Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.

Rapid quality control of black truffles using Direct Analysis in Real Time Mass Spectrometry and Hydrophilic Interaction Liquid Chromatography Mass Spectrometry.

This work is the first to describe the application of Direct Analysis in Real Time Mass Spectrometry for rapid and simple quality control of black truffles. Multivariate analyses (PCA and LDA) of mass spectra provided highly efficient models for the discrimination of four truffle species, namely Tuber melanosporumVittad., Tuber aestivumVittad., Tuber uncinatumChatin and Tuber indicum Cooke etMassee. Full-cross validations showed prediction accuracies up to 99%. Hydrophilic Interaction Liquid Chromatography Mass Spectrometry was used as a reference method and the results of both methods were compared to each other. Multivariate models of the chromatograms also showed excellent results with prediction accuracies of 100%. In a direct comparison of methods, Direct Analysis in Real Time Mass Spectrometry showed minimally lower prediction accuracies, but was significantly faster, more robust and easier to use, making it an excellent choice for rapid quality control of black truffles.

Rapid quantification of cannabidiol from oils by direct analysis in real time mass spectrometry.

This work is the first to describe the use of Direct Analysis in Real Time Mass Spectrometry (DART-MS) for the rapid quantification of cannabidiol (CBD) in CBD oils. For this study, self-prepared samples spiked with CBD in hemp seed oil as well as commercial CBD oils from the Austrian market with different CBD contents were analyzed. CBD concentrations were between 5 and 30% (m/m) for the spiked samples as well as between 5 and 15% (m/m) for the real samples. The performance of quantification by means of DART-MS was assessed against a validated liquid chromatography-mass spectrometry (LC-MS) method. The correlation of the quantification results of both methods was high with a correlation factor greater than 0.98 and a maximum bias of 9.8%. Furthermore, the relative standard deviation values of the DART-MS measurments were below the tolerable limit of 12%. These results demonstrate that quantification of CBD by DART-MS is reliable and hence suitable as a rapid and cost-effective alternative method for quality control of CBD content in CBD oils.

Portable vs. Benchtop NIR-Sensor Technology for Classification and Quality Evaluation of Black Truffle.

Truffles represent the best known and most expensive edible mushroom. Known as Ascomycetes, they belong to the genus Tuber and live in symbiosis with plant host roots. Due to their extraordinary taste and smell, truffles are sold worldwide for high prices of up to 3000-5000 euros per kilogram (Tuber magnatum PICO). Amongst black truffles, the species Tuber melanosporum VITTAD. is highly regarded for its organoleptic properties. Nonetheless, numerous different sorts of black truffle are offered at lower prices, including Tuber aestivum VITTAD., Tuber indicum and Tuber uncinatum, which represent the most frequently consumed types. Because truffles do not differ visually for inexperienced consumers, food fraud is likely to occur. In particular, for the highly prized Tuber melanosporum, which morphologically forms very similar fruiting bodies to those of Tuber indicum, there is a risk of fraud via imported truffles from Asia. In this study, 126 truffle samples belonging to the four mentioned species were investigated by four different NIR instruments, including three miniaturized devices-the Tellspec Enterprise Sensor, the VIAVI solutions MicroNIR 1700 and the Consumer Physics SCiO-working on different technical principles. Three different types of measurement techniques were applied for all instruments (outer shell, rotational device and fruiting body) in order to identify the best results for classification and quality assurance in a non-destructive manner. Results provided differentiation with an accuracy up to 100% for the expensive Tuber melanosporum from Tuber indicum. Classification between Tuber melanosporum, Tuber indicum, Tuber aestivum and Tuber uncinatum could also be achieved with success of 100%. In addition, quality monitoring including discrimination between fresh and frozen/thawed, and prediction of the approximate date of harvesting, was performed. Furthermore, feasibility studies according to the geographical origin of the truffle were attempted. The presented work compares the performance for prediction and quality monitoring of portable vs. benchtop NIR devices and applied measurement techniques in order to be able to present a suitable, accurate, fast, non-destructive and reliable method for consumers.

Rapid discrimination of Curcuma longa and Curcuma xanthorrhiza using Direct Analysis in Real Time Mass Spectrometry and Near Infrared Spectroscopy.

This study describes a newly developed method for the fast and straightforward differentiation of two turmeric species using Direct Analysis in Real Time mass spectrometry and miniaturized Near Infrared spectroscopy. Multivariate analyses (PCA and LDA) were performed on the mass spectrometric data, thus creating a powerful model for the discrimination of Curcumalonga and Curcumaxanthorrhiza. Cross-validation of the model revealed correctness-scores of 100% with 20-fold as well as leave-one-out validation techniques. To further estimate the models prediction power, seven retail samples of turmeric powder were analyzed and assorted to a species. Looking for a fast, non-invasive, cost-efficient and laboratory independent method, miniaturized NIR spectrometers offer an alternative for quality control of turmeric species. However, different technologies implemented to compensate for their small size, lead to different applicability of these spectrometers. Therefore, we investigated the three handheld spectrometers microPHAZIR, MicroNIR 2200 and MicroNIR 1700ES for their application in spice analysis in hyphenation to PCA, LDA and ANN methods used for the discriminant analysis. While microPHAZIR proved to be the most valuable device for differentiating C.longa and C.xanthorrhiza, MicroNIR 1700ES offered the worst results. These findings are interpreted on the basis of a quantum chemical simulation of the NIR spectrum of curcumin as the representative constituent. It was found that the information accessible to MicroNIR 1700ES that is relevant to the analyzed constituents is located in the spectral region prone to interferences with the matrix, likely limiting the performance of this spectrometer in this analytical scenario.

Rapid differentiation and quality control of tobacco products using Direct Analysis in Real Time Mass Spectrometry and Liquid Chromatography Mass Spectrometry.

This work is the first to describe the use of Direct Analysis in Real Time mass spectrometry (DART-MS) for the rapid and cost-effective quality control of cigars. Multivariate analyses (PCA and LDA) on mass spectra led to highly efficient models for the discrimination of tobacco products. In addition to the geographic origin of the cigars, different pretreatments of the tobacco leaves could also be determined. Cross-validations of the models yielded prediction accuracies up to 92.18%. All results were compared using LC-MS as reference method. Multivariate analyses of chromatograms also yielded powerful models with correctness scores up to 88.1%. However, the models generated with LC-MS were neither able to determine the geographic origin of the tobacco leaves nor different pretreatments. In addition to qualitative analysis, the nicotine content of tobacco leaves was also determined via both methods. While precise quantification was not possible with DART-MS, a correlation coefficient of >0.96 was achieved in direct comparison with LC-MS, which allows semi-quantitative statements about the nicotine content without any problems.