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The guest-host interactions of poly(amidoamine) (PAMAM) dendrimers and porous silica surfaces were investigated by near-infrared (NIR) diffuse reflection spectroscopy. G0-G7 of amine-terminated PAMAM (PAMAM-N[H.sub.2]) dendrimers were analyzed comprising early, mid, and late generations. For early stages, the adsorption process of the partly protonated dendrimers to the negatively charged silica surface strongly depends on the size/shape characteristics of the guest (PAMAM-N[H.sub.2] dendrimers) and host (porous silica) materials. G0-G4 (15-45 [Angstrom]) show smaller particle sizes than the pore diameter of the silica (60 [Angstrom]) and thus have access to the interior surface of the host material. For mid and later stages (G5-G7; 54-81 [Angstrom]) only low amounts of the dendrimers adsorb to the silica surface due to the inaccessibility to the interior surface. The loading capacity of the silica material with adsorbed PAMAM-N[H.sub.2] was evaluated by means of capillary zone electrophoresis (CZE), whereas deviations from the theoretical to the effective particle size and molecular weight (MW) was determined by gas-phase electrophoretic mobility molecular analysis (GEMMA) and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-lin TOF-MS). Deviations from the theoretical to the actual values showed a maximum of 13.8% and 28.0% for the particle size and MW, respectively. The NIR absorption spectra show a distinct band at 4932 [cm.sup.-1] ([v.sub.sym] (NH) + amide II) due to the adsorbed dendrimers. It was found that the absorbance tends to increase with decreasing generation number. On this basis multivariate calibration was performed with the theoretical data and the data obtained by GEMMA and MALDI-lin TOF-MS. All in all, the calculated partial least-squares regression (PLSR) model containing the GEMMA/MALDI-lin TOF-MS reference values showed better results than the models exclusively calculated from the theoretical values. This indicates that the theoretical values do not imply the structural imperfections arising during the synthesis that may be present in the PAMAM-N[H.sub.2] dendrimers.

Understanding early stage renal malfunctions with regard to the glomerular filtration processes is essential for nephropathological prescreening strategies and intervention at an early stage. Mass spectrometry imaging (MSI) in combination with histopathology can provide an universal analytical approach. Proteomic and lipidomic aspects of glomerular biocompositions were applied for microstructural differentiation in healthy rat kidney samples. Usability of commonly used tissue embedding media and the compatibility of histological staining and fixation methods were of interest. It was demonstrated that ultra-thin tissue samples (500 nm, 1 and 10 µm) can be used for lipid and peptide-based differentiation at the glomerular resolution level in formalin-fixed tissue samples in combination with preceding histological staining for correlating optical and molecular mass images.

Manganese ion homeostasis is vital for bacteria and is achieved via manganese-dependent transcription factors. Manganese mediation of transcription factor attachment to the corresponding oligonucleotide sequences can be investigated, e.g. via electrophoretic mobility shift assays (EMSA). Formation of specific biocomplexes leads to differences in the migration pattern upon gel electrophoresis. Focusing on electrophoresis in the gas-phase, applying a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) also known as nES differential mobility analyzer (nES DMA), and on transcription factors (MntR proteins) from Bacillus subtilis and Mycobacterium tuberculosis, we took interest in the gas-phase electrophoresis of the corresponding biospecific complexes. We compared nES GEMMA, separating analytes in the nanometer regime (a few to several hundred nm in diameter) in the gas-phase in their native state according to particle size, to EMSA data. Indeed we were able to demonstrate manganese-mediated attachment of MntR to target genomic sequences with both analytical techniques. Despite some inherent pitfalls of the nES GEMMA method like analyte/instrument surface interactions, we were able to detect the target complexes. Moreover, we were able to calculate the molecular weight (MW) of the obtained species by application of a correlation function based on nES GEMMA obtained data. As gas-phase electrophoresis also offers the possibility of offline hyphenation to orthogonal analysis techniques, we are confident that nES GEMMA measurements are not just complementary to EMSA, but will offer the possibility of further in-depth characterization of biocomplexes in the future., (© 2024. The Author(s).)

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon., (© 2024. The Author(s).)

