Rapid screening of riot control agents using DART-TD-HRMS.

PURPOSE: Riot Control Agents (RCAs) are chemicals used in law enforcement for non-lethal riot control and use in conflicts between states that violates the Chemical Weapons Convention. OPCW's Scientific Advisory Board has identified sixteen potential RCAs including capsaicinoids, CS, and CR. RCAs may be misused for criminal purposes, so methods for detecting such misuse are needed. This study therefore evaluates the feasibility of a rapid, high throughput screening method of RCAs on surfaces (particularly clothing surfaces) by Direct Analysis in Real Time with a thermal desorption unit coupled to high-resolution mass spectrometry (DART-TD-HRMS). METHODS: A broadly applicable method for detecting potential RCAs was developed and tested on cotton fabric samples sprayed with self-defence sprays from an in-house reference stock. The feasibility of detecting RCAs by direct analysis of surface wipe samples placed in the DART source was also investigated. RESULTS: The method detected all sixteen RCAs and contaminated clothing were successfully screened for active agents in a reference collection of self-defence sprays. A pilot study also showed that RCAs can be detected by holding a sample directly in front of the DART source. CONCLUSION: DART-TD-HRMS enables rapid and simple screening of RCAs on fabric samples enabling a high sample throughput.

Comparison of skin decontamination strategies in the initial operational response following chemical exposures.

In mass casualty incidents including hazardous chemical skin exposure, decontamination is the primary intervention to avoid systemic uptake of the toxic compound. The protocol needs to be both simple and efficient to enable a rapid response and avoid delay of patient management. In the present study, decontamination strategies included in the initial operational response were evaluated following human skin exposure in vitro to four different contaminants. Results demonstrated that the efficacy of selected decontamination procedures was highly dependent on the chemical contaminant used. Dry removal of the sulfur mustard simulant methyl salicylate prior to wet decontamination was found beneficial compared to wet decontamination alone. Rapidly initiated wet decontamination was more efficient compared to dry and wet removal of the industrial chemical 2-butoxyethanol and the nerve agent tabun. Following VX-exposure, all wet decontamination procedures resulted in increased agent penetration compared to the control. In conclusion, challenges in establishing simple and efficient decontamination procedures for a broad-spectrum of chemicals have been demonstrated. The impact of including a dry removal step during decontamination was evidently agent specific. Despite the variation in efficacy, immediately initiated dry removal may facilitate patient management until wet decontamination resources are available and to reduce the risk of secondary contamination.

Psychometric evaluation of the Swedish Multidimensional Psychological Flexibility Inventory (MPFI).

Psychiatric disorders are common, and reliable measures are crucial for research and clinical practice. A cross-diagnostic construct that can be used to index treatment outcomes as well as prevalence of psychological ill health is psychological flexibility. The aim of this study was to validate a Swedish version of the Multidimensional Psychological Flexibility Inventory (MPFI). The MPFI has 12 subscales, six of which measure flexibility, and six that measure inflexibility. Using confirmatory factor analysis in a community sample of 670 participants, we found that a model with two higher order factors had satisfactory fit (CFI = .933) and a 12-factor model had the best fit to the data (CFI = .955). All 12 subscales showed adequate reliability (CRs = .803-.933) and the factor structure was similar across age groups and gender. Findings suggest that the Swedish version of the MPFI is a reliable instrument that can be used to index psychological flexibility. Potential areas for improvement of the instrument are discussed.

Do cold weather temperatures affect the efficacy of skin decontamination?

Skin decontamination in cold weather temperatures might be challenging due to the aggravating circumstances. However, no information is available on the efficacy of commonly used procedures in winter conditions. Therefore, the efficacy of the reactive skin decontamination lotion (RSDL) and soapy water decontamination following skin exposure to the nerve agent VX was evaluated at three ambient air temperatures (-5°C, -15°C and room temperature). Experiments were performed in vitro using human dermatomed skin. The ability of RSDL to degrade VX at the three different air temperatures was separately evaluated. The ambient air temperature in experiments without decontamination did not influence the penetration rate of VX through skin. RSDL decontamination was highly efficient in removing VX from skin when performed in all three ambient temperatures, despite the slower agent degradation rate of VX at the lower temperatures. Decontamination with soapy water at RT resulted in an increased skin penetration of VX compared with the control without decontamination; however, in colder temperatures the VX skin penetration was similar to the corresponding control without decontamination. At RT, dry removal prior to washing with soapy water did not improve decontamination of VX compared with washing solely with soapy water. This study demonstrated high efficacy of RSDL decontamination following skin exposure to VX also at cold temperatures. The previously reported 'wash-in' effect of soapy water on VX skin penetration was reduced at cold temperatures. Altogether, this study found a scientific basis to establish guidelines for skin decontamination of chemical casualties at cold weather temperatures.

