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The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering., 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 © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this phase III randomized double-blinded controlled trial was to investigate the efficacy of a rose geranium in sesame oil (RG) nasal spray compared with an isotonic saline (IS) nasal spray for alleviating nasal vestibulitis symptoms among patients undergoing chemotherapy., Methods: Patients undergoing active chemotherapy who reported associated nasal symptoms were randomized 1:1 to receive RG or IS, administered twice daily for 2 weeks. Consenting participants completed nasal symptom questionnaires at baseline and then weekly while on treatment. The proportion of patients experiencing improvements in their nasal symptoms 2 weeks after initiating the nasal spray, using a six-point global impression of change score, was estimated within and between each randomized arm, and compared between arms, using Fisher's exact test. The estimated odds ratio was determined (95% confidence interval)., Results: One hundred and six patients consented to this study; 43 participants in the RG arm and 41 in the IS arm were evaluable for the primary endpoint. Participants had a mean age of 57.8 years (SD 13.9). Demographic characteristics and baseline nasal symptoms were similar between arms. Of the evaluable participants who received RG, 67.4% reported improved nasal symptoms, compared with 36.6% of the participants who received IS (P = 0.009). Adverse events were sparse and did not differ between arms., Conclusion: Rose geranium in sesame oil significantly improves nasal vestibulitis symptoms among patients undergoing chemotherapy., Trial Registration: NCT04620369., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Fusarium fujikuroi is a pathogen of rice causing diverse disease symptoms such as 'bakanae' or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin., Results: Through the combination of diverse '-omics' methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism., Conclusion: In this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms., (© 2024. The Author(s).)

Fusarium mangiferae causes the mango malformation disease (MMD) on young mango trees and seedlings resulting in economically significant crop losses. In addition, F. mangiferae produces a vast array of secondary metabolites (SMs), including mycotoxins that may contaminate the harvest. Their production is tightly regulated at the transcriptional level. Here, we show that lack of the H3 K9-specific histone methyltransferase, FmKmt1, influences the expression of the F. mangiferae polyketide synthase (PKS) 8 (FmPKS8), a so far cryptic PKS. By a combination of reverse genetics, untargeted metabolomics, bioinformatics and chemical analyses including structural elucidation, we determined the FmPKS8 biosynthetic gene cluster (BGC) and linked its activity to the production of fusamarins (FMN), which can be structurally classified as dihydroisocoumarins. Functional characterization of the four FMN cluster genes shed light on the biosynthetic pathway. Cytotoxicity assays revealed moderate toxicities with IC 50 values between 1 and 50 μM depending on the compound., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The phenylspirodrimanes (PSDs) from Stachybotrys chartarum represent a structurally diverse group of meroterpenoids, which, on the one hand, exhibit a structural exclusivity since their occurrence is not known for any other species and, on the other hand, offer access to chemically and biologically active compounds. In this study, phenylspirodrimanes 1 - 3 were isolated from S. chartarum and their water-mediated Cannizzaro-type transformation was investigated using quantum chemical DFT calculations substantiated by LC-MS and NMR experiments. Considering the inhibitory activity of PSDs against proteolytic enzymes and their modulatory effect on plasminogen, PSDs 1 - 3 were used as a starting material for the synthesis of their corresponding biologically active lactams. To access the library of the PSD derivatives and screen them against physiologically relevant serine proteases, a microscale semisynthetic approach was developed. This allowed us to generate the library of 35 lactams, some of which showed the inhibitory activity against physiologically relevant serine proteases such as thrombin, FXIIa, FXa, and trypsin. Among them, the agmatine-derived lactam 16 showed the highest inhibitory activity against plasma coagulation factors and demonstrated the anticoagulant activity in two plasma coagulation tests. The semisynthetic lactams were significantly less toxic compared to their parental natural PSDs., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Synthesis of azetidine-derived natural products by the opportunistic pathogen Pseudomonas aeruginosa is controlled by quorum sensing, a process involving the production and sensing of diffusible signal molecules that is decisive for virulence regulation. In this study, we engineered P. aeruginosa for the titratable expression of the biosynthetic aze gene cluster, which allowed the purification and identification of two new products, azetidomonamide C and diazetidomonapyridone. Diazetidomonapyridone was shown to have a highly unusual structure with two azetidine rings and an open-chain diimide moiety. Expression of aze genes strongly increased biofilm formation and production of phenazine and alkyl quinolone virulence factors. Further physiological studies revealed that all effects were mainly mediated by azetidomonamide A and diazetidomonapyridone, whereas azetidomonamides B and C had little or no phenotypic impact. The P450 monooxygenase AzeF which catalyzes a challenging, stereoselective hydroxylation of the azetidine ring converting azetidomonamide C into azetidomonamide A is therefore crucial for biological activity. Based on our findings, we propose this group of metabolites to constitute a new class of diffusible regulatory molecules with community-related effects in P. aeruginosa .

