Multivariate regression modelling for gender prediction using volatile organic compounds from hand odor profiles via HS-SPME-GC-MS.

The efficacy of using human volatile organic compounds (VOCs) as a form of forensic evidence has been well demonstrated with canines for crime scene response, suspect identification, and location checking. Although the use of human scent evidence in the field is well established, the laboratory evaluation of human VOC profiles has been limited. This study used Headspace-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) to analyze human hand odor samples collected from 60 individuals (30 Females and 30 Males). The human volatiles collected from the palm surfaces of each subject were interpreted for classification and prediction of gender. The volatile organic compound (VOC) signatures from subjects' hand odor profiles were evaluated with supervised dimensional reduction techniques: Partial Least Squares-Discriminant Analysis (PLS-DA), Orthogonal-Projections to Latent Structures Discriminant Analysis (OPLS-DA), and Linear Discriminant Analysis (LDA). The PLS-DA 2D model demonstrated clustering amongst male and female subjects. The addition of a third component to the PLS-DA model revealed clustering and minimal separation of male and female subjects in the 3D PLS-DA model. The OPLS-DA model displayed discrimination and clustering amongst gender groups with leave one out cross validation (LOOCV) and 95% confidence regions surrounding clustered groups without overlap. The LDA had a 96.67% accuracy rate for female and male subjects. The culminating knowledge establishes a working model for the prediction of donor class characteristics using human scent hand odor profiles.

Salinity Impacts the Functional mcrA and dsrA Gene Abundances in Everglades Marshes.

Coastal wetlands, such as the Everglades, are increasingly being exposed to stressors that have the potential to modify their existing ecological processes because of global climate change. Their soil microbiomes include a population of organisms important for biogeochemical cycling, but continual stresses can disturb the community's composition, causing functional changes. The Everglades feature wetlands with varied salinity levels, implying that they contain microbial communities with a variety of salt tolerances and microbial functions. Therefore, tracking the effects of stresses on these populations in freshwater and brackish marshes is critical. The study addressed this by utilizing next generation sequencing (NGS) to construct a baseline soil microbial community. The carbon and sulfur cycles were studied by sequencing a microbial functional gene involved in each process, the mcrA and dsrA functional genes, respectively. Saline was introduced over two years to observe the taxonomic alterations that occurred after a long-term disturbance such as seawater intrusion. It was observed that saltwater dosing increased sulfite reduction in freshwater peat soils and decreased methylotrophy in brackish peat soils. These findings add to the understanding of microbiomes by demonstrating how changes in soil qualities impact communities both before and after a disturbance such as saltwater intrusion.

Review of the current and potential use of biological and molecular methods for the estimation of the postmortem interval in animals and humans.

We provide here an overview of the state of applied techniques in the estimation of the early period of the postmortem interval (PMI). The biological methods included consist of body cooling, CSF potassium, body cooling combined with CSF potassium, and tissue autolysis. For each method, we present its application in human and veterinary medicine and provide current methodology, strengths, and weaknesses, as well as target areas for improvement. We examine current and future molecular methods as they pertain to DNA and primarily to messenger RNA degradation for the estimation of the PMI, as well as the use of RNA in aging wounds, aging blood stains, and the identification of body fluids. Various types of RNA have different lengths, structures, and functions in cells. These differences in RNAs determine various intrinsic properties, such as their half-lives in cells, and, hence, their decay rate as well as their unique use for specific forensic tests. Future applications and refinements of RNA-based techniques provide opportunities for the use of molecular methods in the estimation of PMI and other general forensic applications.

Preliminary accuracy of COVID-19 odor detection by canines and HS-SPME-GC-MS using exhaled breath samples.

The novel coronavirus SARS-CoV-2, since its initial outbreak in Wuhan, China has led to a worldwide pandemic and has shut down nations. As with any outbreak, there is a general strategy of detection, containment, treatment and/or cure. The authors would argue that rapid and efficient detection is critical and required to successful management of a disease. The current study explores and successfully demonstrates the use of canines to detect COVID-19 disease in exhaled breath. The intended use was to detect the odor of COVID-19 on contaminated surfaces inferring recent deposition of infectious material from a COVID-19 positive individual. Using masks obtained from hospitalized patients that tested positive for COVID-19 disease, four canines were trained and evaluated for their ability to detect the disease. All four canines obtained an accuracy >90% and positive predictive values ranging from ~73 to 93% after just one month of training.

