Proteomic Characterization of Damaged Single Hairs Recovered after an Explosion for Protein-Based Human Identification.

Evidence recovery is challenging where an explosion has occurred. Though hair evidence may be sufficiently robust to be recovered at the site, forensic analysis underutilizes the matrix by relying on morphological analysis. Where DNA is compromised, particularly in hair, protein-based human identification presents a promising alternative. Detection of amino acid polymorphisms in hair proteins as genetically variant peptides (GVPs) permits the inference of individualizing single nucleotide polymorphisms for identification. However, an explosive blast may damage hair proteins and compromise GVP identification. This work assesses effects of an explosive blast on the hair proteome and GVP identification, investigates microscopy as a predictor of proteome profiling success in recovered hairs to improve analysis throughput, and quantifies discriminative power in damaged hairs. The proteomics dataset has been deposited into the ProteomeXchange Consortium (PXD017427). With the exception of degradation in keratins K75, K80, K40, and keratin-associated protein KAP10-11 as markers of hair cuticular damage, corroborated by scanning electron microscopic analysis, minimal hair proteome degradation following explosion allowed successful proteome profiling of single hairs regardless of morphological damage. Finally, GVP identification remained independent of explosion conditions, permitting similar discriminative power between exploded and undamaged hairs. These findings lend greater confidence to GVP analysis in one-inch hairs for forensic identification and provide information about hair protein localization.

Integrating the MasSpec Pen to the da Vinci Surgical System for In Vivo Tissue Analysis during a Robotic Assisted Porcine Surgery.

Minimally invasive robotic-assisted surgeries have been increasingly used as a first-line of treatment for patients undergoing oncologic surgeries. In-situ tissue identification is critical to guide tissue resection and assist decision-making. Traditional intraoperative histopathologic analysis of frozen tissue sections can be time-consuming and present logistical challenges which interrupt surgical workflows. We report the development and implementation of a laparoscopic, drop-in version of the MasSpec Pen device integrated into the da Vinci Xi Surgical system for in vivo tissue analysis in a robotic-assisted porcine surgery. We evaluated the performance of the drop-in MasSpec Pen during surgery by introducing the device into the animal upper gastrointestinal system and performing in vivo analyses of the stomach and liver, including charred and bloody tissues after electrocauterization. The molecular profiles obtained included ions tentatively identified as metabolites and lipids typically observed with MasSpec Pen analysis, without causing observable tissue damage. Statistical classifiers built to distinguish porcine liver and stomach tissues using the in vivo data yielded an overall tissue identification accuracy of 98% (n = 53 analyses). The results provide evidence that the drop-in MasSpec Pen developed can be used to acquire mass spectra in vivo during a robotic-assisted surgery and might be used as an in vivo tissue assessment tool to help guide surgical resections and streamline surgical workflows.

Rapid and mild silylation of ß-amino alcohols at room temperature mediated by N-methylimidazole for enhanced detectability by gas chromatography/electron ionization mass spectrometry.

RATIONALE: In this work, we expand the use of in situ activation of chloro(dimethyl)phenylsilane using N-methylimidazole (NMI) for the effective derivatization of ß-aminoethyl alcohols. Due to its enhanced nucleophilic character, NMI is expected to act as an efficient activator in these reactions. METHODS: The derivatization of a panel of ß-aminoethyl alcohols was accomplished by reacting the analyte with chloro(dimethyl)phenylsilane in the presence of either NMI or pyridine. After the addition of chloro(dimethyl)phenylsilane, the vials were gently tumbled for 1 h at ambient temperature. The phenyldimethylsilyl derivatives were identified using gas chromatography/electron ionization mass spectrometry (GC/EI-MS). RESULTS: A total of ten ß-aminoethyl alcohols were successfully derivatized via in situ activation of chloro(dimethyl)-phenylsilane with NMI. Derivatization with NMI was significantly more efficient than with pyridine by a factor of 3-6 for the studied alcohols. The derivatizations in the presence of NMI were found to occur in just 1 h and were conveniently executed at ambient temperature. CONCLUSIONS: The use of the nitrogenous base NMI in order to activate chloro(dimethyl)phenylsilane for the efficient silylation of a panel of ß-aminoethyl alcohols has been demonstrated. The present work shows that NMI is an efficient base for the smooth derivatization of these types of alcohols. Furthermore, the installation of the bulky PDMS group onto these alcohols adds to the certainty that this is a viable approach for the installation of the more commonly employed, trimethylsilyl group. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Multidrug-resistant Stenotrophomonas maltophilia in residential aged care facilities: An emerging threat.