Common B7 biodiesels consist of mixtures of mineral oil-based diesel and 7% fatty acid methyl ester (FAME). While biocontent increase can be achieved with these blends at high-quality levels during cold temperature periods, fuel filter blocking events are reported from time to time. Based on a preliminary study on fuel filters, the selection of compounds responsible for filter blocking could be narrowed down to saturated monoglycerides (SMGs). The most abundant SMGs in Europe are 1- and 2-monopalmitin (1-C16:0, 2-C16:0) and 1-monostearin (C18:0), based on the FAME origin. Until now, there has been no simple, precise, and accurate method to quantitatively detect those SMGs in the B7 matrix, which was the aim of the following work. An improved gas chromatography electron ionization tandem mass spectrometry method was developed for the quantitative detection of 1-C16:0, 2-C16:0, C18:0, and C20:0 SMGs. During the method improvement, (a) the sample preparation and (b) the calibration were optimized for low concentrations. (c) The samples were analyzed by multiple reaction monitoring focusing on specific qualifier and quantifier ions with optimized collision energies, (d) time segments and improved scan time were implemented, and (e) limits of detection and limits of quantification were determined. The time-stability of SMG standards in CHCl 3 with 4% neat biodiesel and the discrimination effects of the standard components were evaluated to assess method reliability. Overall, a highly sensitive and precise method for the improved detection of SMGs in biodiesel is presented., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Parkinson's disease (PD) associated state of neuroinflammation due to the aggregation of aberrant proteins is widely reported. One type of post-translational modification involved in protein stability is glycosylation. Here, we aimed to characterize the human Parkinsonian nigro-striatal N -glycome, and related transcriptome/proteome, and its correlation with endoplasmic reticulum (ER) stress and unfolded protein response (UPR), providing a comprehensive characterization of the PD molecular signature. Significant changes were seen upon a PD: a 3% increase in sialylation and 5% increase in fucosylation in both regions, and a 2% increase in oligomannosylated N -glycans in the substantia nigra. In the latter, a decrease in the mRNA expression of sialidases and an upregulation in the UPR pathway were also seen. To show the correlation between these, we also describe a small in vitro study where changes in specific glycosylation trait enzymes (inhibition of sialyltransferases) led to impairments in cell mitochondrial activity, changes in glyco-profile, and upregulation in UPR pathways. This complete characterization of the human nigro-striatal N -glycome provides an insight into the glycomic profile of PD through a transversal approach while combining the other PD "omics" pieces, which can potentially assist in the development of glyco-focused therapeutics., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress., (© 2023. The Author(s).)

Collagen fibrils are the basic structural building blocks that provide mechanical properties such as stiffness, toughness, and strength to tissues from the nano- to the macroscale. Collagen fibrils are highly hydrated and transient deformation mechanisms contribute to their mechanical behavior. One approach to describe and quantify the apparent viscoelastic behavior of collagen fibrils is to find rheological models and fit the resulting empirical equations to experimental data. In this study, we consider a nonlinear rheological Maxwell model for this purpose. The model was fitted to tensile stress-time data from experiments conducted in a previous study on hydrated and partially dehydrated individual collagen fibrils via AFM. The derivative tensile modulus, estimated from the empirical equation, increased for decreasing hydration of the collagen fibril. The viscosity is only marginally affected by hydration but shows a dependency with strain rate, suggesting thixotropic behavior for low strain rates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many studies aim at maximizing fungal secondary metabolite production but the influence of light during cultivation has often been neglected. Here, we combined an untargeted isotope-assisted liquid chromatography-high-resolution mass spectrometry-based metabolomics approach with standardized cultivation of Trichoderma atroviride under three defined light regimes (darkness (PD), reduced light (RL) exposure, and 12/12 h light/dark cycle (LD)) to systematically determine the effect of light on secondary metabolite production. Comparative analyses revealed a similar metabolite profile upon cultivation in PD and RL, whereas LD treatment had an inhibiting effect on both the number and abundance of metabolites. Additionally, the spatial distribution of the detected metabolites for PD and RL was analyzed. From the more than 500 detected metabolites, only 25 were exclusively produced upon fungal growth in darkness and 85 were significantly more abundant in darkness. The majority were detected under both cultivation conditions and annotation revealed a cluster of substances whose production followed the pattern observed for the well-known T. atroviride metabolite 6-pentyl-alpha-pyrone. We conclude that cultivation of T. atroviride under RL can be used to maximize secondary metabolite production.

Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte's pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle's shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution.

From the moment of production, artworks are constantly exposed to changing environmental factors potentially inducing degradation. Therefore, detailed knowledge of natural degradation phenomena is essential for proper damage assessment and preservation. With special focus on written cultural heritage, we present a study on the degradation of sheep parchment employing accelerated aging with light (295-3000 nm) for one month, 30/50/80% relative humidity (RH) and 50 ppm sulfur dioxide with 30/50/80%RH for one week. UV/VIS spectroscopy detected changes in the sample surface appearance, showing browning after light-aging and increased brightness after SO 2 -aging. Band deconvolution of ATR/FTIR and Raman spectra and factor analysis of mixed data (FAMD) revealed characteristic changes of the main parchment components. Spectral features for degradation-induced structural changes of collagen and lipids turned out to be different for the employed aging parameters. All aging conditions induced denaturation (of different degrees) indicated by changes in the secondary structure of collagen. Light treatment resulted in the most pronounced changes for collagen fibrils in addition to backbone cleavage and side chain oxidations. Additional increased disorder for lipids was observed. Despite shorter exposure times, SO 2 -aging led to a weakening of protein structures induced by transitions of stabilizing disulfide bonds and side chain oxidations.