Resolving human DNA mixtures with F-108 polymer and capillary electrophoresis single-strand conformational polymorphism analysis.

Current technologies have increased the sensitivity for analyzing forensic DNA samples, especially those considered "touch samples." Because of this, there has been an increase in the number of forensic mixtures-two or more contributors within a single sample-submitted to the crime laboratories. Therefore, the need to resolve these mixtures has increased as well. Several technologies are currently utilized, but many of them are time consuming and do not resolve the entire profile. Therefore, CE-Single-Strand Conformational Polymorphisms coupled with the Pluronic F-108 polymer was assessed for its ability to resolve human forensic mixtures. This technique has been able to detect sequence variation, such as single nucleotide polymorphism in short tandem repeat loci, such as D7S820 and vWA. Samples were first analyzed with the Performance Optimized Polymer-7, and mixtures created from samples that shared alleles. These samples were sequenced to detect single base-pair mutations and evaluated with the F-108 and CE-Single Strand Conformational Polymorphism analysis. Results from this study indicated the method would serve as a valuable screening tool to detect base sequence variation between individuals when they share alleles in a mixture and before using Massive Parallel Sequencing technology to distinguish which bases differ.

An interdisciplinary review of the thanatomicrobiome in human decomposition.

Death does not occur instantaneously and organs do not decompose at the same rate or in the same way. Nulligravid human uteri and prostate glands are the last internal organs to deteriorate during decomposition; however, the reason for this very important observation is still enigmatic. Recent studies have elucidated that the composition and abundance of microbes in the human thanatomicrobiome (microbiome of death) varies by organ and changes as a function of time and temperature. The ileocecal area has the largest absolute postmortem burden that spreads to the liver and spleen and continues to the heart and brain depending on the cause of death. To truly understand the mechanisms of microbial assembly during decomposition, a thorough examination of different strategies utilized by the trillions of microbes that colonize decaying tissues is needed from a multi-organ and multidisciplinary approach. In this review, we highlight interdisciplinary research and provide an overview of human decomposition investigations of thanatomicrobiomic changes in internal organs.

Bioinformatics Approach to Assess the Biogeographical Patterns of Soil Communities: The Utility for Soil Provenance.

Soil DNA profiling has potential as a forensic tool to establish a link between soil collected at a crime scene and soil recovered from a suspect. However, a quantitative measure is needed to investigate the spatial/temporal variability across multiple scales prior to their application in forensic science. In this study, soil DNA profiles across Miami-Dade, FL, were generated using length heterogeneity PCR to target four taxa. The objectives of this study were to (i) assess the biogeographical patterns of soils to determine whether soil biota is spatially correlated with geographic location and (ii) evaluate five machine learning algorithms for their predictive ability to recognize biotic patterns which could accurately classify soils at different spatial scales regardless of seasonal collection. Results demonstrate that soil communities have unique patterns and are spatially autocorrelated. Bioinformatic algorithms could accurately classify soils across all scales with Random Forest significantly outperforming all other algorithms regardless of spatial level.

Chemical and canine analysis as complimentary techniques for the identification of active odors of the invasive fungus, Raffaelea lauricola.

Raffaelea lauricola, a fungus causing a vascular wilt (laurel wilt) in Lauraceae trees, was introduced into the United States in the early 2000s. It has devastated forests in the Southeast and has now moved into the commercial avocado groves in southern Florida. Trained detection canines are currently one of the few successful methods for early detection of pre-symptomatic diseased trees. In order to achieve the universal and frequent training required to have successful detection canines, it is desirable to create accessible, safe, and long-lasting training aids. However, identification of odorants and compounds is limited by several factors, including both the availability of chemicals and the need to present chemicals individually and in combination to detection canines. A method for the separation and identification of volatile organic compounds (VOCs) from environmental substances for the creation of such a canine training aid is presented here. Headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to identify the odors present in avocado trees infected with the R. lauricola phytopathogen. Twenty-eight compounds were detected using this method, with nine present in greater than 80% of samples. The majority of these compounds were not commercially available as standard reference materials, and a canine trial was designed to identify the active odors without the need of pure chemical compounds. To facilitate the creation of a canine training aid, the VOCs above R. lauricola were separated by venting a 0.53mm ID solgel-wax gas chromatography column to the atmosphere. Ten minute fractions of the odor profile were collected on cotton gauze in glass vials and presented to the detection canines in a series of field trials. The canines alerted to the VOCs from the vials that correspond to a portion of the chromatogram containing the most volatile species from R. lauricola. This innovative fractionation and collection method can be used to develop reliable and cost effective canine training aids.

