortem magnetic resonance imaging. RESULTS: GPR39 is expressed in aged human dorsolateral prefrontal cortex, localized to microglia and peri-capillary cells resembling pericytes. GPR39-capillary colocalization, and density of GPR39-expressing microglia was increased in aged brains compared to young. SNP distribution was equivalent between groups; however, homozygous SNP carriers were present only in the MCI group, and had higher WMH volume than wild-type or heterozygous SNP carriers. DISCUSSION: GPR39 may play a role in aging-related VCI, and may serve as a therapeutic target and biomarker for the risk of developing VCI.

Microbiomes in forensic botany: a review.

Fragments of botanical material can often be found at crime scenes (on live and dead bodies, or on incriminating objects) and can provide circumstantial evidence on various aspects of forensic investigations such as determining crime scene locations, times of death or possession of illegal species. Morphological and genetic analysis are the most commonly applied methods to analyze plant fragment evidence but are limited by their low capacity to differentiate between potential source locations, especially at local scales. Here, we review the current applications and limitations of current plant fragment analysis for forensic investigations and introduce the potential of microbiome analysis to complement the existing forensic plant fragment analysis toolkit. The potential for plant fragment provenance identification at geographic scales meaningful to forensic investigations warrants further investigation of the phyllosphere microbiome in this context. To that end we identify three key areas of future research: 1) Retrieval of microbial DNA of sufficient quality and quantity from botanical material; 2) Variability of the phyllosphere microbiome at different taxonomic and spatial scales, with explicit reference to assignment capacity; 3) Impacts on assignment capacity of time, seasonality and movement of fragments between locations. The development of robust microbiome analysis tools for forensic purposes in botanical material could increase the evidentiary value of the botanical evidence commonly encountered in casework, aiding in the identification of crime scene locations.

Increased plant species richness associates with greater soil bacterial diversity in urban green spaces.

The vegetation and soil microbiome within urban green spaces is increasingly managed to help conserve biodiversity and improve human health concurrently. However, the effects of green space management on urban soil ecosystems is poorly understood, despite their importance. Across 40 urban green spaces in metropolitan Adelaide, South Australia, we show that soil bacterial communities are strongly affected by urban green space type (incl. sport fields, community gardens, parklands and revegetated areas), and that plant species richness is positively associated with soil bacterial diversity. Importantly, these microbiome trends were not affected by geographic proximity of sample sites. Our results provide early evidence that urban green space management can have predictable effects on the soil microbiome, at least from a diversity perspective, which could prove important to inform policy development if urban green spaces are to be managed to optimise population health benefits.

Ancestry and phenotype predictions from touch DNA using massively parallel sequencing.

Direct PCR can be used to successfully generate full STR profiles from DNA present on the surface of objects. STR profiles are only of use in cases where a potential donor profile is available for comparison, and DNA is of sufficient DNA quality and quantity to generate a reliable profile. Often, no donor information is available and only trace DNA is present on items. As a result, alternative techniques are required to generate genetic data that can provide investigative leads. Massively parallel sequencing (MPS) offers the ability to detect trace levels of DNA and improve DNA analysis success from touched items. Here, we present the first application of direct PCR coupled with MPS to generate forensic intelligence SNP data from latent DNA. The panels assessed are (1) the HIrisplex System that targets 24 SNPs to simultaneously predict hair and eye, and (2) the Precision ID Ancestry Panel that targets 165 autosomal SNPs indicative of biogeographic ancestry. For each panel, we analysed 60 touched samples across five individuals and four substrates (glass slide, fuse, zip-lock bag and wire) using Ion AmpliSeq Library Preparation Kit on the automated Ion Chef System and Ion Torrent PGM. We examine the SNP recovery, concordance with reference samples and the genotype reproducibility from different substrates and donors. The results demonstrate the application of this approach for obtaining informative genetic from trace amounts of DNA.

Use of a Spray Device to Locate Touch DNA on Casework Samples.

The use of a fluorescent dye to visualize cellular material on surfaces offers a targeted sampling approach for locating touch DNA on casework items. However, the current application of such dye is not feasible for examination of relatively large items. As a result, development of an efficient dye application system is required to translate this approach into practice. Here, the spray pattern (area covered, intensity, and evenness) of 15 different commercial spray devices was examined visually using food coloring. From this, five devices were selected to apply Diamond Nucleic Acid Dye (DD) to three substrates (glass slide, plastic sheet, and brown packing tape) seeded with saliva and touch DNA. The cellular material was visualized using the Dino-lite Microscope and Polilight. The inhibitory effects of DD afforded by each spray device were examined using Identifiler Plus® DNA profiling kit and a DNA input of 800 pg. The two most promising devices were further tested on a range of mock casework items seeded with touch DNA. The results presented demonstrate the feasibility of a spray system to apply DD to large surfaces and subsequently detect cellular material at both micro and macroscale. Specifically, the data suggest that a pressurized continuous-spray system is favorable and that droplet size influences the intensity of fluorescence and surface coverage. Furthermore, this study indicates that full STR profiles can be obtained following spraying with DD solution, even with excessive application, which is essential for the widespread use of these devices in casework.

