Clostridioides difficile Modifies its Aromatic Compound Metabolism in Response to Amidochelocardin-Induced Membrane Stress.

Amidochelocardin is a broad-spectrum antibiotic with activity against many Gram-positive and Gram-negative bacteria. According to recent data, the antibiotic effect of this atypical tetracycline is directed against the cytoplasmic membrane, which is associated with the dissipation of the membrane potential. Here, we investigated the effect of amidochelocardin on the proteome of Clostridioides difficile to gain insight into the membrane stress physiology of this important anaerobic pathogen. For the first time, the membrane-directed action of amidochelocardin was confirmed in an anaerobic pathogen. More importantly, our results revealed that aromatic compounds potentially play an important role in C. difficile upon dissipation of its membrane potential. More precisely, a simultaneously increased production of enzymes required for the synthesis of chorismate and two putative phenazine biosynthesis proteins point to the production of a hitherto unknown compound in response to membrane depolarization. Finally, increased levels of the ClnAB efflux system and its transcriptional regulator ClnR were found, which were previously found in response to cationic antimicrobial peptides like LL-37. Therefore, our data provide a starting point for a more detailed understanding of C. difficile's way to counteract membrane-active compounds. IMPORTANCE C. difficile is an important anaerobe pathogen causing mild to severe infections of the gastrointestinal tract. To avoid relapse of the infection following antibiotic therapy, antibiotics are needed that efficiently eradicate C. difficile from the intestinal tract. Since C. difficile was shown to be substantially sensitive to membrane-active antibiotics, it has been proposed that membrane-active antibiotics might be promising for the therapy of C. difficile infections. Therefore, we studied the response of C. difficile to amidochelocardin, a membrane-active antibiotic dissipating the membrane potential. Interestingly, C. difficile's response to amidochelocardin indicates a role of aromatic metabolites in mediating stress caused by dissipation of the membrane potential.

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum.

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo - an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.

Differential effects of Helenalin, an anti-inflammatory sesquiterpene lactone, on the proteome, metabolome and the oxidative stress response in several immune cell types.

Extracts of Arnica spp. are traditionally used due to their anti-inflammatory effects for the topical treatment of e.g. haematoma or muscle distortions. One of the main active compounds is Helenalin, a sesquiterpene lactone that can be found in various Asteraceae. However, immunotoxic effects of the compound are only poorly analysed. In this study, a 2D gel electrophoresis based proteomic approach together with a membrane based proteomic assay, metabolomics and the detection of intracellular reactive oxygen species (iROS) were used to investigate potential immunotoxic properties of Helenalin on the human immune cell lines Jurkat and THP-1 and on human peripheral blood mononuclear cells (PBMC). The study revealed a dose-dependent cytotoxicity towards both tested cell lines and the PBMC. However, the cell lines were less sensitive to the Helenalin treatment than the PBMC. The proteomic assays showed strong effects on the carbohydrate metabolism and the protein folding in THP-1 cells but only weak impact on Jurkat cells. Metabolomic studies as well as iROS detection in THP-1 cells verified the results of the proteomic analysis. In summary, the approaches used in this study were able to identify target pathways of Helenalin especially in THP-1 monocytes and thus enable a risk assessment of the substance.

A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

A one-stage cultivation process for lipid- and carbohydrate-rich biomass of Scenedesmus obtusiusculus based on artificial and natural water sources.

A one-stage cultivation process of the microalgae Scenedesmus obtusiusculus with medium based on natural water sources was developed to enhance lipids and carbohydrates. A medium based on artificial sea water, Baltic Sea water and river water with optimized nutrient concentrations compared to the standard BG-11 for nitrate (-75%), phosphate and iron (-90%) was used for cultivation. Although nitrate exhaustion over cultivation resulted in nitrate limitation, growth of the microalgae was not reduced. The lipid content increased from 6.0% to 19.9%, an increase in oleic and stearic acid was observed. The unsaponifiable matter of the lipid fraction was reduced from 19.5% to 11.4%. The carbohydrate yield rose from 45% to 50% and the protein content decreased from 32.4% to 15.9%. Using natural water sources with optimized nutrient concentrations could open the opportunity to modulate biomass composition and to reduce the cultivation costs.