Chemotyping the temporal volatile organic compounds of an invasive fungus to the United States, Raffaelea lauricola.

Volatile organic compounds (VOCs) in the headspace of the fungus Raffaelea lauricola have been monitored and identified over a twenty-eight day growth period. R. lauricola is an invasive and phytopathogenic fungus that was first identified in the United States in the mid-2000s. It is believed to be spread by a host beetle, Xyleborus glabratus, and is detrimental both to wild members of the Lauraceae family and to commercial avocado groves particularly in the Southeastern region of the country. The fungus causes the fatal laurel wilt disease, a result of the host tree shutting down its vascular system in order to halt the spread of the fungus. The current study identified the VOCs present in the headspace of R. lauricola over the initial growth stage using headspace solid phase microextracion-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Results revealed the VOC dynamics of the fungus in culture, indicating that the initial growth period of the fungus may coincide with potential responses from the host trees that may recognize and respond to the pathogen when the fungal VOCs are produced as a result of primary metabolic processes. As fungal growth progresses past initial growth phases, the predominant compounds seen in the odor profile are hydrocarbons and terpenes, produced from secondary metabolic processes. The odor profile pattern for the twenty-eight day growth period did change with the stages of growth. Based on the information learned from this pilot study, a discussion is presented of possible host tree reactions to R. lauricola and implications for future experiments.

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis.

Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of this study was to expand the detection of coat color SNPs in horses. The genes for the solute carrier family member 2 (slc45a2/matp), type III receptor protein-tyrosine kinase (kit) and mc1r genes using CE-SSCP and F-108 polymer were compared to mini-sequencing with the SNaPshotTM kit. The F-108 polymer reproducibly resolved homozygous and heterozygous individuals for the mc1r and kit markers, but was unable to resolve heterozygous individuals for slc45a2 at 38ºC. The need for temperatures <15ºC, the SNP position being close to the 5'-end, and conformational structures/free energy with similar values resulted in the inability to resolve the secondary structures. Despite this limitation, the CE-SSCP method could be used to provide a rapid phenotypic description for equine forensic investigations.

An investigation into the concurrent collection of human scent and epithelial skin cells using a non-contact sampling device.

In criminal investigations, the collection of human scent often employs a non-contact, dynamic airflow device, known as the Scent Transfer Unit 100 (STU-100), to transfer volatile organic compounds (VOCs) from an object/person onto a collection material that is subsequently presented to human scent discriminating canines. Human scent is theorized to be linked to epithelial skin cells that are shed at a relatively constant rate allowing both scent and cellular material to be deposited into the environment and/or onto objects. Simultaneous collection of cellular material, with adequate levels of nuclear deoxyribonucleic acid (nDNA), and human scent using a non-invasive methodology would facilitate criminal investigations. This study evaluated the STU-100 for the concurrent collection of human scent and epithelial skin cells from a porous (paper) and non-porous (stainless steel bar) object that was held for a specified period of time in the dominant hand of twenty subjects (10 females and 10 males). Human scent analysis was performed using headspace static solid-phase microextraction with gas chromatography-mass spectrometry (HS-SPME/GC-MS). A polycarbonate filter was used to trap epithelial skin cells which, upon extraction, were subsequently analyzed, inter-laboratory, using the quantitative polymerase chain reaction (qPCR). The STU-100 proved to be inadequate for collecting the minimum number of epithelial skin cells required to obtain nuclear DNA concentrations above the limit of detection for the qPCR kit. With regard to its use for human scent collection, a reduction in the number and mass of compounds was observed when compared to samples that were directly collected. However, when the indirect collection of human scent from the two different objects was compared, a greater number and mass of compounds was observed from the non-porous object than from the porous object. This outcome suggests that the matrix composition of the scent source could affect the efficacy of the human scent collected when using a non-contact, dynamic airflow sampling device. The findings from this study are of importance because although the STU-100 proved to not be suitable for collecting epithelial skin cells for DNA analysis, its non-contact capability allows for the possibility of other potential forensic evidence, like that of human scent, to be obtained.