Naturally-diverse airborne environmental microbial exposures modulate the gut microbiome and may provide anxiolytic benefits in mice.

Growing epidemiological evidence links natural green space exposure with a range of health benefits, including for mental health. Conversely, greater urbanisation associates with increased risk of mental health disorders. Microbiomes are proposed as an important but understudied link that may help explain many green space-human health associations. However, there remains a lack of controlled experimental evidence testing possible beneficial effects from passive exposure to natural biodiversity via airborne microbiota. Previous mouse model studies have used unrealistic environmental microbial exposures-including excessive soil and organic matter contact, feed supplements and injections-to demonstrate host microbiota, immune biomarker, and behavioural changes. Here, in a randomised controlled experiment, we demonstrate that realistic exposures to trace-level dust from a high biodiversity soil can change mouse gut microbiota, in comparison to dust from low biodiversity soil or no soil (control) (n = 54 total mice, comprising 3 treatments × 18 mice, with 9 females + 9 males per group). Furthermore, we found a nominal soil-derived anaerobic spore-forming butyrate-producer, Kineothrix alysoides, was supplemented to a greater extent in the gut microbiomes of high biodiversity treatment mice. Also, increasing relative abundance of this rare organism correlated with reduced anxiety-like behaviour in the most anxious mice. Our results point to an intriguing new hypothesis: that biodiverse soils may represent an important supplementary source of butyrate-producing bacteria capable of resupplying the mammalian gut microbiome, with potential for gut health and mental health benefits. Our findings have potential to inform cost-effective population health interventions through microbiome-conscious green space design and, ultimately, the mainstreaming of biodiversity into health care.

Broadening the taxonomic scope of coral reef palaeoecological studies using ancient DNA.

Marine environments face acute pressures from human impacts, often resulting in substantial changes in community structure. On the inshore Great Barrier Reef (GBR), palaeoecological studies show the collapse of the previously dominant coral Acropora from the impacts of degraded water quality associated with European colonization. Even more dramatic impacts can result in the replacement of corals by fleshy macroalgae on modern reefs, but their past distribution is unknown because they leave no fossil record. Here, we apply DNA metabarcoding and high-throughput sequencing of the 18S rDNA gene on palaeoenvironmental DNA (aeDNA) derived from sediment cores at two sites on Pandora Reef (GBR), to enhance palaeoecological studies by incorporating key soft-bodied taxa, including macroalgae. We compared temporal trends in this aeDNA record with those of coral genera derived from macrofossils. Multivariate analysis of 12 eukaryotic groups from the aeDNA community showed wide variability over the past 750 years. The occurrence of brown macroalgae was negatively correlated only with the dominant coral at both sites. The occurrence of coralline and green macroalgae was positively correlated with only the dominant coral at one of the sites, where we also observed a significant association between the whole coral community and the occurrence of each of the three macroalgae groups. Our results demonstrate that reef sediments can provide a valuable archive for understanding the past distribution and occurrence of important soft-bodied reef dwellers. Combining information from fossils and aeDNA provides an enhanced understanding of temporal changes of reefs ecosystems at decadal to millennial timescales.

High-throughput Sequencing of Trace Quantities of Soil Provides Reproducible and Discriminative Fungal DNA Profiles.

High-throughput sequencing (HTS) offers improved resolution between forensic soil samples by characterizing individual taxa present; however, the heterogeneous distribution of taxa in soils, and limited quantity of material available, may hinder the reliability of HTS in casework. Using HTS of the internal transcribed spacer, we examined the effect of soil mass (50, 150, and 250 mg) on fungal DNA profiles, focusing on reproducibility and discriminatory power between close proximity soils, and samples with similar textural classification. The results show that reduced soil mass had no significant effect on sample differentiation and that 150 mg soil provides the most reproducible DNA profiles across different soil types. In addition, Ascomycota was identified as a robust fungal target for forensic intelligence as this phylum was detected consistently across all samples regardless of sample quantity. Overall, this study highlights the value of trace quantities of soil for use in forensic casework.

Predicting the origin of soil evidence: High throughput eukaryote sequencing and MIR spectroscopy applied to a crime scene scenario.

