Merging Flow Synthesis and Enzymatic Maturation to Expand the Chemical Space of Lasso Peptides.

Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.

Rapid identification of pyoverdines of fluorescent Pseudomonas spp. by UHPLC-IM-MS.

Siderophores are iron-chelating molecules produced by bacteria and other microbes. They are involved with virulence in infections and play key roles in bacterial community assembly and as plant protectants due to their pathogen control properties. Although assays exist to screen whether newly isolated bacteria can produce siderophores, the chemical structures of many of these bio-active molecules remain unidentified due to the lack of rapid analytical procedures. An important group of siderophores are pyoverdines. They consist of a structurally diverse group of chromopeptides, whose amino acid sequence is characteristic for the fluorescent Pseudomonas species that secrets them. Although over 60 pyoverdine structures have been described so far, their characterization is cumbersome and several methods (isoelectrofocusing, iron uptake measurement, mass determination) are typically combined as ambiguous results are often achieved by a single method. Those additional experiments consume valuable time and resources and prevent high-throughput analysis. In this work, we present a new pyoverdine characterisation option by recording their collision cross sections (CCS) using trapped ion mobility spectrometry. This can be done simultaneously in combination with UHPLC and high-resolution MS resulting in a rapid identification of pyoverdines. The high specificity of CCS values is presented for 17 pyoverdines secreted by different Pseudomonas strains. The pyoverdine mass determination by full scan MS was supported by fragments obtained from broadband collision induced dissociation (bbCID). As iron contaminations in laboratories are not uncommon, CCS values of ferripyoverdines were also evaluated. Thereby, unusual and highly characteristic ion mobility patterns were obtained that are suitable as an alternative identification marker.

Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.

In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

Pyoverdine Analysis - From High-resolution MS/MS Fragmentation to Ion Mobility Measurements.

Microorganisms produce iron chelators called siderophores that are a rich source for drug discovery or plant protective agents. Pyoverdines are a class of siderophores from fluorescent Pseudomonas members and consist of different peptide chains specific to each bacterial species. The structural elucidation and characterization of pyoverdines require comprehensive analytical methods as bacterial extracts are complex mixtures. Here, we present a high-throughput UHPLC-MS/MS pipeline and the application of ion mobility spectrometry to facilitate research in the field of medicine and agriculture.

Selective and Fast Analysis of Chlorinated Paraffins in the Presence of Chlorinated Mono-, Di-, and Tri-Olefins with the R-Based Automated Spectra Evaluation Routine (RASER).

Chlorinated paraffins (CPs) are complex mixtures consisting of various C homologues (nC ≈ 10-30) and Cl homologues (nCl ≈ 2-20). Technical CP mixtures are produced on a large scale (>106 t/y) and are widely used such as plasticizers in plastic and coolants in metalwork. Since 2017, short-chain CPs (C10-C13) are classified as persistent organic pollutants (POPs) by the Stockholm Convention but longer-chain CPs are not regulated. Analysis of technical CP mixtures is challenging because they consist of hundreds of homologues and millions of constitutional isomers and stereoisomers. Furthermore, such mixtures can also contain byproducts and transformation products such as chlorinated olefins (COs). We applied a liquid-chromatography method coupled to an atmospheric pressure chemical ionization technique with a high-resolution mass detector (LC-APCI-Orbitrap-MS) to study CP and CO homologues in two plastic materials. Respective mass spectra can contain up to 23,000 signals from 1320 different C-Cl homologue classes. The R-based automated spectra evaluation routine (RASER) was developed to efficiently search for characteristic ions in these complex mass spectra. With it, the time needed to evaluate such spectra was reduced from weeks to hours, compared to manual data evaluation. Unique sets of homologue distributions could be obtained from the two plastic materials. CPs were found together with their transformation products, the chlorinated mono-olefins (COs), di-olefins (CdiOs), and tri-olefins (CtriOs) in both plastic materials. Based on these examples, it can be shown that RASER is an efficient and selective tool for evaluating high-resolution mass spectra of CP mixtures containing hundreds of homologues.