Nasal Lavage Fluid as a Biomedical Sample for Verification of Chlorine Exposure.

Chlorine is a toxic chemical that has been used as a chemical warfare agent in recent armed conflicts. There is an urgent need for methods to verify alleged uses of chlorine, and phospholipid chlorohydrins (PL-HOCl) derived from the pulmonary surfactant of exposed victims have previously been proposed as biomarkers of chlorine exposure. Here, we describe an improved protocol for the chemical analysis of these biomarkers and its applicability to biomedical samples from chlorine-exposed animals. By the use of a polymeric solid-phase-supported transesterification of PL-HOCl using ethanolamine, a common biomarker, oleoyl ethanolamide chlorohydrin (OEA-HOCl), was derived from all the diverse oleoyl PL-HOCl that may be formed by chlorine exposure. Compared to native lipid biomarkers, OEA-HOCl represents a larger biomarker pool and is better suited for nano-liquid chromatography--tandem mass spectrometry (nLC-MS-MS analysis), generating 3 amol Limit of Detection (LOD) and a reduced sample carry-over. With the improved protocol, significantly elevated levels of OEA-HOCl were identified in bronchoalveolar lavage fluid (BALF) of chlorine-exposed rats, 2-48 hours after exposure. The difficulty of BALF sampling from humans limits the methods usefulness as a verification tool of chlorine exposure. Conversely, nasal lavage fluid (NLF) is readily collected without advanced equipment. In NLF from chlorine-exposed rats, PL-HOCl were identified and significantly elevated levels of the OEA-HOCl biomarker were detected 2-24 hours after exposure. In order to test the potential of NLF as a biomedical sample for verification of human exposure to chlorine, in-vitro chlorination of human NLF samples was performed. All human in-vitro chlorinated NLF samples exhibited elevated OEA-HOCl biomarker levels, following sample derivatization. These data indicate the potential of human NLF as a biomedical sample for the verification of chlorine exposure, but further work is required to develop and validate the method for the use on real-world samples.

Improved thermoelectric performance of Bi-deficient BiCuSeO material doped with Nb, Y, and P.

Thermoelectric materials convert waste heat into electric energy. Oxyselenide-based material, specifically, p-type BiCuSeO, is one of the most promising materials for these applications. There are numerous approaches to improve the heat-to-electricity conversion performance. Usually, these approaches are applied individually, starting from the pure intrinsic material. Higher performance could, however, be reached by combining a few strategies simultaneously. In the current work, yttrium, niobium, and phosphorous substitutions on the bismuth sites in already bismuth-deficient Bi1-xCuSeO systems were investigated via density functional theory. The bismuth-deficient system was used as the reference system for further introduction of substitutional defects. The substitution with phosphorous showed a decrease of up to 40 meV (11%) in the energy gap between conduction and valence bands at the highest substitution concentration. Doping with niobium led to the system changing from a p-type to an n-type conductor, which provides a possible route to obtain n-type BiCuSeO systems.

Route Determination of Sulfur Mustard Using Nontargeted Chemical Attribution Signature Screening.

Route determination of sulfur mustard was accomplished through comprehensive nontargeted screening of chemical attribution signatures. Sulfur mustard samples prepared via 11 different synthetic routes were analyzed using gas chromatography/high-resolution mass spectrometry. A large number of compounds were detected, and multivariate data analysis of the mass spectrometric results enabled the discovery of route-specific signature profiles. The performance of two supervised machine learning algorithms for retrospective synthetic route attribution, orthogonal partial least squares discriminant analysis (OPLS-DA) and random forest (RF), were compared using external test sets. Complete classification accuracy was achieved for test set samples (2/2 and 9/9) by using classification models to resolve the one-step routes starting from ethylene and the thiodiglycol chlorination methods used in the two-step routes. Retrospective determination of initial thiodiglycol synthesis methods in sulfur mustard samples, following chlorination, was more difficult. Nevertheless, the large number of markers detected using the nontargeted methodology enabled correct assignment of 5/9 test set samples using OPLS-DA and 8/9 using RF. RF was also used to construct an 11-class model with a total classification accuracy of 10/11. The developed methods were further evaluated by classifying sulfur mustard spiked into soil and textile matrix samples. Due to matrix effects and the low spiking level (0.05% w/w), route determination was more challenging in these cases. Nevertheless, acceptable classification performance was achieved during external test set validation: chlorination methods were correctly classified for 12/18 and 11/15 in spiked soil and textile samples, respectively.

