Mass Spectrometric Interrogation of Earwax: Toward the Detection of Ménière's Disease.

Many diseases remain difficult to identify because the occurrence of characteristic biomarkers within traditional matrices such as blood and urine remain unknown. Disease diagnosis could, therefore, benefit from the analysis of readily accessible, non-traditional matrices that have a high chemical content and contain distinguishing biomarkers. One such matrix is cerumen (i.e., earwax), whose chemical complexity can pose challenges when analyzed by conventional methods. A combination of cerumen chemical profiles analyzed by gas chromatography-mass spectrometry (GC-MS) and direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) were investigated to ascertain the possible presence of the rare otolaryngological disorder Ménière's disease. This illness is currently identified via "diagnosis by exclusion" in which the disease is distinguished from others with overlapping symptoms by the process of elimination. GC-MS revealed a chemical profile difference between those with and without a Ménière's disease diagnosis by a visually apparent diminution of the compounds present in the Ménière's disease samples. DART-HRMS revealed that the two classes could be differentiated using three fatty acids: cis-9-hexadecenoic acid, cis-10-heptadecenoic acid, and cis-9-octadecenoic acid. These compounds were subsequently quantified by GC-MS and overall, the amounts of these fatty acids were decreased in Ménière's disease patients. The average levels for non-Ménière's disease samples were 7.89 µg/mg for cis-9-hexadecenoic acid, 0.87 µg/mg for cis-10-heptadecenoic acid, and 4.94 µg/mg for cis-9-octadecenoic acid. The average levels for Ménière's disease samples were 1.70 µg/mg for cis-9-hexadecenoic acid, 0.13 µg/mg for cis-10-heptadecenoic acid, and 2.07 µg/mg for cis-9-octadecenoic acid. The confidence levels for cis-9-hexadecenoic acid, cis-10-heptadecenoic acid, and cis-9-octadecenoic acid were 98.7%, 99.9%, and 95.4%, respectively. The results suggest that assessment of the concentrations of these fatty acids could be a useful clinical tool for the more rapid and accurate detection of Ménière's disease.

Investigation of Small-Molecule Constituents in Voacanga africana Seeds and Mapping of Their Spatial Distributions Using Laser Ablation Direct Analysis in Real-Time Imaging-Mass Spectrometry (LADI-MS).

Plant seeds are a renewable resource that can furnish access to medicinal natural products that can only otherwise be isolated from aerial or root parts, the harvest of which may be destructive to the plant or threaten its viability. However, optimization of the isolation of such compounds from seeds would be greatly assisted if the spatial distribution of the molecules of interest within the plant tissue were known. For example, iboga alkaloids that hold promise for the treatment of opioid use disorder are typically isolated from the leaves, bark, or roots of Tabernanthe or Voacanga spp. trees, but it would be more environmentally sustainable to isolate such compounds from their seeds. Here, we leveraged the unique capabilities of the ambient mass spectral imaging technique termed laser ablation direct analysis in real-time imaging-mass spectrometry (LADI-MS) to reveal the spatial distributions of a range of molecules, including alkaloids within V. africana seeds. In addition to six compounds previously reported in these seeds, namely, tetradecanoic acid, n-hexadecanoic acid, (Z,Z)-9,12-octadecadienoic acid, (Z)-9-octadecenoic acid, octadecanoic acid, and Δ14-vincamine, an additional 31 compounds were newly identified in V. africana seeds. The compound classes included alkaloids, terpenes, and fatty acids. The ion images showed that the fatty acids were localized in the embryo of the seed. The alkaloids, which were mainly localized in the seed endosperm, included strictamine, akuammidine, polyneruidine, vobasine, and Δ14-vincamine. This information can be exploited to enhance the efficiency of secondary metabolite isolation from V. africana seeds while eliminating the destruction of other plant parts.

Distinguishing Infested Flour from Uninfested Flour through Chemometric Processing of DART-HRMS Data─Revealing the Presence of Tribolium castaneum, the Red Flour Beetle.

Insect infestation of agricultural stored products is a significant challenge to food security across the globe. One common pest is Tribolium castaneum (red flour beetle). In a new approach to addressing the threat of these beetles, Direct Analysis in Real Time-High-Resolution Mass Spectrometry was used to examine infested and uninfested flour samples. These samples were then distinguished through statistical analysis techniques, including EDR-MCR, in order to highlight the important m/z values contributing to the differences in the flour profiles. A subset of these values responsible for the identification of infested flour (nominal m/z 135, 136, 137, 163, 211, 279, 280, 283, 295, 297, and 338) were further investigated, and compounds responsible for these masses included 2-(2-ethoxyethoxy)ethanol, 2-ethyl-1,4-benzoquinone, palmitic acid, linolenic acid and oleic acid. These results have the potential to lead to a rapid technique by which flour and other grains can be tested for insect infestation.

