Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin.

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva. The availability of mass spectrometry-based tools for the detection of less abundant, but more specific, protein targets (e.g., human statherin) has enabled the development of high confidence assays for human saliva. Sample throughput, however, has traditionally been low due to multi-step workflows for protein extraction, quantitation, enzymatic digestion, solid phase cleanup, and nano-/capillary-based chromatography. Here, we present two novel "direct" single-stage extraction strategies for sample preparation. These feature immunoaffinity purification and reversed-phase solid-phase microextraction in conjunction with intact mass analysis of human statherin for saliva identification. Mass analysis was performed on the Thermo Scientific Q-Exactive™ Orbitrap mass spectrometer with a 10-min analytical run time. Data analysis was performed using Byos® from Protein Metrics. Two sample sets were analyzed with a population of 20 individuals to evaluate detection reliability. A series of casework-type samples were then assayed to evaluate performance in an authentic forensic context. Statherin was confidently identified in 92% and 71% of samples extracted using the immunoaffinity purification and solid phase microextraction approaches, respectively. Overall, immunoaffinity purification outperformed the solid phase microextraction, especially with complex mixtures. In toto, robotic extraction and intact mass spectrometry enable the reliable identification of trace human saliva in a variety of sample types.

Evolution of gnathostome prodynorphin and proenkephalin: characterization of a shark proenkephalin and prodynorphin cDNAs.

Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA. The sequence identity within the opioid positions was 59% at the amino acid level. Shark α-neo-endorphin, dynorphin A, and dynorphin B have amino acid motifs in common with shark met-enkephalin-8, and shark proenkephalin opioid positions E and F, respectively, which have not been observed in other gnathostome prodynorphin and proenkephalin precursor sequences. Shark prodynorphin encodes both kappa (α-neo-endorphin, dynorphin A, and dynorphin B) and delta (met-enkephalin and leu-enkephalin) opioid sequences. Mixed function prodynorphin precursors (encoding both enkephalins and dynorphins) are also found in representatives of the teleost fishes, lungfishes, and amphibians. It appears that only mammals evolved a prodynorphin precursor that exclusively encodes kappa opioid agonists (dynorphins).

Developmental validation of a multiplex proteomic assay for the identification of forensically relevant biological fluids.

The aim of this study was to validate a multiplex proteomic assay for the identification of high-specificity protein biomarkers by multiple reaction monitoring mass spectrometry on a triple quadrupole mass spectrometer for the accurate, reliable, and confirmatory identification of bodily fluids commonly encountered in a forensic context. This includes the identification of peripheral blood, semen, saliva, urine, and vaginal/menstrual fluid. The assay is able to efficiently identify pure or mixed stains through the identification of target peptide fragments originating from tissue-specific proteins including: uromodulin from urine; prostatic acid phosphatase, prostate specific antigen and semenogelin-II for semen; statherin, submaxillary gland androgen-regulated protein 3B and amylase for saliva; cornulin, martrigel-induced gene C4 protein, suprabasin and neutrophil gelatinase-associated lipocalin for vaginal/menstrual fluid; and alpha-1 antitrypsin, hemopexin, and hemoglobin subunit beta for peripheral blood. Based on the results of the developmental validation studies which included an assessment of reproducibility and repeatability, sensitivity, species specificity, carryover, mixtures, as well as a series of casework type samples. Only a small selection of case samples was unable to unambiguously identify the target fluid including urine recovered from substrates as well as semen when mixed with personal lubricants. Overall, the mass spectrometry-based workflow offers significant advantages compared to existing serological methods.

Effects of organic acids and common household products on the occurrence of false positive test results using immunochromatographic assays.

Forensic serological analyses often rely on lateral flow immunochromatographic assays to detect proteins that are characteristic of forensically relevant body fluids. In this study, we demonstrate that a positive result, however, is not limited to target protein binding. Citric and lactic acids at various pH levels were tested using 9 different commercial immunochromatographic assays. Varying rates of false positive results were observed with commercial serological assays irrespective of brand or target biological fluid over a wide pH range. The use of kit specific buffers were only partially effective in mitigating the occurrence of organic acid-associated false positive results. Common household products containing organic acids were also tested and found to produce non-specific binding events. This is not to suggest that immunochromatographic assays are not useful as presumptive indicators of bodily fluids. Rather, this study provides a cautionary demonstration of the ease with which organic acids in common household products can generate false positives results. This finding underscores the presumptive nature of these antibody-based lateral flow assay systems.

Validation of reduced-scale reactions for the Quantifiler Human DNA kit.