In vitro human skin decontamination efficacy of MOF-808 in decontamination lotion following exposure to the nerve agent VX.

Metal-organic frameworks (MOFs) have shown promising properties for removal of chemical warfare agents, in particular for material decontamination and functionalized fabrics. The MOF-properties could also be beneficial for skin decontamination, especially when exposed to highly toxic and low volatile nerve agents. In such exposures, efficient decontamination is crucial for adequate medical management. In the present study, seven zirconium-based MOFs were evaluated for their ability to degrade VX and subsequently tested in vitro for decontamination of VX on human dermatomed skin. Of the MOFs evaluated, MOF-808 showed the greatest ability to degrade VX in an alkaline buffer with complete degradation of VX within 5 min. PCN-777, Zr-NDC and NU-1000 displayed degradation half-lives of approximately 10 min. When including MOF-808 in a skin friendly carrier with slightly acidic pH, a decreased agent degradation rate was observed, requiring over 24 h to reach complete degradation. In skin decontamination experiments, MOF-808 enhanced the efficacy compared to the carrier alone, essentially by improved agent absorption. Adding MOF-808 to Reactive Skin Decontamination Lotion (RSDL) did not improve the high effectiveness of RSDL alone. The present study showed that including MOF in skin decontamination lotions could be beneficial. Further studies should include optimizing the particulates and formulations.

Skin penetration and decontamination efficacy following human skin exposure to fentanyl.

Unintentional exposure to potent synthetic opioids during law enforcement seizures and rescue operations can potentially result in incapacitating effects or life-threatening respiratory depression. The hazard comes mainly from inhalation exposure, however, the skin contact risk should be considered. In the present study, the skin penetration of fentanyl and the efficacy of different decontamination protocols were evaluated by applying two forms of fentanyl on dermatomed human skin mounted in a diffusion cell. Studies were performed on dry skin or skin moistened by water, sweat or hand sanitizer. The free base of fentanyl displayed greater skin penetration ability than the hydrochloride salt and a higher steady state penetration rate of fentanyl in solution compared to powder on dry skin. Sweaty skin increased the penetration rate, both when applied in solution and as powder. The hand sanitizer increased skin penetration of the free base fentanyl but not the hydrochloride salt. Of the evaluated decontamination procedures, only soapy water demonstrated a general efficacy. In conclusion, the skin contact hazard of fentanyl is highly dependent on the exposure conditions and contamination density. The risk for physiological effects of fentanyl is assessed to occur only at very high exposures on sweaty skin. In such events, skin decontamination using soap and water is estimated to be a sufficient decontamination procedure.

Attribution of fentanyl analogue synthesis routes by multivariate data analysis of orthogonal mass spectral data.

Chemical attribution signatures (CAS) can be used to obtain useful forensic information and evidence from illicit drug seizures. A CAS is typically generated using hyphenated chemical analysis techniques and consists of a fingerprint of the by-products and additives present in a sample. Among other things, it can provide information on the sample's origin, its method of production, and the sources of its precursors. This work investigates the possibility of using multivariate CAS analysis to identify the synthetic methods used to prepare seized fentanyl analogues, independently of the analogues' acyl derivatization. Three chemists working in two labs synthesized three different fentanyl analogues, preparing each one in duplicate by six different routes. The final collection of analogues (96 samples) and two intermediates (16 + 32 samples) were analysed by GC-MS and UHPLC-HRMS, and the resulting analytical data were used for multivariate modelling. Independently of analogue structure, the tested fentanyls could be classified based on the method used in the first step of their synthesis. The multivariate model's ability to classify unknown samples was then evaluated by applying it to six new fentanyl analogues. Additionally, seized fentanyl samples was analysed and classified by the model.

Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay.

Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification in Plasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in the P. falciparum parasite lysate and intact infected red blood cells, we found P. falciparum purine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme's active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA for P. falciparum constitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs.

Backbone resonance assignment for the N-terminal region of bacterial tRNA-(N1G37) methyltransferase.