Plants from the Solanaceae family are known to be sources of several nutritionally relevant steroidal glycoalkaloids (SGAs). With the aim of quantitatively investigating the occurrence of the main SGA from tomatoes, eggplants, and potatoes in various food samples and evaluating their relevance in the human diet, a rapid single-step extraction liquid chromatography-tandem mass spectrometry method was developed. Over the course of isolating several commercially unavailable SGAs from tomato products to use them as reference standards, a previously unknown derivative was detected, structurally characterized, and identified as a novel isomer of esculeoside B-1 and B-2. After validation of the method, 36 food items exclusively derived from Solanaceae plants were analyzed for their SGA contents and a specific occurrence of each alkaloid in tomato, eggplant, or potato products was revealed. This is the first study reporting quantitative data on the occurrence of esculeoside A, B-1, B-2, and iso -esculeoside B in tomato products obtained by using appropriate reference compounds rather than applying a semi-quantitative approach based on α-tomatine as a reference. Some of the analyzed tomato products contained the esculeosides in concentrations of >500 mg/kg, clearly indicating their relevance in the human diet and the need of investigating their potential bioactivities in the future.

Scope: In this study, the applicability of several β-carboline, imidazole, and steroidal alkaloids as biomarkers for tomato juice intake is evaluated., Methods and Results: Over the course of a 2-week crossover dietary intervention study, 14 volunteers were given low and high doses of tomato juice after 3 days of avoiding tomato-based products. On the day of consumption and the following days, volunteers provided urine samples that were quantitatively analyzed by high-performance liquid chromatography-tandem mass spectrometry. Herein, glucose-derived β-carboline alkaloids are determined as supporting, yet non-specific dietary biomarkers for tomato juice intake. Several imidazole alkaloids represent further biomarkers, which are shown to specifically indicate consumption of tomato juice for 24 h and partly >24 h. Additionally, steroidal alkaloids derived from esculeogenin B are determined to be specific biomarkers for tomato juice detectable for at least 48 h after consumption. The intake of low and high amounts of tomato juice is significantly distinguishable based on the urinary excretion of all determined biomarkers as well., Conclusions: The dietary intake of tomato juice is conclusively traceable based on urinary excretion of multiple β-carboline, imidazole, and steroidal alkaloids, and can be determined for up to 48 h after consumption. Furthermore, different intake doses can clearly be distinguished based on their urinary excretion., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study aimed at the identification of novel imidazole alkaloids derived from histamine or histidinol and generally investigating the occurrence of suchlike alkaloids in a variety of foodstuffs. Herein, N-caprylhistamine was synthesized and the glucosidic derivative N-caprylhistamine-β-glucoside was isolated from ripe tomato fruits and structurally characterized. The obtained reference standards were used for the extension of an established LC-MS/MS-based method for the quantitation of several imidazole alkaloids in tomato products. After validation for the two additional analytes and demonstrating the applicability of the method to nine other food matrices, 104 food items were screened for the occurrence of the described imidazole alkaloids. Remarkably, all of the investigated alkaloids were only quantifiable in tomato-based products and the occurrence of N-caprylhistamine and N-caprylhistamine-β-glucoside was reported for the first time. These imidazole alkaloids could thus be applicable as specific intake biomarkers for tomatoes and their biological activities as well as metabolic fate should be investigated in future research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Unlabelled: Objective of this study was the improvement of selected parameters of udder health by mastitis vaccination in a dairy herd with elevated bulk milk somatic cell counts and Staphylococcus (S.) aureus as predominant mastitis causing pathogen., Material and Methods: On a dairy farm, pregnant heifers (status group [SG] 1; n = 181) as well as cows stratified for their udder health state (classification based on results of cytobacteriological investigations of quarter milk samples obtained before dry cow therapy [MS0]) (SG 2-4; n = 416) were randomly assigned to one of the following vaccination groups (VG): Startvac® (VG SV), Bestvac® Rind Mastitis (containing herd-specific S. aureus-strains; VG BV) and the unvaccinated control (VG Co, placebo), respectively. The collected data (5 [MS5] and 52 [MS52] days in milk [DIM]: quarter milk somatic cell count [QSCC] and bacteriological investigation of quarter milk samples; dairy herd improvement test [DHIT] days 1-10: milk yield and individual cow somatic cell count; until 305 DIM: clinical mastitis cases) were compared between the VG within their SG., Results: S. aureus prevalences were significantly lower in VG SV (p < 0.001) and VG BV (p = 0.006) within SG 3 and in VG SV (p = 0.008) within SG 4, respectively, in comparison to VG Co. Milk yields (DHIT days [p = 0.042] and 305-day milk yield [p = 0.040]) were significantly less in VG SV within SG 4 compared to VG Co. Significant different changes over time in comparison to VG Co indicating a vaccine effect during lactation were only observed for QSCC within SG 4 for VG BV (p = 0.017; increase towards MS52) and for S. aureus prevalence within SG 3 for VG BV (p < 0.001; opposing trends from MS0 towards MS52). All other interactions of time and VG under investigation were not significant in any of the SG. Furthermore, there were no descriptive differences in the incidence of clinical mastitis and duration of a necessary mastitis therapy, respectively, between the VG within their SG., Conclusion: In this field study, the application of two different mastitis vaccines was not an appropriate tool to improve the considered parameters of udder health sustainably.