In the last years, LA-ICP-MS has become an attractive technique for analyzing solid samples from various research fields. However, application in material science is often hampered by the limited availability of appropriate certified reference materials, which are a precondition for accurate quantification. Thus, frequently in-house prepared standards are used, which match the sample's composition and include all elements of interest at the required concentration levels. However, preparing and characterizing such standards is often labor-intensive and time-consuming. This work proposes a new approach for the fabrication of matrix-matched standards based on the concept of standard addition. In the first step, the analytes of interest are homogeneously deposited onto the sample surface using liquid standards and a spraying device. For analysis, the generated thin layer is ablated simultaneously with the underlying sample. Thereby deviations in the ablation process and particle transport can be avoided. It could be shown that the developed method is highly versatile and could be easily adapted to the actual needs. Using silicon, silicon carbide, copper, aluminum, and glass as a matrix, excellent linear correlations between observed signal intensities and deposited amounts were found for the elements Zn, Ag, In, and Pb (R 2 - values greater than 0.99). The method was applied to determine the content of sulfur, zinc, silver, indium, and lead in a commercial Kapton® polyimide film. The obtained results could be verified based on the homogeneously distributed sulfur by conventional liquid ICP-MS analysis after sample digestion, showing similar precision and accuracy. Lead was found to show a very inhomogeneous distribution in the Kapton® film, with concentration below the LOD at most measured locations and irregularly occurring spots with significantly higher concentrations. Finally, a quantitative depth profile of sulfur in a Kapton® film has been measured to assess the uptake of SO 2 after a weathering experiment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The properties of biogenic aerosol strongly depend on the particle's proteinaceous compounds. Proteins from primary biological aerosol particles (PBAPs) can cause allergic reactions in the human respiratory system or act as ice and condensation nuclei in clouds. Consequently, these particles have high impact on human health and climate. The detection of biogenic aerosol is commonly performed with fluorescence-based techniques. However, many PBAPs (i.e., pollen of birch, mugwort, or ragweed) show weak or rather low fluorescence signals in the particular protein region (λ ex  ~ 255-280 nm, λ em  ~ 280-350 nm). We hypothesize that the fluorescence signal of proteins present in birch pollen is being distorted within its native matrix. In this study, we conducted in vitro quenching experiments and employed UV/Vis spectroscopy, capillary zone electrophoresis (CZE), liquid chromatography (LC), electrospray ionization mass spectrometry (ESI-MS), and multistage MS (MS 2 and MS 3 ) to target major components in birch pollen washing water (BPWW) possibly quenching the fluorescence activity of proteins and thus explaining the lack of corresponding protein fluorescent signals. We identified quercetin-3-O-sophoroside (Q3OS, MW 626 g mol -1 ) to be the main UV/Vis absorbing component in BPWW. Our results point out that Q3OS suppresses the fluorescence of proteins in our samples predominantly due to inner filter effects. In general, when applying fluorescence spectroscopy to analyze and detect PBAPs in the laboratory or the atmosphere, it is important to critically scrutinize the obtained spectra., (© 2022. The Author(s).)

Extracellular vesicles (EVs) are in the scientific spotlight due to their potential application in the medical field, ranging from medical diagnosis to therapy. These applications rely on EV stability during isolation and purification-ideally, these steps should not impact vesicle integrity. In this context, we investigated EV stability and particle numbers via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) and nanoparticle tracking analysis (NTA). In nES GEMMA, native, surface-dry analytes are separated in the gas-phase according to the particle size. Besides information on size and particle heterogeneity, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU, 18 October 2011). Likewise, and in contrast to NTA, nES GEMMA enables detection of co-purified proteins. On the other hand, NTA, yielding data on hydrodynamic size distributions, is able to relate particle concentrations, omitting electrolyte exchange (and resulting EV loss), which is prerequisite for nES GEMMA. Focusing on EVs of different origin, we compared vesicles concentrations and stability, especially after electrolyte exchange and size exclusion chromatography (SEC). Co-isolated proteins were detected in most samples, and the vesicle amount varied in dependence on the EV source. We found that depletion of co-purified proteins was achievable via SEC, but was associated with a loss of EVs and-most importantly-with decreased vesicle stability, as detected via a reduced nES GEMMA measurement repeatability. Ultimately, we propose the repeatability of nES GEMMA to yield information on EV stability, and, as a result, we propose that nES GEMMA can yield additional valuable information in EV research.

MALDI mass spectrometry imaging (MALDI MSI) is a powerful analytical method for achieving 2D localization of compounds from thin sections of typically but not exclusively biological samples. The dynamically harmonized ICR cell (ParaCell) was recently introduced to achieve extreme spectral resolution capable of providing the isotopic fine structure of ions detected in complex samples. The latest improvement in the ICR technology also includes 2ω detection, which significantly reduces the transient time while preserving the nominal mass resolving power of the ICR cell. High-resolution MS images acquired on FT-ICR instruments equipped with 7T and 9.4T superconducting magnets and the dynamically harmonized ICR cell operating at suboptimal parameters suffered severely from the pixel-to-pixel shifting of m / z peaks due to space-charge effects. The resulting profile average mass spectra have depreciated mass measurement accuracy and mass resolving power under the instrument specifications that affect the confidence level of the identified ions. Here, we propose an analytical workflow based on the monitoring of the total ion current to restrain the pixel-to-pixel m / z shift. Adjustment of the laser parameters is proposed to maintain high spectral resolution and mass accuracy measurement within the instrument specifications during MSI analyses. The optimized method has been successfully employed in replicates to perform high-quality MALDI MS images at resolving power (FWHM) above 1,000,000 in the lipid mass range across the whole image for superconducting magnets of 7T and 9.4T using 1 and 2ω detection. Our data also compare favorably with MALDI MSI experiments performed on higher-magnetic-field superconducting magnets, including the 21T MALDI FT-ICR prototype instrument of the NHMFL group at Tallahassee, Florida.