Bacterial tRNA (guanine37-N1)-methyltransferase (TrmD) is an important antibacterial target due to its essential role in translation. TrmD has two domains connected with a flexible linker. The N-terminal domain (NTD) of TrmD contains the S-adenosyl-L-methionine (SAM) cofactor binding site and the C-terminal domain is critical for tRNA binding. Here we report the backbone NMR resonance assignments for NTD of Pseudomonas aeruginosa TrmD. Its secondary structure was determined based on the assigned resonances. Relaxation analysis revealed that NTD existed as dimers in solution. NTD also exhibited thermal stability in solution. Its interactions with SAM and other compounds suggest it can be used for evaluating SAM competitive inhibitors by NMR.

An efficient proteome-wide strategy for discovery and characterization of cellular nucleotide-protein interactions.

Metabolite-protein interactions define the output of metabolic pathways and regulate many cellular processes. Although diseases are often characterized by distortions in metabolic processes, efficient means to discover and study such interactions directly in cells have been lacking. A stringent implementation of proteome-wide Cellular Thermal Shift Assay (CETSA) was developed and applied to key cellular nucleotides, where previously experimentally confirmed protein-nucleotide interactions were well recaptured. Many predicted, but never experimentally confirmed, as well as novel protein-nucleotide interactions were discovered. Interactions included a range of different protein families where nucleotides serve as substrates, products, co-factors or regulators. In cells exposed to thymidine, a limiting precursor for DNA synthesis, both dose- and time-dependence of the intracellular binding events for sequentially generated thymidine metabolites were revealed. Interactions included known cancer targets in deoxyribonucleotide metabolism as well as novel interacting proteins. This stringent CETSA based strategy will be applicable for a wide range of metabolites and will therefore greatly facilitate the discovery and studies of interactions and specificities of the many metabolites in human cells that remain uncharacterized.

Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice.

Glucose regulated protein 78 kDa (GRP78) is a recently emerged target for cancer therapy and a biomarker for cancer prognosis. Overexpression of GRP78 is observed in many types of cancers, with the cell-surface GRP78 being preferentially present in cancer cells and cancer blood vessel endothelial cells. Isthmin (ISM) is a secreted high-affinity proapoptotic protein ligand of cell-surface GRP78 that suppresses angiogenesis and tumor growth in mice. The C-terminal AMOP (adhesion-associated domain in MUC4 and other proteins) domain of ISM is critical in mediating its interaction with human umbilical vein endothelial cells (HUVECs). In this work, we report novel cyclic peptides harboring the RKD motif in the ISM AMOP domain that function as proapoptotic ligands of cell-surface GRP78. The most potent peptide, BC71, binds to GRP78 and converge to tumor in mice. Intravenous administration of BC71 suppressed xenograft tumor growth in mice as a single agent, with significant reduction in tumor angiogenesis and upsurge in apoptosis. Fluorescent-labeled BC71 accumulates in tumor in mice by targeting cell-surface GRP78. We show that BC71 triggers apoptosis via cell-surface GRP78 and activates caspase-8 and p53 signaling pathways in HUVECs. Using amide hydrogen-deuterium exchange mass spectrometry (HDXMS), we identified that BC71 preferentially binds to ATP-bound GRP78 via amino acid residues 244-257 of GRP78. Hence, BC71 serves as a valuable prototype for further development of peptidomimetic anticancer drugs targeting cell-surface GRP78 as well as PET imaging agents for cancer prognosis.

Synthesis route attribution of sulfur mustard by multivariate data analysis of chemical signatures.

A multivariate model was developed to attribute samples to a synthetic method used in the production of sulfur mustard (HD). Eleven synthetic methods were used to produce 66 samples for model construction. Three chemists working in both participating laboratories took part in the production, with the aim to introduce variability while reducing the influence of laboratory or chemist specific impurities in multivariate analysis. A gas chromatographic/mass spectrometric data set of peak areas for 103 compounds was subjected to orthogonal partial least squares - discriminant analysis to extract chemical attribution signature profiles and to construct multivariate models for classification of samples. For one- and two-step routes, model quality allowed the classification of an external test set (16/16 samples) according to synthesis conditions in the reaction yielding sulfur mustard. Classification of samples according to first-step methodology was considerably more difficult, given the high purity and uniform quality of the intermediate thiodiglycol produced in the study. Model performance in classification of aged samples was also investigated.

On the use of spectra from portable Raman and ATR-IR instruments in synthesis route attribution of a chemical warfare agent by multivariate modeling.