Developmental validation of the ParaDNA(®) Intelligence System-A novel approach to DNA profiling.

DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).

Microbiota shifts in the surface mucopolysaccharide layer of corals transferred from natural to aquaria settings.

Bacteria associated with the surface mucopolysaccharide layer (SML) of corals have been proposed to be paramount to coral health, and are occasionally studied in aquaria. Using automated ribosomal intergenic spacer analysis (ARISA), this study examined the temporal changes in the SML microbiota of coral fragments (Siderastrea siderea) transferred from the reef to aquaria. In total, 460 amplicon peaks were detected, 155 of which were unique. Extensive microbiota shifts occurred one day after transfer, with stabilization between 14 and 28days. These results suggest that studies examining coral in laboratory settings should consider the observed temporal dynamics in the SML microbiota.

F-108 polymer and capillary electrophoresis easily resolves complex environmental DNA mixtures and SNPs.

Ecological studies of microbial communities often use profiling methods but the true community diversity can be underestimated in methods that separate amplicons based on sequence length using performance optimized polymer 4. Taxonomically, unrelated organisms can produce the same length amplicon even though the amplicons have different sequences. F-108 polymer has previously been shown to resolve same length amplicons by sequence polymorphisms. In this study, we showed F-108 polymer, using the ABI Prism 310 Genetic Analyzer and CE, resolved four bacteria that produced the same length amplicon for the 16S rRNA domain V3 but have variable nucleotide content. Second, a microbial mat community profile was resolved and supported by NextGen sequencing where the number of peaks in the F-108 profile was in concordance with the confirmed species numbers in the mat. Third, equine DNA was analyzed for SNPs. The F-108 polymer was able to distinguish heterozygous and homozygous individuals for the melanocortin 1 receptor coat color gene. The method proved to be rapid, inexpensive, reproducible, and uses common CE instruments. The potential for F-108 to resolve DNA mixtures or SNPs can be applied to various sample types-from SNPs to forensic mixtures to ecological communities.

Developmental Validation of the ParaDNA® Screening System - A presumptive test for the detection of DNA on forensic evidence items.

Current assessment of whether a forensic evidence item should be submitted for STR profiling is largely based on the personal experience of the Crime Scene Investigator (CSI) and the submissions policy of the law enforcement authority involved. While there are chemical tests that can infer the presence of DNA through the detection of biological stains, the process remains mostly subjective and leads to many samples being submitted that give no profile or not being submitted although DNA is present. The ParaDNA(®) Screening System was developed to address this issue. It consists of a sampling device, pre-loaded reaction plates and detection instrument. The test uses direct PCR with fluorescent HyBeacon™ detection of PCR amplicons to identify the presence and relative amount of DNA on an evidence item and also provides a gender identification result in approximately 75 minutes. This simple-to-use design allows objective data to be acquired by both DNA analyst and non-specialist personnel, to enable a more informed submission decision to be made. The developmental validation study described here tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Screening System on a range of mock evidence items. The data collected demonstrates that the ParaDNA Screening System identifies the presence of DNA on a variety of evidence items including blood, saliva and touch DNA items.

Fluorescent Random Amplified Microsatellites (F-RAMS) analysis of mushrooms as a forensic investigative tool.

The capability of Fluorescent Random Amplified Microsatellites (F-RAMS) to profile hallucinogenic mushrooms to species and sub-species level was assessed. Fifteen samples of Amanita rubescens and 22 samples of other hallucinogenic and non-hallucinogenic mushrooms of the genera Amanita and Psilocybe were profiled using two fluorescently-labeled, 5'degenerate primers, 5'-6FAM-SpC3-DD (CCA)5 and 5'-6FAM-SpC3-DHB (CGA)5, which target different microsatellite repeat regions. Among the two primers, 5'-6FAM-SpC3-DHB (CGA)5 provided more reliable data for identification purposes, by grouping samples of the same species and clustering closely related species together in a dendrogram based on amplicon similarities. A high degree of intra-specific variation between the 15 A. rubescens samples was shown with both primers and the amplicons generated for all A. rubescens samples were organized into three classes of amplicons (discriminant, private, and marker) based on their individualizing potential.