The forensic scenario, on which the round robin study was based, simulated a suspected intentional manipulation of a real estate rental agreement consisting of a total of three pages. The aims of this study were to (i) establish the amount and reliability of information extractable from a single type of evidence and to (ii) provide suggestions on the most suitable combination of compatible techniques for a multi-modal imaging approach to forgery detection. To address these aims, seventeen laboratories from sixteen countries were invited to answer the following tasks questions: (i) which printing technique was used? (ii) were the three pages printed with the same printer? (iii) were the three pages made from the same paper? (iv) were the three pages originally stapled? (v) were the headings and signatures written with the same ink? and (vi) were headings and signatures of the same age on all pages? The methods used were classified into the following categories: Optical spectroscopy, including multispectral imaging, smartphone mapping, UV-luminescence and LIBS; Infrared spectroscopy, including Raman and FTIR (micro-)spectroscopy; X-ray spectroscopy, including SEM-EDX, PIXE and XPS; Mass spectrometry, including ICPMS, SIMS, MALDI and LDIMS; Electrostatic imaging, as well as non-imaging methods, such as non-multimodal visual inspection, (micro-)spectroscopy, physical testing and thin layer chromatography. The performance of the techniques was evaluated as the proportion of discriminated sample pairs to all possible sample pairs. For the undiscriminated sample pairs, a distinction was made between undecidability and false positive claims. It was found that none of the methods used were able to solve all tasks completely and/or correctly and that certain methods were a priori judged unsuitable by the laboratories for some tasks. Correct results were generally achieved for the discrimination of printer toners, whereas incorrect results in the discrimination of inks. For the discrimination of paper, solid state analytical methods proved to be superior to mass spectrometric methods. None of the participating laboratories deemed addressing ink age feasible. It was concluded that correct forensic statements can only be achieved by the complementary application of different methods and that the classical approach of round robin studies to send standardised subsamples to the participants is not feasible for a true multimodal approach if the techniques are not available at one location., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ochratoxin A (OTA) is one of the major mycotoxins causing severe effects on the health of humans and animals. Ochratoxin alpha (OTα) is a metabolite of OTA, which is produced through microbial or enzymatic hydrolysis, and one of the preferred routes of OTA detoxification. The methods described here are applicable for the extraction and quantification of OTA and OTα in several pig and poultry matrices such as feed, feces/excreta, urine, plasma, dried blood spots, and tissue samples such as liver, kidney, muscle, skin, and fat. The samples are homogenized and extracted. Extraction is either based on a stepwise extraction using ethyl acetate/sodium hydrogencarbonate/ethyl acetate or an acetonitrile/water mixture. Quantitative analysis is based on reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Method validation was successfully performed and the linearity, limit of quantification, accuracy, precision as well as the stability of the samples, were evaluated. The analyte recovery of the spiked samples was between 80 and 120% (80-150% for spiked concentrations ≤ 1 ng/g or ng/mL) and the relative standard deviation was ≤ 15%. Therefore, we provide a toolbox for the extraction and quantification of OTA and OTα in all relevant pig and poultry matrices.

Abbreviations: ATG7: autophagy related 7; BODIPY: boron dipyrromethene; DAG: diacyl glycerides; DBI: diazepam binding inhibitor; GFP: green fluorescent protein; KRT14: keratin 14; HPLC-MS: high performance liquid chromatography-mass spectrometry; LD: lipid droplet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MSI: mass spectrometric imaging; ORO: Oil Red O; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: preputial gland; PLIN2: perilipin 2; PtdIns: phosphatidylinositol; PL: phospholipids; POPC: 1-palmitoyl-2-oleoyl-PC; PS: phosphatidylserine; qRT-PCR: quantitative reverse transcribed PCR; SG: sebaceous gland; scRNAseq: single-cell RNA sequencing; TAG: triacylglycerides; TLC: thin layer chromatography.