The dynamic range and low stoichiometry of protein phosphorylation frequently demands the enrichment of phosphorylated peptides from protein digests prior to mass spectrometry. Several techniques have been reported in literature for phosphopeptide enrichment, including metal oxides such as TiO(2) and ion metal affinity chromatography (IMAC). While the metal oxides have been used with reasonable success, IMAC has suffered from reduced selectivity and poor reproducibility. In this report, we present the first demonstration of the use of immobilized zirconium on a phosphonate-terminated self-assembled monolayer (SAM) for specific phosphopeptide capture and direct analysis by MALDI MS. By using the herein described functionalized-surface-based technology, efficient enrichment of phosphopeptides in different standard test systems such as alpha- or beta-casein digests or synthetic phosphopeptides spiked in nonphosphorylated protein digest has been demonstrated. The limit of detection for a beta-casein phosphopeptide was assessed to be at the low femtomole level. Compared to other state-of the-art technologies, like use of TiO(2) and Fe-IMAC, the presented technique demonstrated a superior performance with respect to specificity and bias with respect to singly or multiply phosphorylated peptides. Additionally, this platform was also successfully applied for ESI sample preparation, providing detailed sequence information of the investigated phosphopeptide. This technology was also proven to be applicable for real life samples such as phosphorylation site analysis of recombinant human MAPK1 and HSP B1 isolated from a 2D-gel spot by phosphopeptide enrichment and direct MALDI MS/MS.

The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches. In these cases higher effective temperatures could be measured accompanied by increased analyte intensities, which can be explained by stronger volatilization processes caused by a hindered heat dissipation resulting in a raised analyte transfer into the gas phase. For all sublimed compounds, a strong increase of the initial ion velocity compared with DD preparations could be measured. Higher initial ion velocities correlate with a decrease in internal ion energy which might be attributed to the very uniform crystal morphology exhibited by sublimed compounds. For sublimed matrices without reduced grain size, at least slightly higher analyte intensities could be detected at raised laser fluences. Analyte accumulation in the uppermost matrix layers or the detected higher ion stability can be explanations for these results.