Stenotrophomonas maltophilia is a multidrug-resistant (MDR), Gram-negative bacterium intrinsically resistant to beta-lactams, including last-resort carbapenems. As an opportunistic pathogen, it can cause serious healthcare-related infections. This study assesses the prevalence, resistance profiles, and genetic diversity of S. maltophilia isolated from residential aged care facilities (RACFs). RACFs are known for their overuse and often inappropriate use of antibiotics, creating a strong selective environment that favors the development of bacterial resistance. The study was conducted on 73 S. maltophilia isolates recovered from wastewater and facility swab samples obtained from three RACFs and a retirement village. Phenotypic and genotypic assessments of the isolates revealed high carbapenem resistance, exemplifying their intrinsic beta-lactam resistance. Alarmingly, 49.3% (36/73) of the isolates were non-wild type for colistin, with minimum inhibitory concentration values of > 4 mg/L, and 11.0% (8/73) were resistant to trimethoprim-sulfamethoxazole. No resistance mechanisms were detected for either antimicrobial. Genotypic assessment of known lineages revealed isolates clustering with Sm17 and Sm18, lineages not previously reported in Australia, suggesting the potential ongoing spread of MDR S. maltophilia. Lastly, although only a few isolates were biocide tolerant (2.7%, 2/73), their ability to grow in high concentrations (64 mg/L) of triclosan is concerning, as it may be selecting for their survival and continued dissemination.

Resistome Analysis of Klebsiella pneumoniae Complex from Residential Aged Care Facilities Demonstrates Intra-facility Clonal Spread of Multidrug-Resistant Isolates.

Antimicrobial-resistant Klebsiella pneumoniae is one of the predominant pathogens in healthcare settings. However, the prevalence and resistome of this organism within residential aged care facilities (RACFs), which are potential hotspots for antimicrobial resistance, remain unexplored. Here, we provide a phenotypic and molecular characterization of antimicrobial-resistant K. pneumoniae isolated from RACFs. K. pneumoniae was isolated from urine, faecal and wastewater samples and facility swabs. The antimicrobial susceptibility profiles of all the isolates were determined and the genomic basis for resistance was explored with whole-genome sequencing on a subset of isolates. A total of 147 K. pneumoniae were isolated, displaying resistance against multiple antimicrobials. Genotypic analysis revealed the presence of beta-lactamases and the ciprofloxacin-resistance determinant QnrB4 but failed to confirm the basis for the observed cephalosporin resistance. Clonal spread of the multidrug-resistant, widely disseminated sequence types 323 and 661 was observed. This study was the first to examine the resistome of K. pneumoniae isolates from RACFs and demonstrated a complexity between genotypic and phenotypic antimicrobial resistance. The intra-facility dissemination and persistence of multidrug-resistant clones is concerning, given that residents are particularly vulnerable to antimicrobial resistant infections, and it highlights the need for continued surveillance and interventions to reduce the risk of outbreaks.

The scope of antimicrobial resistance in residential aged care facilities determined through analysis of Escherichia coli and the total wastewater resistome.

IMPORTANCE: Antimicrobial resistance (AMR) is a global threat that imposes a heavy burden on our health and economy. Residential aged care facilities (RACFs), where frequent inappropriate antibiotic use creates a selective environment that promotes the development of bacterial resistance, significantly contribute to this problem. We used wastewater-based epidemiology to provide a holistic whole-facility assessment and comparison of antimicrobial resistance in two RACFs and a retirement village. Resistant Escherichia coli, a common and oftentimes problematic pathogen within RACFs, was isolated from the wastewater, and the phenotypic and genotypic AMR was determined for all isolates. We observed a high prevalence of an international high-risk clone, carrying an extended-spectrum beta-lactamase in one facility. Analysis of the entire resistome also revealed a greater number of mobile resistance genes in this facility. Finally, both facilities displayed high fluoroquinolone resistance rates-a worrying trend seen globally despite measures in place aimed at limiting their use.

31P-edited diffusion-ordered 1H NMR spectroscopy for the spectral isolation and identification of organophosphorus compounds related to chemical weapons agents and their degradation products.