The growing importance of fluoropolymers in high-tech applications and green technologies results in the rising need for their characterization. In contrast to conventional methods used for this task, laser-induced breakdown spectroscopy (LIBS) provides the advantage of a spatially resolved analysis. Nevertheless, the high excitation energy of fluorine results in low sensitivity of the atomic F(I) lines, which limits the feasibility of its LIBS-based analysis. This work presents a novel approach for quantitative mapping of fluorine in fluoropolymer samples. It bases on monitoring of molecular emission bands (CuF or CaF) arising from fluorine containing molecules. These species were generated during later stages of the LIBS plasma by a recombination of fluorine atoms originating from fluoropolymer sample with a molecule-forming partner (Cu or Ca) stemming from a surface coating. This approach enables F detection limits in the parts per million (μg g -1 ) range and elemental imaging using single shot measurements. The elements required for molecular formation are deposited on the sample surface prior to analysis. We evaluate two techniques - spray coating and sputter coating - with regards to their effects on sensitivity and spatial resolution in elemental mapping. Overall, both methods proved to be suitable for a spatially resolved analysis of fluorine: whereas sputter-coating of copper yielded a better sensitivity, spray coating of calcium provided a higher spatial resolution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Although lipids are crucial molecules for cell structure, metabolism, and signaling in most organs, they have additional specific functions in the skin. Lipids are required for the maintenance and regulation of the epidermal barrier, physical properties of the skin, and defense against microbes. Analysis of the lipidome-the totality of lipids-is of similar complexity to those of proteomics or other omics, with lipid structures ranging from simple, linear, to highly complex structures. In addition, the ordering and chemical modifications of lipids have consequences on their biological function, especially in the skin. Recent advances in analytic capability (usually with mass spectrometry), bioinformatic processing, and integration with other dermatological big data have allowed researchers to increasingly understand the roles of specific lipid species in skin biology. In this paper, we review the techniques used to analyze skin lipidomics and epilipidomics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to the fast growing importance of monoclonal antibodies in biomedical research, bioanalytics and human therapy, sensitive, fast and reliable methods are needed to monitor their production, target their characteristics, and for their final quality control. Application of a nano electrospray (nES) with soft X-ray radiation (SXR) based charge reduction and differential mobility analysis (DMA, aka nano electrospray gas-phase electrophoretic mobility molecular analysis, nES GEMMA) allows the size-separation and detection of macromolecules and (bio-)nanoparticles from a few nm up to several hundreds of nm in diameter in a native-like environment. The current study focuses on the analysis of a 148 kDa recombinant monoclonal antibody (rmAb) with the above mentioned instrumental setup and applying an universal detector, i.e. a water-based condensation particle detector (CPC). Next to the intact rmAb, its aggregates and fragment products after digestion with IdeS protease were analyzed. Additionally, influence of temperature treatment and pH variation on the stability of the rmAb was monitored. In this context, changes in electrophoretic mobility diameter (EMD) values, peak shape, and signal intensity based on particle numbers were of interest. Molecular weights calculated by application of a correlation derived from respective standard protein compounds were compared to mass spectrometric values and were found to be in good accordance. To conclude, we demonstrate that nES-DMA is a valuable tool in the characterization and quality control of rmABs., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agriculture.

Gas-phase electrophoresis yields size distributions of polydisperse, aerosolized analytes based on electrophoretic principles. Nanometer-sized, surface-dry, single-charged particles are separated in a high laminar sheath flow of particle-free air and an orthogonal tunable electric field. Additionally, nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) data are particle-number based. Therefore, small particles can be detected next to larger ones without a bias, for example, native proteins next to their aggregates. Analyte transition from the liquid to the gas phase is a method inherent prerequisite. In this context, nonvolatile sample buffers influence results. In the worst case, the (bio-)nanoparticle signal is lost due to an increased baseline and unspecific clustering of nonvolatile components. We present a novel online hyphenation of liquid chromatography and gas-phase electrophoresis, coupling a size-exclusion chromatography (SEC) column to an advanced nES GEMMA. Via this novel approach, it is possible to (i) separate analyte multimers already present in liquid phase from aggregates formed during the nES process, (ii) differentiate liquid phase and spray-induced multimers, and (iii) to remove nonvolatile buffer components online before SEC-nES GEMMA analysis. Due to these findings, SEC-nES GEMMA has the high potential to help to understand aggregation processes in biological buffers adding the benefit of actual size determination for noncovalent assemblies formed in solution. As detection and characterization of protein aggregation in large-scale pharmaceutical production or sizing of noncovalently bound proteins are findings directly related to technologically and biologically relevant situations, we proposed the presented method to be a valuable addition to LC-MS approaches., (© 2021 The Authors. Electrophoresis published by Wiley-VCH GmbH.)