Integral membrane proteins are notoriously difficult to identify and analyze by mass spectrometry because of their low abundance and limited number of trypsin cleavage sites. Our strategy to address this problem is based on a novel technology for MALDI-MS peptide sample preparation that increases the success rate of membrane protein identification by increasing the sensitivity of the MALDI-TOF system. For this, we used sample plates with predeposited matrix spots of CHCA crystals prepared by vacuum sublimation onto an extremely low wettable (ultraphobic) surface. In experiments using standard peptides, an up to 10-fold gain of sensitivity was found for on-chip preparations compared with classical dried-droplet preparations on a steel target. In order to assess the performance of the chips with membrane proteins, three model proteins (bacteriorhodopsin, subunit IV(a) of ATP synthase, and the cp47 subunit from photosystem II) were analyzed. To mimic realistic analysis conditions, purified proteins were separated by SDS-PAGE and digested with trypsin. The digest MALDI samples were prepared either by dried-droplet technique on steel plates using CHCA as matrix, or applied directly onto the matrix spots of the chip surface. Significantly higher signal-to-noise ratios were observed for all of the spectra resulting from on-chip preparations of different peptides.In a second series of experiments, the membrane proteome of Rhodococcus jostii RHA1 was investigated by AIEC/SDS-PAGE in combination with MALDI-TOF MS/MS. As in the first experiments, Coomassie-stained SDS-PAGE bands were digested and the two different preparation methods were compared. For preparations on the Mass.Spec.Turbo Chip, 43 of 60 proteins were identified, whereas only 30 proteins were reliably identified after classical sample preparation. Comparison of the obtained Mascot scores, which reflect the confidence level of the protein identifications, revealed that for 70% of the identified proteins, higher scores were obtained by on-chip sample preparation. Typically, this gain was a consequence of higher sequence coverage due to increased sensitivity.

By using automation and affinity-tag technologies, analysis of the large number of ORFs generated by genome-sequencing projects is greatly accelerated. Protocols describing culture of E. coli in automation-compatible formats and subsequent micro-to large-scale automated purification of 6xHis-tagged proteins are presented.

Methods based on immobilized-metal affinity chromatography (IMAC) technology for the isolation of 6xHis-tagged proteins offer a one-step purification process that is both robust and meets the challenge of quantitatively purifying thousands of proteins with differing structures and characteristics. To perform this method in a high-throughput, automated format, protocols have been developed that can be run on lab automation workstations. Ready-to-run protocols covering a wide range of protein purification and assay applications, and which rely on the well-established 6xHis-Ni-NTA IMAC technology, are available. An Ni-NTA magnetic bead-based protocol allows micro-scale purification of up to 15 microg of 6xHis-tagged protein per well. If larger amounts of purified protein are needed, a vacuum-controlled Ni-NTA resin-based process provides a convenient medium-scale method for purification of up to 300 microg of 6xHis-tagged protein in a 96-well format. This protocol has been adapted to further increase the yield of 6xHis-tagged proteins allowing purification of up to several milligrams of highly pure protein per well. The protocol can process 96 samples, each derived from up to 25 mL culture volume (E. coli), in less than 3 h. Examples of purification and assay applications using the protocols mentioned above, and data on reproducibility and cross-contamination-free processing are shown.

The TAGZyme system allows efficient and precise exoproteolytic cleavage of N-terminal affinity tags, such as the 6xHis tag, from recombinant proteins. In combination with Ni-NTA technology, the TAGZyme system provides high-purity proteins free of vector-encoded amino acids for use in applications that demand recombinant reagents, an absence of nonspecific cleavage, and a complete removal of all impurities from the target protein preparation. We present results of recent studies on the use of the TAGZyme system. The N-terminal affinity tag sequences encoded by the TAGZyme pQE expression vectors are optimized with respect to their cleavage using TAGZyme exoproteases. The vectors have multiple cloning site sequences designed to allow complete exoproteolytic removal of the encoded N-terminal affinity tag regardless of the restriction site used for cloning. The efficient cleavage reaction and removal of the TAGZyme enzymes by subtractive Ni-NTA chromatography is demonstrated for 6xHis-interleukin-1beta and 6xHis-TNF. In both cases, more than 99.8% of TAGZyme proteolytic activity was separated from recovered, detagged proteins. N-terminal analyses by Edman degradation revealed the predicted sequences of the native proteins and indicated a purity in excess of 99%. For cleavage of both 6xHis-tagged GFP and IL-1beta, DAPase enzyme gave an average cleavage rate of 1.5 +/- 0.5 min per amino acid.

