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Seventeen bacterial strains able to suppress plant pathogens have been isolated from healthy Vietnamese crop plants and taxonomically assigned as members of the Bacillus cereus group. In order to prove their potential as biocontrol agents, we perform a comprehensive analysis that included the whole-genome sequencing of selected strains and the mining for genes and gene clusters involved in the synthesis of endo- and exotoxins and secondary metabolites, such as antimicrobial peptides (AMPs). Kurstakin, thumolycin, and other AMPs were detected and characterized by different mass spectrometric methods, such as MALDI-TOF-MS and LIFT-MALDI-TOF/TOF fragment analysis. Based on their whole-genome sequences, the plant-associated isolates were assigned to the following species and subspecies: B. cereus subsp. cereus (6), B. cereus subsp. bombysepticus (5), Bacillus tropicus (2), and Bacillus pacificus. These three isolates represent novel genomospecies. Genes encoding entomopathogenic crystal and vegetative proteins were detected in B. cereus subsp. bombysepticus TK1. The in vitro assays revealed that many plant-associated isolates enhanced plant growth and suppressed plant pathogens. Our findings indicate that the plant-associated representatives of the B. cereus group are a rich source of putative antimicrobial compounds with potential in sustainable agriculture. However, the presence of virulence genes might restrict their application as biologicals in agriculture.
In den Weltmeeren findet rund die Hälfte der jährlichen globalen Kohlenstofffixierung statt, davon ein großer Anteil in küstennahen Regionen. Hier kommt es zu wiederkehrenden saisonalen Algenblüten, die durch eine zeitlich begrenzte explosionsartige Vermehrung von Mikroalgen (hauptsächlich Diatomeen und Coccolithophoren) charakterisiert sind. Vor allem Frühjahrsblüten (März-Mai) haben aufgrund ihrer zeitlichen und räumlichen Vorhersagbarkeit einen hohen Stellenwert als Modellsysteme, anhand deren sich der Kohlenstoffkreislauf der Meere untersuchen lässt.
Mikroalgen produzieren eine große Vielfalt an Makromolekülen, die für die mit ihnen vergesellschafteten Bakterien als Nahrungsgrundlage dienen. Besonders im Fokus stehen hier die für den Kohlenstoffkreislauf relevanten Polysaccharide. Im Gegensatz zu anderen natürlichen Makromolekülen wie DNA oder Proteinen können Polysaccharide aus vielen verschiedenen Monomeren mit unterschiedlichsten Bindungen bestehen. Zusätzlich finden sich an diesen Zuckermonomeren viele Modifikationen wie Acetylierungen, Methylierungen oder Sulfatierungen, die die Komplexität weiter erhöhen. Diese Variabilität bedingt eine hohe strukturelle und funktionale Diversität. So können Polysaccharide Speicherstoffe, Zellwandbestandteile oder Teile der extrazellulären Matrix darstellen.
Komplementär hierzu besitzen Polysaccharid-verwertende Bakterien entsprechend komplexe, enzymatische Abbaumechanismen. Besonders hervorzuheben sind hier die Bakterien des Phylums Bacteroidota, die sich in verschiedensten Nischen auf den Abbau von Polysacchariden spezialisiert haben. Sie finden sich in Bodenproben, als Teil der menschlichen Darmflora, oder eben auch als bedeutende Begleiter von Algenblüten.
Bacteroidota (und in marinen Systemen hauptsächlich die zu ihnen gehörenden Flavobakterien) besitzen zum Abbau diverser Polysaccharide sogenannte Polysaccharide utilization loci (PULs), genomische Inseln, die alle notwendigen Proteine zur Aufnahme und Abbau eines bestimmten Polysaccharids codieren. Hierzu gehören hochspezifische Enzyme (Carbohydrate-active enzymes, CAZymes), transkriptionelle Regulatoren sowie Transportersysteme, die initial gespaltene Oligosaccharide über die Membran in das Bakterium transportieren, wo sie von weiteren Enzymen vollständig abgebaut werden. Diese Co-Lokalisation der benötigten Gene und deren gemeinsame Regulation stellt einen enormen Selektionsvorteil der Bacteroidota dar und ist der Grund, warum sie, ähnlich wie Algen, einer jährlich wiederkehrenden Sukzession folgen, die sich gut untersuchen lässt.Die Forschungsartikel, die Teil dieser Doktorarbeit sind, untersuchen das Zusammenspiel von Polysaccharid-produzierenden Algen mit den Bakterien, die sie abbauen, aber auch darauf basierende Beziehungen der Bakterien untereinander. Die erste Publikation beschäftigt sich mit dem weit verbreiteten Speicherpolysaccharid α-Glucan, für das der Großteil der blütenbegleitenden Bakterien einen spezifischen aktiven PUL besitzt. Eine Untersuchung der in der Blüte vorhandenen Algenarten bestätigte, dass die Blüte von β-Glucan-produzierenden Algen dominiert wird. Da Bakterien aber selbst α-Glucane als Speicherpolysaccharide verwenden, konnte gezeigt werden, dass nicht die Algen selbst, sondern die Bakterien Hauptproduzent dieser Polysaccharide während einer Phytoplanktonblüte sind. Bakterielle Proteine, die dem Abbau von Algen-β-Glucan und dem daraus folgenden Aufbau von bakteriellem α-Glucan dienen, waren in Umweltproben und in Laborkulturen unter ähnlichen Bedingungen abundant. Die Untersuchung von extrahiertem bakteriellem Polysaccharid bewies, dass dieses nicht nur α-Glucan enthält, sondern dass dieses Polysaccharid auch in der Lage war, α-Glucan PULs mariner Bakterien zu induzieren. Hier zeigte sich ein innerhalb des marinen Kohlenstoffkreislaufs bisher wenig berücksichtigter Kreislauf, indem Bakterien Polysaccharide anderer Bakterien nutzen, die z.B. durch Viren lysiert wurden.