In this study, we have aimed at developing a novel electrochemical sensing approach capable of detecting dopamine, the main biomarker in Parkinson's disease, within the highly complex cell culture matrix of human midbrain organoids in a non-invasive and label-free manner. With its ability to generate organotypic structures in vitro, induced pluripotent stem cell technology has provided the basis for the development of advanced patient-derived disease models. These include models of the human midbrain, the affected region in the neurodegenerative disorder Parkinson's disease. Up to now, however, the analysis of so-called human midbrain organoids has relied on time-consuming and invasive strategies, incapable of monitoring organoid development. Using a redox-cycling approach in combination with a 3-mercaptopropionic acid self-assembled monolayer modification enabled the increase of sensor selectivity and sensitivity towards dopamine, while simultaneously reducing matrix-mediated interferences. In this work, we demonstrate the ability to detect and monitor even small differences in dopamine release between healthy and Parkinson`s disease-specific midbrain organoids over prolonged cultivation periods, which was additionally verified using liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, the detection of a phenotypic rescue in midbrain organoids carrying a pathogenic mutation in leucine-rich repeat kinase 2, upon treatment with the leucine-rich repeat kinase 2 inhibitor II underlines the practical implementability of our sensing approach for drug screening applications as well as personalized disease modelling.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Lipids are highly diverse biomolecules crucial for the formation and function of cellular membranes, for metabolism, and for cellular signaling. In the mammalian skin, lipids additionally serve for the formation of the epidermal barrier and as surface lipids, together regulating permeability, physical properties, acidification and the antimicrobial defense. Recent advances in accuracy and specificity of mass spectrometry have allowed studying enzymatic and non-enzymatic modifications of lipids-the epilipidome-multiplying the known diversity of molecules in this class. As the skin is an organ that is frequently exposed to oxidative-, chemical- and thermal stress, and to injury and inflammation, it is an ideal organ to study epilipidome dynamics, their causes, and their biological consequences. Recent studies uncover loss or gain in biological function resulting from either specific modifications or the sum of the modifications of lipids. These studies suggest an important role for the epilipidome in stress responses and immune regulation in the skin. In this minireview we provide a short survey of the recent developments on causes and consequences of epilipidomic changes in the skin or in cell types that reside in the skin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gruber, Marchetti-Deschmann, Kremslehner and Schosserer.)

Whole IgG antivenoms are prepared from hyperimmune animal plasma by various refinement strategies. The ones most commonly used at industrial scale are precipitation by sodium or ammonium sulphate (ASP), and caprylic acid precipitation (CAP) of non-immunoglobulin proteins. The additional procedures, which have so far been used for experimental purposes only, are anion-exchange (AEX) and cation-exchange chromatography (CEX), as well as affinity chromatography (AC) using IgG's Fc-binding ligands. These protocols extract the whole IgG fraction from plasma, which contains both venom-specific and therapeutically irrelevant antibodies. Such preparations represent a complex mixture of various IgG subclasses whose functional and/or structural properties, as well as relative distribution, might be affected differently, depending on employed purification procedure. The aim of this work was to compare the influence of aforementioned refinement strategies on the IgG subclass distribution, venom-specific protective efficacy, thermal stability, aggregate formation and retained impurity profile of the final products. A unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma was used as a starting material, enabling direct comparison of five purification approaches. The highest purity was achieved by CAP and AC (above 90% in a single step), while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated IgG sample from AEX processing. Alpha-1B-glycoprotein was present in CAP IgG fraction, as well as in those from ASP- and AEX-based procedures. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparation as measured in vivo. AC and CEX remarkably affected drug's venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. Presented data might improve the process of designing and establishing novel downstream processing strategies and give guidance for optimization of the current ones by providing information on potency-protecting and purity-increasing properties of each purification principle.

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 10 4 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI)., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab') 2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness., Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab') 2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio ( w / w ) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab') 2 product against the venom's lethal toxicity was determined., Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab') 2 preparation with respect to the plasma was increased 3.9-fold., Conclusion: A highly streamlined mode for production of equine F(ab') 2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Gas-phase electrophoresis of single-charged analytes (nanoparticles) enables their separation according to the surface-dry particle size (Electrophoretic Mobility Diameter, EMD), which corresponds to the diameter of spherical shaped particles. Employing a nano Electrospray Differential Mobility Analyzer (nES DMA), also known as nES Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA), allows sizing/size-separation and determination of particle-number concentrations. Separations are based on a constant high laminar sheath flow and a tunable, orthogonal electric field enabling scanning of EMDs in the nanometer size range. Additionally, keeping the voltage constant, only nanoparticles of a given EMD pass the instrument and can be collected on corresponding supporting materials for subsequent nanoparticle analyses applying e.g. microscopic, immunologic or spectroscopic techniques. In our proof-of-concept study we now focus for the first time on mass spectrometric (MS) characterization of DMA size-selected material. We carried out size-selection of liposomes, vesicles consisting of a lipid bilayer and an aqueous lumen employed as carriers in e.g. pharmaceutic, cosmetic or nutritional applications. Particles of 85 nm EMD were collected on gold-coated silicon wafers. Subsequently, matrix was applied and Matrix-Assisted Laser Desorption / Ionization (MALDI) MS carried out. However, we not only focused on plain liposomes but also demonstrated the applicability of our approach for very heterogeneous low density lipoprotein (VLDL) particles, a transporter of lipid metabolism. Our novel offline hyphenation of gas-phase electrophoresis (termed nES DMA or nES GEMMA) and MALDI-MS opens the avenue to the molecular characterization of size-select nanoparticles of complex nature., (Copyright © 2019. Published by Elsevier B.V.)