Organophosphorus compounds represent a large class of molecules that include pesticides, flame-retardants, biologically relevant molecules, and chemical weapons agents (CWAs). The detection and identification of organophosphorus molecules, particularly in the cases of pesticides and CWAs, are paramount to the verification of international treaties by various organizations. To that end, novel analytical methodologies that can provide additional support to traditional analyses are important for unambiguous identification of these compounds. We have developed an NMR method that selectively edits for organophosphorus compounds via (31)P-(1)H heteronuclear single quantum correlation (HSQC) and provides an additional chromatographic-like separation based on self-diffusivities of the individual species via (1)H diffusion-ordered spectroscopy (DOSY): (1)H-(31)P HSQC-DOSY. The technique is first validated using the CWA VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) by traditional two-dimensional DOSY spectra. We then extend this technique to a complex mixture of VX degradation products and identify all the main phosphorus-containing byproducts generated after exposure to a zinc-cyclen organometallic homogeneous catalyst.

Worldwide distribution and environmental origin of the Adelaide imipenemase (AIM-1), a potent carbapenemase in Pseudomonas aeruginosa.

Carbapenems are potent broad-spectrum ß-lactam antibiotics reserved for the treatment of serious infections caused by multidrug-resistant bacteria such as Pseudomonas aeruginosa. The surge in P. aeruginosa resistant to carbapenems is an urgent threat, as very few treatment options remain. Resistance to carbapenems is predominantly due to the presence of carbapenemase enzymes. The assessment of 147 P. aeruginosa isolates revealed that 32 isolates were carbapenem non-wild-type. These isolates were screened for carbapenem resistance genes using PCR. One isolate from wastewater contained the Adelaide imipenemase gene (blaAIM-1) and was compared phenotypically with a highly carbapenem-resistant clinical isolate containing the blaAIM-1 gene. A further investigation of wastewater samples from various local healthcare and non-healthcare sources as well as river water, using probe-based qPCR, revealed the presence of the blaAIM-1 gene in all the samples analysed. The widespread occurrence of blaAIM-1 throughout Adelaide hinted at the possibility of more generally extensive spread of this gene than originally thought. A blast search revealed the presence of the blaAIM-1 gene in Asia, North America and Europe. To elucidate the identity of the organism(s) carrying the blaAIM-1 gene, shotgun metagenomic sequencing was conducted on three wastewater samples from different locations. Comparison of these nucleotide sequences with a whole-genome sequence of a P. aeruginosa isolate revealed that, unlike the genetic environment and arrangement in P. aeruginosa, the blaAIM-1 gene was not carried as part of any mobile genetic elements. A phylogenetic tree constructed with the deduced amino acid sequences of AIM-1 suggested that the potential origin of the blaAIM-1 gene in P. aeruginosa might be the non-pathogenic environmental organism, Pseudoxanthomonas mexicana.

Assessing Single-Source Reproducibility of Human Head Hair Peptide Profiling from Different Regions of the Scalp.

Neither microscopical hair comparisons nor mitochondrial DNA sequencing alone, or together, constitutes a basis for personal identification. Due to these limitations, a complementary technique to compare questioned and known hair shafts was investigated. Recently, scientists from Lawrence Livermore National Laboratory's Forensic Science Center and other collaborators developed a peptide profiling technique, which can infer non-synonymous single nucleotide polymorphisms (SNPs) preserved in hair shaft proteins as single amino acid polymorphisms (SAPs). In this study, peptide profiling was evaluated to determine if it can meet forensic expectations when samples are in limited quantities with the possibility that hair samples collected from different areas of a single donor's scalp (i.e., single source) might not exhibit the same SAP profile. The average dissimilarity, percent differences in SAP profiles within each source, ranged from 0% difference to 29%. This pilot study suggests that more work is needed before peptide profiling of hair can be considered for forensic comparisons.

Automated analysis of scanning electron microscopic images for assessment of hair surface damage.

Mechanical damage of hair can serve as an indicator of health status and its assessment relies on the measurement of morphological features via microscopic analysis, yet few studies have categorized the extent of damage sustained, and instead have depended on qualitative profiling based on the presence or absence of specific features. We describe the development and application of a novel quantitative measure for scoring hair surface damage in scanning electron microscopic (SEM) images without predefined features, and automation of image analysis for characterization of morphological hair damage after exposure to an explosive blast. Application of an automated normalization procedure for SEM images revealed features indicative of contact with materials in an explosive device and characteristic of heat damage, though many were similar to features from physical and chemical weathering. Assessment of hair damage with tailing factor, a measure of asymmetry in pixel brightness histograms and proxy for surface roughness, yielded 81% classification accuracy to an existing damage classification system, indicating good agreement between the two metrics. Further ability of the tailing factor to score features of hair damage reflecting explosion conditions demonstrates the broad applicability of the metric to assess damage to hairs containing a diverse set of morphological features.