A comprehensive method to elucidate pyoverdines produced by fluorescent Pseudomonas spp. by UHPLC-HR-MS/MS.

Microbial secondary metabolites represent a rich source for drug discovery, plant protective agents, and biotechnologically relevant compounds. Among them are siderophores, iron-chelating molecules, that show a great influence on bacterial community assembly and the potential to control pathogen invasions. One of such a siderophore is pyoverdine that is produced by fluorescent Pseudomonas members and consists of different peptide chains specific to each bacterial species. The identification and structural elucidation of such suites of siderophores remain widely underexplored as general high-throughput analytical protocols are missing. Therefore, a dedicated method was established allowing a rapid localization and structural elucidation of pyoverdines. Liquid bacterial culture samples were purified by an easy small-scale solid-phase extraction (SPE). Ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) separated highly polar pyoverdines and their derivatives. All ion fragmentation (AIF) generated mass spectra containing the characteristic fragments of the biological precursor of pyoverdine, ferribactin. This led to the revelation of the mass of secreted pyoverdines. Targeted MS/MS experiments at multiple collision energies accomplished the full structure elucidation of the pyoverdine peptide chain. A mass calculator and a fragmentation predictor facilitated greatly the interpretation of MS/MS spectra by providing accurate masses for a straightforward comparison of measured and theoretical values. The method was successfully validated using four well-known pyoverdines with various peptide chains. Finally, the applicability was proven by the analysis of 13 unknown pyoverdines secreted by sampled bacterial cultures. Among these, 4 novel pyoverdine peptide chains were discovered and are herein reported for the first time.

Bioactive Nitrosylated and Nitrated N-(2-hydroxyphenyl)acetamides and Derived Oligomers: An Alternative Pathway to 2-Amidophenol-Derived Phytotoxic Metabolites.

Incubation of Aminobacter aminovorans, Paenibacillus polymyxa, and Arthrobacter MPI764 with the microbial 2-benzoxazolinone (BOA)-degradation-product 2-acetamido-phenol, produced from 2-aminophenol, led to the recently identified N-(2-hydroxy-5-nitrophenyl) acetamide, to the hitherto unknown N-(2-hydroxy-5-nitrosophenyl)acetamide, and to N-(2-hydroxy-3-nitrophenyl)acetamide. As an alternative to the formation of phenoxazinone derived from aminophenol, dimers- and trimers-transformation products have been found. Identification of the compounds was carried out by LC/HRMS and MS/MS and, for the new structure N-(2-hydroxy-5-nitrosophenyl)acetamide, additionally by 1D- and 2D-NMR. Incubation of microorganisms, such as the soil bacteria Pseudomonas laurentiana, Arthrobacter MPI763, the yeast Papiliotrema baii and Pantoea ananatis, and the plants Brassica oleracea var. gongylodes L. (kohlrabi) and Arabidopsis thaliana Col-0, with N-(2-hydroxy-5-nitrophenyl) acetamide, led to its glucoside derivative as a prominent detoxification product; in the case of Pantoea ananatis, this was together with the corresponding glucoside succinic acid ester. In contrast, Actinomucor elegans consortium synthesized 2-acetamido-4-nitrophenyl sulfate. 1 mM bioactive N-(2-hydroxy-5-nitrophenyl) acetamide elicits alterations in the Arabidopsis thaliana expression profile of several genes. The most responsive upregulated gene was pathogen-inducible terpene synthase TPS04. The bioactivity of the compound is rapidly annihilated by glucosylation.

Anti-microtubule activity of the traditional Chinese medicine herb Northern Ban Lan (Isatis tinctoria) leads to glucobrassicin.