Lysobacter spp. are common bacterial inhabitants of the rhizosphere of diverse plant species. However, the impact of the rhizosphere conditions on their physiology is still relatively understudied. To provide clues on the behaviour of Lysobacter spp. in this ecological niche, we investigated the physiology of L. capsici AZ78 (AZ78), a biocontrol strain isolated from tobacco rhizosphere, on a common synthetic growth medium (LBA) and on a growth medium containing components of the plant rhizosphere (RMA). The presence of a halo surrounding the AZ78 colony on RMA was a first visible effect related to differences in growth medium composition and it corresponded to the formation of a large outer ring. The lower quantity of nutrients available in RMA as compared with LBA was associated to a higher expression of a gene encoding cAMP-receptor-like protein (Clp), responsible for cell motility and biofilm formation regulation. AZ78 cells on RMA were motile, equipped with cell surface appendages and organised in small groups embedded in a dense layer of fibrils. Metabolic profiling by mass spectrometry imaging revealed increased diversity of analytes produced by AZ78 on RMA as compared with LBA. In particular, putative cyclic lipodepsipeptides, polycyclic tetramate macrolactams, cyclic macrolactams and other putative secondary metabolites with antibiotic activity were identified. Overall, the results obtained in this study shed a light on AZ78 potential to thrive in the rhizosphere by its ability to move, form biofilm and release secondary metabolites., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness., Competing Interests: The authors have declared that no competing interests exist.

Animals use adhesive secretions in highly diverse ways, such as for settlement, egg anchorage, mating, active or passive defence, etc. One of the most interesting functions is the use of bioadhesives to capture prey, as the bonding has to be performed within milliseconds and often under unfavourable conditions. While much is understood about the adhesive and biomechanical properties of the threads of other hunters such as spiders, barely anything is documented about those of the New Zealand glowworm Arachnocampa luminosa. We analysed tensile properties of the fishing lines of the New Zealand glowworm Arachnocampa luminosa under natural and dry conditions and measured their adhesion energy to different surfaces. The capture system of A. luminosa is highly adapted to the prevailing conditions (13-15 °C, relative humidity of 98%) whereby the wet fishing lines only show a bonding ability at high relative humidity (>80%) with a mean adhesive energy from 20-45 N/m and a stronger adhesion to polar surfaces. Wet threads show a slightly higher breaking strain value than dried threads, whereas the tensile strength of wet threads was much lower. The analyses show that breaking stress and strain values in Arachnocampa luminosa were very low in comparison to related Arachnocampa species and spider silk threads but exhibit much higher adhesion energy values. While the mechanical differences between the threads of various Arachnocampa species might be consequence of the different sampling and handling of the threads prior to the tests, differences to spiders could be explained by habitat differences and differences in the material ultrastructure. Orb web spiders produce viscid silk consisting of β-pleated sheets, whereas Arachnocampa has cross-β-sheet crystallites within its silk. As a functional explanation, the low tear strength for A. luminosa comprises a safety mechanism and ensures the entire nest is not pulled down by prey which is too heavy.

Background: Measles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs., Methods: MUV, MEV and ECVs were purified by ultracentrifugation, hydrophobic interaction chromatography and immunoaffinity chromatography, proteins in the samples were resolved by SDS-PAGE and subjected to identification by MALDI-TOF/TOF-MS. A comparative analysis of HCPs present in all samples was carried out., Results: By proteomics approach, it was verified that almost all virus-coded proteins are present in MEV and MUV particles. Protein C in MEV which was until now considered to be non-structural viral protein, was found to be present inside the MeV virions. Results on the presence of HCPs in differently purified virus preparations imply that actin, annexins, cyclophilin A, moesin and integrin β1 are part of the virions., Conclusions: All HCPs detected in the viruses are present in ECVs as well, indicating their possible function in vesicle formation, or that most of them are only present in ECVs. Only five HCPs were constantly present in purified virus preparations, regardless of the purification method used, implying they are likely the integral part of the virions. The approach described here is helpful for further investigation of HCPs in other virus preparations.

Highly fused, fully conjugated aromatic compounds are interesting candidates for organic electronics. With higher crystallinity their electronic properties improve. It is shown here that the crystallization of three archetypes of such molecules-pentacenetetrone, indigo, and perinone-can be achieved hydrothermally. Given their molecular structure, this is a truly startling finding. In addition, it is demonstrated that perinone can also be synthesized in solely high-temperature water from the starting compounds naphthalene bisanhydride and o-phenylene diamine without the need for co-solvents or catalysts. The transformation can be drastically accelerated by the application of microwave irradiation. This is the first report on the hydrothermal generation of two fused heterocycles., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Laterally resolved chemical analysis (chemical imaging) has increasingly attracted attention in the Life Sciences during the past years. While some developments have provided improvements in lateral resolution and speed of analysis, there is a trend toward the combination of two or more analysis techniques, so-called multisensor imaging, for providing deeper information into the biochemical processes within one sample. In this work, a human malignant pleural mesothelioma sample from a patient treated with cisplatin as a cytostatic agent has been analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). While LA-ICPMS was able to provide quantitative information on the platinum distribution along with the distribution of other elemental analytes in the tissue sample, MALDI MS could reveal full information on lipid distributions, as both modes of polarity, negative and positive, were used for measurements. Tandem MS experiments verified the occurrence of distinct lipid classes. All imaging analyses were performed using a lateral resolution of 40 μm, providing information with excellent depth of details. By analyzing the very same tissue section, it was possible to perfectly correlate the obtained analyte distribution information in an evaluation approach comprising LA-ICPMS and MALDI MS data. Correlations between platinum, phosphorus, and lipid distributions were found by the use of advanced statistics. The present proof-of-principle study demonstrates the benefit of data combination for outcomes beyond one method imaging modality and highlights the value of advanced chemical imaging in the Life Sciences.