Efflux Pump-Driven Antibiotic and Biocide Cross-Resistance in Pseudomonas aeruginosa Isolated from Different Ecological Niches: A Case Study in the Development of Multidrug Resistance in Environmental Hotspots.

Pseudomonas aeruginosa is an opportunistic pathogen displaying high intrinsic antimicrobial resistance and the ability to thrive in different ecological environments. In this study, the ability of P. aeruginosa to develop simultaneous resistance to multiple antibiotics and disinfectants in different natural niches were investigated using strains collected from clinical samples, veterinary samples, and wastewater. The correlation between biocide and antimicrobial resistance was determined by employing principal component analysis. Molecular mechanisms linking biocide and antimicrobial resistance were interrogated by determining gene expression using RT-qPCR and identifying a potential genetic determinant for co- and cross-resistance using whole-genome sequencing. A subpopulation of P. aeruginosa isolates belonging to three sequence types was resistant against the common preservative benzalkonium chloride and showed cross-resistance to fluoroquinolones, cephalosporins, aminoglycosides, and multidrug resistance. Of these, the epidemiological high-risk ST235 clone was the most abundant. The overexpression of the MexAB-OprM drug efflux pump resulting from amino acid mutations in regulators MexR, NalC, or NalD was the major contributing factor for cross-resistance that could be reversed by an efflux pump inhibitor. This is the first comparison of antibiotic-biocide cross-resistance in samples isolated from different ecological niches and serves as a confirmation of laboratory-based studies on biocide adapted isolates. The isolates from wastewater had a higher incidence of multidrug resistance and biocide-antibiotic cross-resistance than those from clinical and veterinary settings.

Development of a Protein-based Human Identification Capability from a Single Hair.

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high-resolution nanoflow liquid chromatography-based mass spectrometry and a novel exome-driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10-2 and 6.0 × 10-9 . The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.

Hair Proteome Variation at Different Body Locations on Genetically Variant Peptide Detection for Protein-Based Human Identification.

Human hair contains minimal intact nuclear DNA for human identification in forensic and archaeological applications. In contrast, proteins offer a pathway to exploit hair evidence for human identification owing to their persistence, abundance, and derivation from DNA. Individualizing single nucleotide polymorphisms (SNPs) are often conserved as single amino acid polymorphisms in genetically variant peptides (GVPs). Detection of GVP markers in the hair proteome via high-resolution tandem mass spectrometry permits inference of SNPs with known statistical probabilities. To adopt this approach for forensic investigations, hair proteomic variation and its effects on GVP identification must first be characterized. This research aimed to assess variation in single-inch head, arm, and pubic hair, and discover body location-invariant GVP markers to distinguish individuals. Comparison of protein profiles revealed greater body location-specific variation in keratin-associated proteins and intracellular proteins, allowing body location differentiation. However, robust GVP markers derive primarily from keratins that do not exhibit body location-specific differential expression, supporting GVP identification independence from hair proteomic variation at the various body locations. Further, pairwise comparisons of GVP profiles with 8 SNPs demonstrated greatest interindividual variation and high intraindividual consistency, enabling similar differentiative potential of individuals using single hairs irrespective of body location origin.

Combined DNA Typing and Protein Identification from Unfired Brass Cartridges,,.

Biological evidence analysis from contact traces is adversely affected by low quantity and quality of DNA. Proteins in these samples contain potentially individualizing information and may be particularly important for difficult surfaces such as brass, where DNA may yield incomplete profiles. In this study, touched unfired brass cartridges were sampled using dry tape or wet swabs and analyzed by separating DNA and protein from the same collected material, thus producing both genomic and proteomic information. DNA recovery was similar for both collection methods, with tape yielding an average of 1.36 ± 1.87 ng and swabs, 1.34 ± 3.04 ng. Analysis by mass spectrometry identified 95 proteins, with the two collection methods showing no significant difference (p = 0.76) in the average number of collected proteins: 44.5 ± 10.9, (tape) versus 47.9 ± 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.