Traditional Chinese medicine (TCM) belongs to the most elaborate and extensive systems of plant-based healing. The herb Northern Ban Lan (Isatis tinctoria) is famous for its antiviral and anti-inflammatory activity. Although numerous components isolated from I. tinctoria have been characterized so far, their modes of action have remained unclear. Here, we show that extracts from I. tinctoria exert anti-microtubular activity. Using time-lapse microscopy in living tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cells expressing green fluorescent protein-tubulin, we use activity-guided fractionation to screen out the biologically active compounds of I. tinctoria. Among 54 fractions obtained from either leaves or roots of I. tinctoria by methanol (MeOH/H2 O 8:2), or ethyl acetate extraction, one specific methanolic root fraction was selected, because it efficiently and rapidly eliminated microtubules. By combination of further purification with ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry most of the bioactivity could be assigned to the glucosinolate compound glucobrassicin. Glucobrassicin can also affect microtubules and induce apoptosis in HeLa cells. In the light of these findings, the antiviral activity of Northern Ban Lan is discussed in the context of microtubules being hijacked by many viral pathogens for cell-to-cell spread.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

7-Rhamnosylated Flavonols Modulate Homeostasis of the Plant Hormone Auxin and Affect Plant Development.

Flavonols are a group of secondary metabolites that affect diverse cellular processes. They are considered putative negative regulators of the transport of the phytohormone auxin, by which they influence auxin distribution and concomitantly take part in the control of plant organ development. Flavonols are accumulating in a large number of glycosidic forms. Whether these have distinct functions and diverse cellular targets is not well understood. The rol1-2 mutant of Arabidopsis thaliana is characterized by a modified flavonol glycosylation profile that is inducing changes in auxin transport and growth defects in shoot tissues. To determine whether specific flavonol glycosides are responsible for these phenotypes, a suppressor screen was performed on the rol1-2 mutant, resulting in the identification of an allelic series of UGT89C1, a gene encoding a flavonol 7-O-rhamnosyltransferase. A detailed analysis revealed that interfering with flavonol rhamnosylation increases the concentration of auxin precursors and auxin metabolites, whereas auxin transport is not affected. This finding provides an additional level of complexity to the possible ways by which flavonols influence auxin distribution and suggests that flavonol glycosides play an important role in regulating plant development.

Developmental trajectories of amphibian microbiota: response to bacterial therapy depends on initial community structure.

Improving host health through microbial manipulation requires untangling factors that shape the microbiome. There is currently little understanding of how initial community structure may drive the microbiota trajectory across host development or influence bacterial therapy outcomes. Probiotic baths of surface symbionts, Pseudomonas fluorescens and Flavobacterium johnsoniae were administered to 240 tadpoles of the midwife toad, Alytes obstetricans in semi-natural outdoor mesocosms originating from geographically and genetically distinct populations in Switzerland. Host bacterial and fungal assemblages were compared in tadpoles from the pond of origin, across metamorphosis, and in toadlets via microbial fingerprinting. Bacterial and fungal community structures differed significantly among populations and a microbial population signature persisted from the tadpole stage, through metamorphosis, and following probiotic treatment. A minimal core surface microbiota is described by persistence through development and by shared membership across populations. The impact of F. johnsoniae on the tadpole surface microbiome was assessed with shotgun metagenomics. Bacterial therapy reduced abundance, diversity, and functional repertoire compared to untreated controls. A correlation between host skin peptides and microbiota suggests a mechanism of host-directed symbiosis throughout development. Early developmental stages are ideal targets for amphibian bacterial therapy that can govern a microbiome trajectory at critical timepoints and may impact susceptibility to disease.

The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley.

The wheat gene Lr34 encodes an ABCG-type transporter which provides durable resistance against multiple pathogens. Lr34 is functional as a transgene in barley, but its mode of action has remained largely unknown both in wheat and barley. Here we studied gene expression in uninfected barley lines transgenic for Lr34. Genes from multiple defense pathways contributing to basal and inducible disease resistance were constitutively active in seedlings and mature leaves. In addition, the hormones jasmonic acid and salicylic acid were induced to high levels, and increased levels of lignin as well as hordatines were observed. These results demonstrate a strong, constitutive re-programming of metabolism by Lr34. The resistant Lr34 allele (Lr34res) encodes a protein that differs by two amino acid polymorphisms from the susceptible Lr34sus allele. The deletion of a single phenylalanine residue in Lr34sus was sufficient to induce the characteristic Lr34-based responses. Combination of Lr34res and Lr34sus in the same plant resulted in a reduction of Lr34res expression by 8- to 20-fold when the low-expressing Lr34res line BG8 was used as a parent. Crosses with the high-expressing Lr34res line BG9 resulted in an increase of Lr34sus expression by 13- to 16-fold in progenies that inherited both alleles. These results indicate an interaction of the two Lr34 alleles on the transcriptional level. Reduction of Lr34res expression in BG8 crosses reduced the negative pleiotropic effects of Lr34res on barley growth and vigor without compromising disease resistance, suggesting that transgenic combination of Lr34res and Lr34sus can result in agronomically useful resistance.

Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids.

Most plastid isoprenoids, including photosynthesis-related metabolites such as carotenoids and the side chain of chlorophylls, tocopherols (vitamin E), phylloquinones (vitamin K), and plastoquinones, derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. Seven out of 10 functional GGPPS isozymes in Arabidopsis thaliana reside in plastids. We aimed to address the function of different GGPPS paralogues for plastid isoprenoid biosynthesis. We constructed a gene co-expression network (GCN) using GGPPS paralogues as guide genes and genes from the upstream and downstream pathways as query genes. Furthermore, knock-out and/or knock-down ggpps mutants were generated and their growth and metabolic phenotypes were analyzed. Also, interacting protein partners of GGPPS11 were searched for. Our data showed that GGPPS11, encoding the only plastid isozyme essential for plant development, functions as a hub gene among GGPPS paralogues and is required for the production of all major groups of plastid isoprenoids. Furthermore, we showed that the GGPPS11 protein physically interacts with enzymes that use GGPP for the production of carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. GGPPS11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. Both gene co-expression and protein-protein interaction likely contribute to the channeling of GGPP by GGPPS11.

Petunia hybrida PDR2 is involved in herbivore defense by controlling steroidal contents in trichomes.

As a first line of defense against insect herbivores many plants store high concentrations of toxic and deterrent secondary metabolites in glandular trichomes. Plant Pleiotropic Drug Resistance (PDR)-type ABC transporters are known secondary metabolite transporters, and several have been implicated in pathogen or herbivore defense. Here, we report on Petunia hybrida PhPDR2 as a major contributor to trichome-related chemical defense. PhPDR2 was found to localize to the plasma membrane and be predominantly expressed in multicellular glandular trichomes of leaves and stems. Down-regulation of PhPDR2 via RNA interference (pdr2) resulted in a markedly higher susceptibility of the transgenic plants to the generalist foliage feeder Spodoptera littoralis. Untargeted screening of pdr2 trichome metabolite contents showed a significant decrease in petuniasterone and petuniolide content, compounds, which had previously been shown to act as potent toxins against various insects. Our findings suggest that PhPDR2 plays a leading role in controlling petuniasterone levels in leaves and trichomes of petunia, thus contributing to herbivory resistance.

Ditechnetium Heptoxide Revisited: Solid-State, Gas-Phase, and Theoretical Studies.

Ditechnetium heptoxide was synthesized from the oxidation of TcO2 with O2 at 450 °C and characterized by single-crystal X-ray diffraction, electron-impact mass spectrometry (EI-MS), and theoretical methods. Refinement of the structure at 100 K indicates that Tc2O7 crystallizes as a molecular solid in the orthorhombic space group Pbca [a = 7.312(3) Å, b = 5.562(2) Å, c = 13.707(5) Å, and V = 557.5(3) Å3]. The Tc2O7 molecule can be described as corner-sharing TcO4 tetrahedron [Tc---Tc = 3.698(1) Å and Tc-OBri-Tc = 180.0°]. The EI-MS spectrum of Tc2O7 consists of both mononuclear and dinuclear species. The main dinuclear species in the gas-phase are Tc2O7 (100%) and Tc2O5 (56%), while the main mononuclear species are TcO3 (33.9%) and TcO2 (42.8%). The difference in the relative intensities of the M2O5 (M = Tc, Re) fragments (1.7% for Re) indicates that these group 7 elements exhibit different gas-phase chemistry. The solid-state structure of Tc2O7 was investigated by density functional theory methods. The optimized structure of the Tc2O7 molecule is in good agreement with the experimental one. Simulations indicate that the more favorable geometry for the Tc2O7 molecule in the gas-phase is bent (Tc-OBri-Tc = 156.5°), while a linear geometry (Tc-OBri-Tc = 180.0°) is favored in the solid-state.