Background: The well-known inflammatory and fibrogenic changes of the lung upon crystalline silica are accompanied by early changes of the phospholipid composition (PLC) as detected in broncho-alveolar lavage fluid (BALF). Amorphous silica nanoparticles (NPs) evoke transient lung inflammation, but their effect on PLC is unknown. Here, we compared effects of unmodified and phosphonated amorphous silica NP and describe, for the first time, local changes of the PLC with innovative bioimaging tools., Methods: Unmodified (SiO 2 -n), 3-(trihydroxysilyl) propyl methylphosphonate coated SiO 2 -n (SiO 2 -p) as well as a fluorescent surrogate of SiO 2 -n (SiO 2 -FITC) nanoparticles were used in this study. In vitro toxicity was tested with NR8383 alveolar macrophages. Rats were intratracheally instilled with SiO 2 -n, SiO 2 -p, or SiO 2 -FITC, and effects on lungs were analyzed after 3 days. BALF from the right lung was analyzed for inflammatory markers. Cryo-sections of the left lung were subjected to fluorescence microscopy and PLC analyses by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MS), Fourier transform infrared microspectroscopy (FT-IR), and tandem mass spectrometry (MS/MS) experiments., Results: Compared to SiO 2 -p, SiO 2 -n NPs were more cytotoxic to macrophages in vitro and more inflammatory in the rat lung, as reflected by increased concentration of neutrophils and protein in BALF. Fluorescence microscopy revealed a typical patchy distribution of SiO 2 -FITC located within the lung parenchyma and alveolar macrophages. Superimposable to this particle distribution, SiO 2 -FITC elicited local increases of phosphatidylglycerol (PG) and phosphatidylinositol (PI), whereas phoshatidylserine (PS) and signals from triacylgyceride (TAG) were decreased in the same areas. No such changes were found in lungs treated with SiO 2 -p or particle-free instillation fluid., Conclusions: Phosphonate coating mitigates effects of silica NP in the lung and abolishes their locally induced changes in PLC pattern. Bioimaging methods based on MALDI-MS may become a useful tool to investigate the mode of action of NPs in tissues.

The formation of networks through light-initiated radical polymerization allows little freedom for tailored network design. The resulting inhomogeneous network architectures and brittle material behavior of such glassy-type networks limit the commercial application of photopolymers in 3D printing, biomedicine, and microelectronics. An ester-activated vinyl sulfonate ester (EVS) is presented for the rapid formation of tailored methacrylate-based networks. The chain transfer step induced by EVS reduces the kinetic chain length of the photopolymer, thus shifting the gel point to higher conversion, which results in reduced shrinkage stress and higher overall conversion. The resulting, more homogeneous network is responsible for the high toughness of the material. The unique property of EVS to promote nearly retardation-free polymerization can be attributed to the fact that after the transfer step no polymerizable double bond is formed, as is usually seen in classical chain transfer agents. Laser flash photolysis, theoretical calculations, and photoreactor studies were used to elucidate the fast chain transfer reaction and exceptional regulating ability of EVS. Final photopolymer networks exhibit improved mechanical performance making EVS an outstanding candidate for the 3D printing of tough photopolymers., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationale: Although the fruiting-body of the fungi of the genus Xylaria shows a great variety of morphological characteristics, their mycelial forms are always very similar, imposing difficulties for their identification. Intact cell mass spectrometry (ICMS) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be a fast and reliable strategy to support the differentiation/identification of Xylaria species in those cases where fruit-bodies are not available., Methods: Many experimental parameters such as sample preparation and culture media are crucial for filamentous fungi analysis by MALDI-TOFMS. For the purposes of this study, we used four matrices (CHCA, DHB, FA and SA) with five different concentrations (0.1, 0.3, 0.5, 1.0 and 2.5%) of TFA in the matrix, the influence of six different culture media (solid and liquid), and three mycelium peptide/protein extraction protocols (acid, basic and thymol-supported solution) to optimize the sample preparation of the endophytic fungus X. arbuscula., Results: It was observed that sinapinic acid (30 mg/mL) dissolved in acetonitrile/0.1% TFA and PDA were the best matrix solution and culture medium, respectively, for the ICMS of X. arbuscula. The formic acid and ammonium bicarbonate (AB) protocols provided similar mass spectra; however, a higher number of peaks were observed using AB extraction. Mass spectra obtained from different thymol-containing solutions (EtOH/aqueous 0.1% TFA and ACN/aqueous 0.1% TFA) show increasing peak abundances at m/z 3000-6500., Conclusions: X. arbuscula could be analyzed by ICMS. However, an extraction step was required to provide suitable MALDI mass spectra. Formic acid-, AB- and thymol-containing solutions were demonstrated to be good cocktails for the extraction of peptide/protein biomarkers from these fungi., (Copyright © 2018 John Wiley & Sons, Ltd.)