Chemical vapor discrimination using a compact and low-power array of piezoresistive microcantilevers.

A compact and low-power microcantilever-based sensor array has been developed and used to detect various chemical vapor analytes. In contrast to earlier micro-electro-mechanical systems (MEMS) array sensors, this device uses the static deflection of piezoresistive cantilevers due to the swelling of glassy polyolefin coatings during sorption of chemical vapors. To maximize the sensor response to a variety of chemical analytes, the polymers are selected based on their Hildebrand solubility parameters to span a wide range of chemical properties. We utilize a novel microcontact spotting method to reproducibly coat a single side of each cantilever in the array with the polymers. To demonstrate the utility of the sensor array we have reproducibly detected 11 chemical vapors, representing a breadth of chemical properties, in real time and over a wide range of vapor concentrations. We also report the detection of the chemical warfare agents (CWAs) VX and sulfur mustard (HD), representing the first published report of CWA vapor detection by a polymer-based, cantilever sensor array. Comparisons of the theoretical polymer/vapor partition coefficient to the experimental cantilever deflection responses show that, while general trends can be reasonably predicted, a simple linear relationship does not exist.

Protein-based forensic identification using genetically variant peptides in human bone.

Bone tissue contains organic material that is useful for forensic investigations and may contain preserved endogenous protein that can persist in the environment for extended periods of time over a range of conditions. Single amino acid polymorphisms in these proteins reflect genetic information since they result from non-synonymous single nucleotide polymorphisms (SNPs) in DNA. Detection of genetically variant peptides (GVPs) - those peptides that contain amino acid polymorphisms - in digests of bone proteins allows for the corresponding SNP alleles to be inferred. Resulting genetic profiles can be used to calculate statistical measures of association between a bone sample and an individual. In this study proteomic analysis on rib cortical bone samples from 10 recently deceased individuals demonstrates this concept. A straight-forward acidic demineralization protocol yielded proteins that were digested with trypsin. Tryptic digests were analyzed by liquid chromatography mass spectrometry. A total of 1736 different proteins were identified across all resulting datasets. On average, individual samples contained 454±121 (x¯±σ) proteins. Thirty-five genetically variant peptides were identified from 15 observed proteins. Overall, 134 SNP inferences were made based on proteomically detected GVPs, which were confirmed by sequencing of subject DNA. Inferred individual SNP genetic profiles ranged in random match probability (RMP) from 1/6 to 1/42,472 when calculated with European population frequencies in the 1000 Genomes Project, Phase 3. Similarly, RMPs based on African population frequencies were calculated for each SNP genetic profile and likelihood ratios (LR) were obtained by dividing each European RMP by the corresponding African RMP. Resulting LR values ranged from 1.4 to 825 with a median value of 16. GVP markers offer a basis for the identification of compromised skeletal remains independent of the presence of DNA template.

Static deflection measurements of cantilever arrays reveal polymer film expansion and contraction.

An optical static method of detection is used to interpret surface stress induced bending related to cantilevers coated on one side with poly(vinyl alcohol), poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate), and poly(vinyl chloride-co-vinyl acetate-co-2-hydroxypropyl acrylate), or respectively, PVA, PVB, and PVC, and exposed to various solvent vapors. Results indicate that the adsorption and surface interactions of the different solvent vapors that cause polymer swelling and shrinking lead to rearrangements, which have been shown to change the elastic properties of the polymer film, and subsequently, the spring constant of the polymer coated cantilever. Static deflection measurements allow the direction of cantilever bending to be determined, which adds a new dimension of usefulness for surface functionalized cantilevers as transducers in the development of novel microelectromechanical systems (MEMS).

Adenocarcinoma of the distal pancreas presenting as an intrathoracic mass.

Tumors of the pancreatic head may present with early heralding symptoms such as obstructive jaundice. Pancreatic masses within the body or tail usually have delayed diagnosis secondary to the lack of any early findings, which, in turn, leads to a higher incidence of involvement of adjacent structures, such as the superior mesenteric artery, portal vein, or superior mesenteric vein. Local involvement along with distant metastases or larger tumor size may be contraindications to surgical resection. The authors report a case of advanced pancreatic adenocarcinoma in which the anomalous thoracic location of the organ resulted in the tumor being resectable. This case adds support to an aggressive approach to surgical resection of distal pancreatic tumors.

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome.

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects' DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European-American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.