Isolated membranes of the moderate halophilic bacterium Haloferax volcanii are able to hydrolyze ATP via an ATPase, which needs the presence of Mg2+ or Mn2+, high concentrations of NaCl, a pH value of 9, and high temperatures with an optimum at 60 degrees C. We have not found any phosphatase activity in the preparations. We developed a purification method for the isolated enzyme with an enrichment factor of 90. SDS-gel electrophoresis of the partially purified enzyme of Haloferax volcanii showed putative ATPase subunits of 63, 51, 37, and 12 kDa. N-ethylmaleimide (NEM) a specific inhibitor for V-ATPases, which alkylates cyteines, inhibited the enzyme slightly. Binding of tritiated NEM to the isolated ATPase fractions resulted in labelling of the 63 and 51 kDa peptides. Using PCR with degenerate oligonucleotides, we could clone and sequence a gene cluster encoding the A1 part of the halophilic ATPase. The described genes are organized in an operon in the order D, C, E, B, A, named alphabetically according to their decreasing size. The deduced products of 64.5, 52, 38.7, 22, and 11.6 kDa confirm the results of the partial purification of the ATPase. Biochemical characterization of the Haloferax volcanii ATPase gave the following results: In presence of Mn2+ higher rates of ATP hydrolysis could be observed than in presence of Mg2+, but free manganese ions inhibited the enzyme activity of the ATPase. Calculation of the true concentrations of the complex between ATP and the respective divalent metal ion led to determination of Michaelis-Menten constants for ATP in the hydrolysis direction of 1 mM in presence of MgCl2 and 0.24 mM in presence of MnCl2. Sodium chloride concentrations in the molar range induce changes in KM by a factor of about 10. The enzyme is specific for ATP; other nucleotides including GTP and ADP are competitive inhibitors of ATP hydrolysis.

Using PCR with degenerate oligonucleotides, we amplified two conserved regions of the plasma membrane ATPase A (alpha)- and B (beta)-subunit-encoding genes from Haloferax volcanii, a halophilic archaeon. The amplified fragments were cloned and sequenced, and then used as homologous probes to clone genomic restriction fragments, one of which contained the nearly complete atpA- and atpB-gene. To complete the latter one, we sequenced a region of an overlapping fragment using synthetic oligonucleotides as primers. The deduced amino-acid sequence showed a high degree of similarity with the A and B sequences of Halobacterium halobium-ATPase, and a relatively high degree with Daucus carota V-ATPase, but less similarity to F-ATPase alpha- and beta-subunits. Like in V-ATPases, the A-subunit is more related to the catalytic F-ATPase beta-subunit, whereas B corresponds to alpha. Cross-reaction of antibodies against CF1-alpha (synthetic peptide) with B and CF1-beta with A of Haloferax volcanii confirmed this finding. The ATPase of Haloferax volcanii is a halophilic enzyme, the amino-acid sequences contain about 20% negatively charged residues. This is discussed in terms of adaption to hypersaline conditions. The enzyme is specific for ATP, we determined KM values for ATP in presence of Mn2+ and Mg2+, respectively. Other nucleotides, including GTP and ADP are competitive inhibitors of ATP hydrolysis. Despite of the high degree of similarity to the Halobacterium enzyme, the ATPase showed no sensitivity to most of the tested inhibitors of ATP hydrolysis. NEM, a specific inhibitor for V-ATPases inhibited the enzyme only slightly. Bafilomycin, NBD-Cl and nitrate showed no effect. These results will be discussed in context with some specific differences in the primary structure of the Haloferax A-subunit.