The application of amplicon length heterogeneity PCR (LH-PCR) for monitoring the dynamics of soil microbial communities associated with cadaver decomposition.

The placement of cadavers in shallow, clandestine graves may alter the microbial and geochemical composition of the underlying and adjacent soils. Using amplicon length heterogeneity-PCR (LH-PCR) the microbial community changes in these soils can be assessed. In this investigation, nine different grave sites were examined over a period of 16weeks. The results indicated that measurable changes occurred in the soil bacterial community during the decomposition process. In this study, amplicons corresponding to anaerobic bacteria, not indigenous to the soil, were shown to produce differences between grave sites and control soils. Among the bacteria linked to these amplicons are those that are most often part of the commensal flora of the intestines, mouth and skin. In addition, over the 16week sampling interval, the level of indicator organisms (i.e., nitrogen fixing bacteria) dropped as the body decomposed and after four weeks of environmental exposure they began to increase again; thus differences in the abundance of nitrogen fixing bacteria were also found to contribute to the variation between controls and grave soils. These results were verified using primers that specifically targeted the nifH gene coding for nitrogenase reductase. LH-PCR provides a fast, robust and reproducible method to measure microbial changes in soil and could be used to determine potential cadaveric contact in a given area. The results obtained with this method could ultimately provide leads to investigators in criminal or missing person scenarios and allow for further analysis using human specific DNA assays to establish the identity of the buried body.

Genetic individualization of Cannabis sativa by a short tandem repeat multiplex system.

Cannabis sativa is the most frequently used of all illicit drugs in the USA. Cannabis has been used throughout history for its stems in the production of hemp fiber, seed for oil and food, and buds and leaves as a psychoactive drug. Short tandem repeats (STRs) were chosen as molecular markers owing to their distinct advantages over other genetic methods. STRs are codominant, can be standardized such that reproducibility between laboratories can be easily achieved, have a high discrimination power, and can be multiplexed. In this study, six STR markers previously described for C. sativa were multiplexed into one reaction. The multiplex reaction was able to individualize 98 cannabis samples (14 hemp and 84 marijuana, authenticated as originating from 33 of the 50 states of the USA) and detect 29 alleles averaging 4.8 alleles per loci. The data did not relate the samples from the same state to each other. This is the first study to report a single-reaction sixplex and apply it to the analysis of almost 100 cannabis samples of known geographic origin.

Black band disease microbial community variation on corals in three regions of the wider Caribbean.

Black band disease (BBD) is a pathogenic consortium of microorganisms that primarily affects massive framework-building scleractinian corals on reefs worldwide. There has been considerable debate concerning the microbial community composition of BBD. The aim of this study was to utilize microbial profiling to assess overall patterns of variation in the BBD bacterial community with respect to geographic location, host coral species, time, and nutrient regime. Length heterogeneity polymerase chain reaction (LH-PCR) was employed to differentiate BBD communities based on the natural variation in the sequence lengths within hypervariable domains of the 16S rRNA gene. Analysis of LH-PCR profiles of 97 BBD samples using multivariate ordination methods and analysis of similarity revealed significant clustering with respect to geographic region when comparing BBD sampled from reefs near Lee Stocking Island in the Bahamas' Exuma Chain, the Northern Florida Keys (NFK), and St. John in the US Virgin Islands. There was much variability in BBD community composition on a regional basis, between sites in the NFK, and in terms of coral host species. The observed differences among BBD microbial community profiles were driven primarily by variation in relative abundance of 313-316-bp amplicons, which correspond to cyanobacteria and alpha-proteobacteria. The results obtained in this study support previous reports of intrinsic variability and complexity of the BBD microbial community but also suggest that this variability has biogeographic patterns.

Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea.

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by gamma-proteobacteria (53 to 64%), followed by beta-proteobacteria (18 to 21%) and alpha-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by alpha-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of delta-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.