Soil can serve as powerful trace evidence in forensic casework, because it is highly individualistic and can be characterised using a number of techniques. Complex soil matrixes can support a vast number of organisms that can provide a site-specific signal for use in forensic soil discrimination. Previous DNA fingerprinting techniques rely on variations in fragment length to distinguish between soil profiles and focus solely on microbial communities. However, the recent development of high throughput sequencing (HTS) has the potential to provide a more detailed picture of the soil community by accessing non-culturable microorganisms and by identifying specific bacteria, fungi, and plants within soil. To demonstrate the application of HTS to forensic soil analysis, 18S ribosomal RNA profiles of six forensic mock crime scene samples were compared to those collected from seven reference locations across South Australia. Our results demonstrate the utility of non-bacterial DNA to discriminate between different sites, and were able to link a soil to a particular location. In addition, HTS complemented traditional Mid Infrared (MIR) spectroscopy soil profiling, but was able to provide statistically stronger discriminatory power at a finer scale. Through the design of an experimental case scenario, we highlight the considerations and potential limitations of this method in forensic casework. We show that HTS analysis of soil eukaryotes was robust to environmental variation, e.g. rainfall and temperature, transfer effects, storage effects and spatial variation. In addition, this study utilises novel analytical methodologies to interpret results for investigative purposes and provides prediction statistics to support soil DNA analysis for evidential stages of a case.

Residual soil DNA extraction increases the discriminatory power between samples.

Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.

Peroxisomal biogenesis in ischemic brain.

AIMS: Peroxisomes are highly adaptable and dynamic organelles, adjusting their size, number, and enzyme composition to changing environmental and metabolic demands. We determined whether peroxisomes respond to ischemia, and whether peroxisomal biogenesis is an adaptive response to cerebral ischemia. RESULTS: Focal cerebral ischemia induced peroxisomal biogenesis in peri-infarct neurons, which was associated with a corresponding increase in peroxisomal antioxidant enzyme catalase. Peroxisomal biogenesis was also observed in primary cultured cortical neurons subjected to ischemic insult induced by oxygen-glucose deprivation (OGD). A catalase inhibitor increased OGD-induced neuronal death. Moreover, preventing peroxisomal proliferation by knocking down dynamin-related protein 1 (Drp1) exacerbated neuronal death induced by OGD, whereas enhancing peroxisomal biogenesis pharmacologically using a peroxisome proliferator-activated receptor-alpha agonist protected against neuronal death induced by OGD. INNOVATION: This is the first documentation of ischemia-induced peroxisomal biogenesis in mammalian brain using a combined in vivo and in vitro approach, electron microscopy, high-resolution laser-scanning confocal microscopy, and super-resolution structured illumination microscopy. CONCLUSION: Our findings suggest that neurons respond to ischemic injury by increasing peroxisome biogenesis, which serves a protective function, likely mediated by enhanced antioxidant capacity of neurons.

Role of soluble epoxide hydrolase in age-related vascular cognitive decline.

P450 eicosanoids are important regulators of the cerebral microcirculation, but their role in cerebral small vessel disease is unclear. We tested the hypothesis that vascular cognitive impairment (VCI) is linked to reduced cerebral microvascular eicosanoid signaling. We analyzed human brain tissue from individuals formerly enrolled in the Oregon Brain Aging Study, who had a history of cognitive impairment histopathological evidence of microvascular disease. VCI subjects had significantly higher lesion burden both on premortem MRI and postmortem histopathology compared to age- and sex-matched controls. Mass spectrometry-based eicosanoid analysis revealed that 14,15-dihydroxyeicosatrienoic acid (DHET) was elevated in cortical brain tissue from VCI subjects. Immunoreactivity of soluble epoxide hydrolase (sEH), the enzyme responsible for 14,15-DHET formation, was localized to cerebral microvascular endothelium, and was enhanced in microvessels of affected tissue. Finally, we evaluated the genotype frequency of two functional single nucleotide polymorphisms of sEH gene EPHX2 in VCI and control groups. Our findings support a role for sEH and a potential benefit from sEH inhibitors in age-related VCI.

Forensic soil DNA analysis using high-throughput sequencing: a comparison of four molecular markers.

Soil analysis, such as mineralogy, geophysics, texture and colour, are commonly used in forensic casework to link a suspect to a crime scene. However, DNA analysis can also be applied to characterise the vast diversity of organisms present in soils. DNA metabarcoding and high-throughput sequencing (HTS) now offer a means to improve discrimination between forensic soil samples by identifying individual taxa and exploring non-culturable microbial species. Here, we compare the small-scale reproducibility and resolution of four molecular markers targeting different taxa (bacterial 16S rRNA, eukaryotic18S rRNA, plant trnL intron and fungal internal transcribed spacer I (ITS1) rDNA) to distinguish two sample sites. We also assess the background DNA level associated with each marker and examine the effects of filtering Operational Taxonomic Units (OTUs) detected in extraction blank controls. From this study, we show that non-bacterial taxa in soil, particularly fungi, can provide the greatest resolution between the sites, whereas plant markers may be problematic for forensic discrimination. ITS and 18S markers exhibit reliable amplification, and both show high discriminatory power with low background DNA levels. The 16S rRNA marker showed comparable discriminatory power post filtering; however, presented the highest level of background DNA. The discriminatory power of all markers was increased by applying OTU filtering steps, with the greatest improvement observed by the removal of any sequences detected in extraction blanks. This study demonstrates the potential use of multiple DNA markers for forensic soil analysis using HTS, and identifies some of the standardisation and evaluation steps necessary before this technique can be applied in casework.