Prometabolites of 5-Diphospho-myo-inositol Pentakisphosphate.

Diphospho-myo-inositol phosphates (PP-InsP(y)) are an important class of cellular messengers. Thus far, no method for the transport of PP-InsP(y) into living cells is available. Owing to their high negative charge density, PP-InsP(y) will not cross the cell membrane. A strategy to circumvent this issue involves the generation of precursors in which the negative charges are masked with biolabile groups. A PP-InsP(y) prometabolite would require twelve to thirteen biolabile groups, which need to be cleaved by cellular enzymes to release the parent molecules. Such densely modified prometabolites of phosphate esters and anhydrides have never been reported to date. This study discloses the synthesis of such agents and an analysis of their metabolism in tissue homogenates by gel electrophoresis. The acetoxybenzyl-protected system is capable of releasing 5-PP-InsP5 in mammalian cell/tissue homogenates within a few minutes and can be used to release 5-PP-InsP5 inside cells. These molecules will serve as a platform for the development of fundamental tools required to study PP-InsP(y) physiology.

Production of proteasome inhibitor syringolin A by the endophyte Rhizobium sp. strain AP16.

Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts.

Herbivory and floral signaling: phenotypic plasticity and tradeoffs between reproduction and indirect defense.

Plant defense against herbivores may compromise attraction of mutualists, yet information remains limited about the mechanisms underlying such signaling tradeoffs. Here, we investigated the effects of foliar herbivory by two herbivore species on defense compounds, floral signaling, pollinator and parasitoid attraction, and seed production. Herbivory generally reduced the quantity of many floral volatile organic compounds VOCs) in Brassica rapa. By contrast, floral color, flower diameter, and plant height remained unaffected. The decreased amounts of floral volatiles led to reduced attractiveness of flowers to pollinators, but increased the attractiveness of herbivore-infested plants to parasitoids. Plants infested with the native butterfly Pieris brassicae produced more flowers during early flowering, effectively compensating for the lower olfactory attractiveness. Herbivory by the invasive Spodoptera littoralis increased the amounts of glucobrassicanapin, and led to delayed flowering. These plants tended to attract fewer pollinators and to produce fewer seeds. Our study indicates a tradeoff between pollinator attraction and indirect defense (parasitoid attraction), which can be mitigated by reduced floral VOC emission and production of more early flowers. We suggest that this compensatory mechanism is specific to plant-herbivore associations with a coevolutionary history.

Superficial dopants allow growth of silicone nanofilaments on hydroxyl-free substrates.

We report new types of silicone nanostructures by a gas-phase reaction of trichloromethylsilane: 1-D silicone nanofilaments with a raveled end and silicone nanoteeth. Filaments with a raveled end are obtained on poly(vinyl chloride), which is superficially doped with the detergent Span 20. Silicone nanoteeth grow on sodium chloride using dibutyl phthalate as superficial dopant. Without dopants, no structures are observed. The dopants are identified by mass spectroscopy and the silicone nanostructures are analyzed by infrared spectroscopy and energy-dispersive analysis of X-rays. The growth of silicone nanostructures on a hydrophobic substrate (poly(vinyl chloride)/Span 20) and a substrate free of hydroxyl groups (sodium chloride/dibutyl phthalate) questions the currently discussed mechanisms for the growth of 1-D silicone nanofilaments, which is discussed. We suggest superficial doping as an alternative pretreatment method to oxidizing activation and prove this principle by the successful coating of copper, which is superficially doped with Span 20.

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching.

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5ß-THF, 3ß,5α-THF, 3ß,5ß-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5ß-THE, 3ß,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20ß-DHP, 3α-DHP, 3ß-DHP, 5α-DHP, 5ß-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5ß-THP, 3ß,5α-THP, 3ß,5ß-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 µL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL-1 was achieved for all steroids suitable for the analysis of authentic samples.