Accurate quantification of DNA samples is an important step in obtaining accurate and reproducible short tandem repeat (STR) profiles. Quantitative real-time-PCR has improved the speed and accuracy of DNA quantification over earlier methods, albeit at significantly greater cost per reaction. Here, the performance of reduced volume (10 microL) DNA quantification assays using the Quantifiler Human DNA Quantification Kit was evaluated using commercial standards and single source biological stains (e.g., venous blood, saliva, and semen). In addition, casework-type samples including those subjected to environmental contaminants containing PCR inhibitors and samples having undergone extensive DNA degradation were also quantified. The concentration of DNA in various forensic samples ranged from 0 to 2.9 ng/microL depending on sample source and/or environmental insult. Compared to full-scale reactions, reduced volume assays displayed equivalent to improved amplification efficiency and sample-to-sample reproducibility (+/-0.01-0.17 C(T FAM)). Furthermore, the use of data from reduced-scale Quantifiler reactions facilitated the accurate determination of the amount of sample DNA extract needed to generate quality STR profiles. The use of 10 microL-scale Quantifiler reaction volumes has the practical benefit of increasing the effective number of reactions per kit by 250%; thereby reducing the cost per assay by 60% while consuming less sample. This is particularly advantageous in cases of consumptive testing.

SPERM HY-LITER™ for the identification of spermatozoa from sexual assault evidence.

Accurate microscopic identification of human spermatozoa is important in sexual assault cases. We have compared the results of examinations with (1) a fluorescent microscopy method, SPERM HY-LITER™, and (2) Baecchi's method for identification of human spermatozoa. In 35 artificial, forensic type samples, spermatozoa were identified in 45.7% with SPERM HY-LITER™ in Copenhagen, in 54.3% in the laboratory of the manufacturer of SPERM HY-LITER™, and 40.0% of the samples with Baecchi's staining method. When differences occurred between the two methods, it was significantly more often that SPERM HY-LITER™ detected spermatozoa when Baecchi's method did not (ts=6.567, df=1, P=0.048). This trend was also seen in selected compromised or degraded samples and in selected adjudicative samples. The reactions with spermatozoa from dog, horse, pig and bull were negative with SPERM HY-LITER™, whereas Baecchi's method was non-selective. Data from forensic casework samples in Copenhagen from two years (2008 and 2009) are presented. The samples from 2008 were investigated using Baecchi's method, while those from 2009 were investigated using SPERM HY-LITER™. The frequencies of positive results were similar between the two methods for the two years (27.9% and 32.1% respectively). Analysis of acid phosphatase (ACP) activity for the positive results obtained for these two years does not support the use of a negative ACP result as a prescreen for microscopic analysis for spermatozoa.

Seasonal changes in CRF-I and urotensin I transcript levels in masu salmon: correlation with cortisol secretion during spawning.

Pacific salmon employ a semelparous reproductive strategy where sexual maturation is followed by rapid senescence and death. Cortisol overproduction has been implicated as the central physiologic event responsible for the post-spawning demise of these fish. Cortisol homeostasis is regulated through the action of hormones of the hypothalamus-pituitary-interrenal (HPI) axis. These include corticotropin-releasing factor (CRF) and urotensin-I (UI). In the present study, masu salmon (Oncorhynchus masou) were assayed for changes in the levels CRF-I and UI mRNA transcripts by quantitative real-time PCR (qRT-PCR). These results were compared to plasma cortisol levels in juvenile, adult, and spawning masu salmon to identify specific regulatory factors that appear to be functionally associated with changes in cortisol levels. Intramuscular implantation of GnRH analog (GnRHa) capsules was also used to determine whether GnRH influences stress hormone levels. In both male and female masu salmon, spawning fish experienced a 5- to 7-fold increase in plasma cortisol levels relative to juvenile non-spawning salmon. Changes in CRF-I mRNA levels were characterized by 1-2 distinctive short-term surges in adult masu salmon. Conversely, seasonal changes in UI mRNA levels displayed broad and sustained increases during the pre-spawning and spawning periods. The increases in UI mRNA levels were positively correlated (R(2)=0.21 male and 0.26 female, p<0.0001) with levels of plasma cortisol in the pre-spawning and spawning periods. Despite the importance of GnRH in sexual maturation and reproduction, the administration of GnRHa to test animals failed to produce broad changes in CRF-I, UI or plasma cortisol levels. These findings suggest a more direct role for UI than for CRF-I in the regulation of cortisol levels in spawning Pacific salmon.