Collecting data under field conditions for forensic investigations of chemical warfare agents calls for the use of portable instruments. In this study, a set of aged, crude preparations of sulfur mustard were characterized spectroscopically without any sample preparation using handheld Raman and portable IR instruments. The spectral data was used to construct Random Forest multivariate models for the attribution of test set samples to the synthetic method used for their production. Colored and fluorescent samples were included in the study, which made Raman spectroscopy challenging although fluorescence was diminished by using an excitation wavelength of 1064 nm. The predictive power of models constructed with IR or Raman data alone, as well as with combined data was investigated. Both techniques gave useful data for attribution. Model performance was enhanced when Raman and IR spectra were combined, allowing correct classification of 19/23 (83%) of test set spectra. The results demonstrate that data obtained with spectroscopy instruments amenable for field deployment can be useful in forensic studies of chemical warfare agents.

Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality.

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131's ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a Ras˧PIK3IP1˧PI3K signaling network.

Modulating release of ranibizumab and aflibercept from thiolated chitosan-based hydrogels for potential treatment of ocular neovascularization.

BACKGROUND: This paper describes the synthesis of thiolated chitosan-based hydrogels with varying degrees of crosslinking that has been utilized to modulate release kinetics of two clinically relevant FDA-approved anti-VEGF protein drugs, ranibizumab and aflibercept. These hydrogels have been fabricated into disc shaped structures for potential use as patches on ocular surface. METHODS: Protein conformational changes and aggregation after loading and release was evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, electrophoresis and size-exclusion chromatography (SEC). Finally, the capacity of both released proteins to bind to VEGF was tested by ELISA and surface plasmon resonance (SPR) technology. RESULTS: The study demonstrates the versatility of thiolated chitosan-based hydrogels for delivering proteins. The effect of various parameters of the hydrogel on protein release kinetics and mechanism of protein release was studied using the Korsmeyer-Peppas release model. Furthermore, we have studied the stability of released proteins in detail while comparing it with non-entrapped proteins under physiological conditions to understand the effect of formulation conditions on protein stability. CONCLUSIONS: The disc-shaped thiolated chitosan-based hydrogels provide a potentially useful platform to deliver ranibizumab and aflibercept for the treatments of ocular diseases such as wet AMD, DME and corneal neovascularization.

Structural analyses of human thymidylate synthase reveal a site that may control conformational switching between active and inactive states.

Thymidylate synthase (TS) is the sole enzyme responsible for de novo biosynthesis of thymidylate (TMP) and is essential for cell proliferation and survival. Inhibition of human TS (hTS) has been extensively investigated for cancer chemotherapy, but several aspects of its activity and regulation are still uncertain. In this study, we performed comprehensive structural and biophysical studies of hTS using crystallography and thermal shift assay and provided the first detailed structural information on the conformational changes induced by ligand binding to the hTS active site. We found that upon binding of the antifolate agents raltitrexed and nolatrexed, the two insert regions in hTS, the functions of which are unclear, undergo positional shifts toward the catalytic center. We investigated the inactive conformation of hTS and found that the two insert regions are also involved in the conformational transition between the active and inactive state of hTS. Moreover, we identified a ligand-binding site in the dimer interface, suggesting that the cavity in the dimer interface could serve as an allosteric site of hTS to regulate the conformational switching between the active and inactive states. On the basis of these findings, we propose a regulatory mechanism of hTS activity that involves allosteric regulation of interactions of hTS with its own mRNA depending on cellular demands for TMP.

l-α-Phosphatidylglycerol Chlorohydrins as Potential Biomarkers for Chlorine Gas Exposure.

Chlorine is a widely available toxic chemical that has been repeatedly used in armed conflict globally. The Organization for the Prohibition of Chemical Weapons (OPCW) have on numerous occasions found "compelling confirmation" that chlorine gas has been used against civilians in northern Syria. However, currently, there are no analytical methods available to unambiguously prove chlorine gas exposure. In this study, we describe the screening for chlorinated biomolecules by the use of mass isotope ratio filters followed by the identification of two biomarkers present in bronchoalveolar lavage fluid (BALF) from chlorine gas exposed mice. The relevance of these markers for human exposure was verified by their presence in in vitro chlorinated human BALF. The biomarkers were detectable for 72 h after exposure and were absent in nonexposed control animals. Furthermore, the biomarkers were not detected in humans diagnosed with chronic respiratory diseases. The potential chlorine specific markers were all chlorohydrins of unsaturated pulmonary surfactant phospholipids; phosphatidylglycerols, and phosphatidylcholines. Mass spectrometry fragmentation characteristics were favorable for the phosphatidylglycerol chlorohydrins, and they were therefore proposed as the best biomarker candidates.