Cylindrofridins A-C, Linear Cylindrocyclophane-Related Alkylresorcinols from the Cyanobacterium Cylindrospermum stagnale.

A rapid and exhaustive one-step biomass extraction as well as an enrichment and cleanup procedure has been developed for HPLC-UV detection and quantification of closely related [7.7]paracyclophanes and structural derivatives based on a two-phase solvent system. The procedure has been validated using the biomass of the carbamidocyclophane- and cylindrocyclophane-producing cyanobacterium Nostoc sp. CAVN2 and was utilized to perform a screening comprising 102 cyanobacterial strains. As a result, three new cylindrocyclophane-related alkylresorcinols, cylindrofridins A-C (1-3), and known cylindrocyclophanes (4-6) were detected and isolated from Cylindrospermum stagnale PCC 7417. Structures of 1-3 were elucidated by a combination of 1D and 2D NMR experiments, HRMS, and ECD spectroscopy. Cylindrofridin A (1) is the first naturally occurring [7.7]paracyclophane-related monomeric derivative. In contrast, cylindrofridins B (2) and C (3) represent dimers related to 1. Due to chlorination at the alkyl carbon atom in 1-3, the site of [7.7]paracyclophane macrocycle formation, the cylindrofridins represent linearized congeners of the cylindrocyclophanes. Compounds 1-3 were not toxic against nontumorigenic HaCaT cells (IC50 values >25 µM) compared to the respective cylindrocyclophanes, but 1 was the only cylindrofridin showing moderate activity against methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae with MIC values of 9 and 17 µM, respectively.

Chemical Composition, Antimicrobial and Antioxidant Activities of the Volatile Oil of Ganoderma pfeifferi Bres.

In a first study of the volatile oil of the mushroom basidiomycete Ganoderma pfeifferi Bres., the chemical composition and antimicrobial and antioxidant activities of the oil were investigated. The volatile oil was obtained from the fresh fruiting bodies of Ganoderma pfeifferi Bres. By hydrodistillation extraction and analyzed by GC-MS. The antimicrobial activity of the oil was evaluated against five bacteria strains and two types of fungi strains, using disc diffusion and broth microdilution methods. In addition, the antioxidant activity of the oil was determined using DPPH assay. Four volatile compounds representing 90.5% of the total oil were identified. The majority of the essential oil was dominated by 1-octen-3-ol (amyl vinyl carbinol) 1 (73.6%) followed by 1-octen-3-ol acetate 2 (12.4%), phenylacetaldehyde 3 (3.0%) and 6-camphenol 4 (1.5%). The results showed that the Gram-positive bacteria species are more sensitive to the essential oil than Gram-negative bacteria. The oil showed strong antimicrobial activity against Staphylococcus aureus as well as Candida albicans. Moreover, the oil exhibited strong radical scavenging activity in the DPPH assay. This first report on the chemical composition and biological properties of G. pfeifferi volatile oil makes its pharmaceutical uses rational and provides a basis in the biological and phytochemical investigations of the volatile oils of Ganodermataceae species.

Cytotoxic saponins from the seeds of Pittosporum angustifolium.