Quantification of hordenine in a complex plant matrix by direct analysis in real time-high-resolution mass spectrometry: Application to the "plant of concern" Sceletium tortuosum.

Recently, there has been an increase in the recreational abuse of several psychoactive plants, resulting in the United Nations Office on Drugs and Crime creating a list of "plants of concern." One such material is Sceletium tortuosum and products derived from it. Regulation of these materials is challenging because of their innocuous appearance, the cumbersome sample preparation steps required to render the material into a form amenable to analysis by conventional techniques, the requirement for nuanced sample analysis protocols, and lengthy analysis times. It is demonstrated here that direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) can be used to not only identify S. tortuosum material based on the detection of characteristic biomarkers including hordenine and several mesembrine alkaloids, but also quantify the amount of hordenine present. Using hordenine-d6 as an internal standard, a protocol, validated according to US Food and Drug Administration (FDA) Guidelines for the Development and Validation of Bioanalytical Methods, was devised for the quantification of the psychoactive component hordenine. The method was then applied to the quantification of hordenine in six commercially available products derived from the foliage and stems of S. tortuosum. By this method, the lower limit of quantification (LLOQ) was found to be 1 µg/ml. Observed hordenine concentrations ranged from 0.02738 to 1.071 mg of hordenine per gram of plant material. The developed technique provides an effective and quick means for the detection and quantification of hordenine in S. tortuosum, which can be extended to analysis of other hordenine-containing products.

Random Forest Processing of Direct Analysis in Real-Time Mass Spectrometric Data Enables Species Identification of Psychoactive Plants from Their Headspace Chemical Signatures.

The United Nations Office on Drugs and Crime has designated several "legal highs" as "plants of concern" because of the dangers associated with their increasing recreational abuse. Routine identification of these products is hampered by the difficulty in distinguishing them from innocuous plant materials such as foods, herbs, and spices. It is demonstrated here that several of these products have unique but consistent headspace chemical profiles and that multivariate statistical analysis processing of their chemical signatures can be used to accurately identify the species of plants from which the materials are derived. For this study, the headspace volatiles of several species were analyzed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS). These species include Althaea officinalis, Calea zacatechichi, Cannabis indica, Cannabis sativa, Echinopsis pachanoi, Lactuca virosa, Leonotis leonurus, Mimosa hositlis, Mitragyna speciosa, Ocimum basilicum, Origanum vulgare, Piper methysticum, Salvia divinorum, Turnera diffusa, and Voacanga africana. The results of the DART-HRMS analysis revealed intraspecies similarities and interspecies differences. Exploratory statistical analysis of the data using principal component analysis and global t-distributed stochastic neighbor embedding showed clustering of like species and separation of different species. This led to the use of supervised random forest (RF), which resulted in a model with 99% accuracy. A conformal predictor based on the RF classifier was created and proved to be valid for a significance level of 8% with an efficiency of 0.1, an observed fuzziness of 0, and an error rate of 0. The variables used for the statistical analysis processing were ranked in terms of the ability to enable clustering and discrimination between species using principal component analysis-variable importance of projection scores and RF variable importance indices. The variables that ranked the highest were then identified as m/z values consistent with molecules previously identified in plant material. This technique therefore shows proof-of-concept for the creation of a database for the detection and identification of plant-based legal highs through headspace analysis.

Application of Direct Analysis in Real Time-High Resolution Mass Spectrometry to Investigations of Induced Plant Chemical Defense Mechanisms-Revelation of Negative Feedback Inhibition of an Alliinase.

Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 µM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.

Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.

The HIV-1 nucleocapsid zinc finger protein as a target of antiretroviral therapy.

Despite advances made in its therapeutic management, human immunodeficiency virus (HIV) infection has remained an intractable problem, and complete eradication of the virus an unrealized goal. Experience in the clinical application of combination therapy using a variety of reverse transcriptase and protease inhibitors have revealed a number of challenges, in spite of the observed albeit temporary success in reduction of patient viral loads. Problems with current protocols include poor patient compliance, and the presence of latent reservoirs of virus that ultimately result in the appearance of phenotypic resistance. These considerations necessitate continued research and development into alternative strategies to circumvent the aforementioned problems. One approach to minimizing and/or eliminating the appearance of escape mutants and latent viral reservoirs is the targeting of essential and mutationally intolerant enzymes such as the nucleocapsid protein, which contains two highly conserved zinc knuckles. Concerns have been raised regarding the targeting of this protein, since the ubiquitous occurrence of important mammalian zinc finger proteins implies that drug specificity towards the nucleocapsid protein may be difficult to attain. In this review, strong evidence supporting the hypothesis that this protein can be targeted to the exclusion of other cellular zinc finger proteins is presented. The effects of small molecule induced abrogation of nucleocapsid protein mediated activities, as well as efforts to develop nucleocapsid protein inhibitors as antiretrovirals are also discussed.