Three new acylated R1-barrigenol triterpene glycosides, 1-3, were isolated from the seeds of Pittosporum angustifolium Lodd. together with four known glycosides, 4-7, containing R1- and A1-barrigenol backbones. On the basis of spectroscopic, spectrometric, and chemical analyses the novel compounds were named pittangretosides N-P and established as 21beta-acetoxy-22alpha-angeloyloxy- (1), 21beta-acetoxy-22alpha-(2-acetoxy-2-methylbutyroyloxy)- (2), and 21beta-(2-methylbutyroyloxy)-22alpha-acetoxy-3beta-[beta-D-glucopyranosyl- (1 --> 2)]-[alpha-L-arabinopyranosyl-(1 --> 3)]-[alpha-L-arabinofuranosyl-(1 --> 4)]-beta-D-glucuronopyranosyloxyolean-12-ene-15alpha, 6alpha, 28-triol (3). Evaluation of the in vitro cytotoxicity against three tumour cell lines and one non-tumourigenic cell line revealed antiproliferative effects with IC50 values in a range of 1.74-34.1 microM.

Triterpene glycosides from the leaves of Pittosporum angustifolium.

Phytochemical investigation of the leaves of Pittosporum angustifolium resulted in the isolation and structural elucidation of nine new triterpene saponins, named pittangretosides A-I (1-9), together with a known compound (10). Mainly by NMR and HRESIMS experiments, eight compounds were identified as A1-barrigenol glycosides (1-7, 10), whereas two compounds exhibited an unusual 17,22-seco-backbone of oleanolic acid (8, 9). All compounds were evaluated for their in vitro cytotoxicities against human urinary bladder carcinoma cells (5637). Only compounds with an angeloyl-residue at C-22 of the aglycone (1-4 and 10) showed antiproliferative effects with IC50 values of 4.1, 5.2, 2.1, 17.9, and 2.4 µM, respectively.

Synthesis and antimicrobial activity of 4-hydroxy-4-(pyridyl)alk-3-en-2-ones.

4-Hydroxy-4-(pyridyl)alk-3-en-2-ones were prepared by base-mediated condensation of ketones with pyridinecarboxylates. Several derivatives show weak antimicrobial activity against Gram-positive and Gram-negative bacteria.

Novel beta-lactam antibiotics synthesized by amination of catechols using fungal laccase.

Novel cephalosporins, penicillins, and carbacephems were synthesized by amination of catechols with amino-beta-lactams like cefadroxil, amoxicillin, ampicillin and the structurally related carbacephem loracarbef using laccase from Trametes sp. All isolated monoaminated products inhibited the growth of several Gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant Enterococci. Observed differences in the cytotoxicity and in vivo activity in a "Staphylococcus-infected, immune suppressed mouse" model are discussed.

Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen.

Ninety crude extracts, including dichloromethane, methanol and aqueous extracts from 30 medicinal plants used in the Yemeni ethnomedicine to treat common infections, were screened in vitro for antimicrobial activities against three Gram-positive bacteria and two Gram-negative bacteria, Candida maltosa and five opportunistic human fungal pathogens (two yeasts, three hyphomycetes). Most of the plants showed antibacterial activities. Extracts from Tamarindus indica flowers and Ficus vasta fruits have been the most active. Of the 30 plants tested, 13 showed antifungal activity (40%) against one ore more human pathogenic fungi. The strongest inhibition was exhibited by Azima tetracantha (fruits), Sansevieria ehrenbergii (fruits) and Solanum incanum (fruits). Ten methanol extracts, especially those of Acacia asak barks and Solanum nigrum fruits, showed effective free radical scavenging activities in the DPPH assay. Remarkable cytotoxic activity against FL-cells was shown only for five plants, among them Plicosepalus curviflorus (stems).

Interventional radiology in Carney triad.

Interventional therapeutic methods are presented in Carney triad, which is a syndrome defined as the simultaneous presence of gastric leiomyosarcoma, extra-adrenal paraganglioma, and pulmonary chondroma. The paragangliomas in the carotid bifurcation and the mediastinum were successfully treated via transarterial embolization with particles. Three intrapulmonary chondromas were ablated using MRI-guided laser-induced thermotherapy (LITT) after previous devascularization via transvenous pulmonary particle embolization. In summary, interventional techniques could be a therapeutic option in patients suffering from Carney triad.