Die anderen zwei Artikel dieser Arbeit befassen sich mit dem Abbau von Zellwandpolysacchariden durch blütenassoziierte Modellbakterien. In einer der Studien wird detailliert der Abbau eines β-Mannans (ein Polysaccharid das hauptsächlich aus dem Monosaccharid Mannose besteht) durch ein Bakterium des Genus Muricauda beschrieben. Die PUL-Struktur dieses Bakteriums kam in mehreren anderen Phytoplanktonblüten-assoziierten Bakterien vor. Diese Beobachtung wies darauf hin, dass es sich hier um ein Mannan mit zusätzlichen Galactose- und Glucose-Substitutionen handelte. Proteom-Untersuchungen bestätigten, dass das Bakterium derartige Substrate unter Induktion des β-Mannan-PULs nutzen können. β-Mannan konnte durch Antikörpermarkierung in Blütenproben sowie spezifischen Mikroalgenarten (Chaetoceros, Coscinodiscus) nachgewiesen werden. Die in dieser Publikation charakterisieren β-Mannan-PUL-codierten Enzyme waren in der Lage, dieses Signal zu löschen, was bewies, dass Muricauda sp. Mannan-basierte Zellwandpolysaccharide bestimmter Arten von Mikroalgen abbauen kann.
Die dritte Studie geht näher auf den Abbau von Xylanen (bestehend aus Xylose) durch ein blütenassoziiertes Bakterium des Genus Flavimarina ein. In diesem Bakterium wurden anhand der enthaltenen Xylanasen zwei putative Xylan-PULs annotiert. Wachstumsexperimente und Proteom-Untersuchungen zeigten, dass einer dieser PULs hauptsächlich bei Wachstum auf Glucoronoxylan induziert wird, während der andere PUL aufArabinoxylane stärker reagierte. Untersuchung der PUL-CAZymes bestätigte diese Ergebnisse durch Charakterisierung mehrerer Xylanasen sowie Glucoronidasen und Arabinofuranosidasen. Zusätzlich codierten beide PULs für Esterasen, die eine Modifikation der natürlichen Substrate durch Acetylierungen oder Methylierungen nahelegen. Da all diese Merkmale von terrestrischen Xylanen geteilt werden und in Blütenproben aus Küstennahen Regionen Xylane nachgewiesen wurden, ist es möglich, dass Bakterien aus solchen Regionen sowohl Xylane terrestrischen Ursprungs (z.B. durch Flusseinspeisung) sowie marinen Ursprungs abbauen können.
For the characterization of Kv7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for Kv7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of Kv7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z’-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.
Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.
Flupirtine and retigabine were essential drugs to combat pain and epilepsy. However, the Kv7 potassium channel openers are fraught with hepatotoxicity and tissue discoloration, respectively, limiting their therapeutic value. Both adverse events are likely due to reactive metabolites arising from oxidative metabolism. Designing safer analogues lacking the structural elements leading to described side effects is an active area of current research. One of the main metabolites of flupirtine is the biologically inactive 4-fluorohippuric acid. Hitherto unexplained, the proposed metabolic pathway leading to the formation of 4-fluorohippuric acid from flupirtine is verified here. Through the use of eighteen flupirtine analogues, mechanistic details of this pathway could be elucidated. A possible connection with the in vitro hepatotoxicity of the flupirtine analogues and the levels of 4-fluorobenzoic acid formed in enzyme incubations was examined by correlation analysis. These findings provide important information for the design of new flupirtine analogues as potential drug candidates.
Overexpression of polo-like kinase 1 (PLK1) has been found in many different types of cancers. With its essential role in cell proliferation, PLK1 has been determined to be a broad-spectrum anti-cancer target. In this study, 3D-QSAR, molecular docking, and molecular dynamics (MD) simulations were applied on a series of novel pteridinone derivatives as PLK1 inhibitors to discover anti-cancer drug candidates. In this work, three models—CoMFA (Q² = 0.67, R² = 0.992), CoMSIA/SHE (Q² = 0.69, R² = 0.974), and CoMSIA/SEAH (Q² = 0.66, R² = 0.975)—of pteridinone derivatives were established. The three models that were established gave R²(pred) = 0.683, R²(pred) = 0.758, and R²(pred) = 0.767, respectively. Thus, the predictive abilities of the three proposed models were successfully evaluated. The relations between the different champs and activities were well-demonstrated by the contour chart of the CoMFA and CoMSIA/SEAH models. The results of molecular docking indicated that residues R136, R57, Y133, L69, L82, and Y139 were the active sites of the PLK1 protein (PDB code: 2RKU), in which the more active ligands can inhibit the enzyme of PLK1. The results of the molecular dynamic MD simulation diagram were obtained to reinforce the previous molecular docking results, which showed that both inhibitors remained stable in the active sites of the PLK1 protein (PDB code: 2RKU) for 50 ns. Finally, a check of the ADME-Tox properties of the two most active molecules showed that molecular N° 28 could represent a good drug candidate for the therapy of prostate cancer diseases.
The absorption of drugs with narrow absorption windows in the upper small intestine can be improved with a mucoadhesive drug delivery system such as enteric films. To predict the mucoadhesive behaviour in vivo, suitable in vitro or ex vivo methods can be performed. In this study, the influence of tissue storage and sampling site on the mucoadhesion of polyvinyl alcohol film to human small intestinal mucosa was investigated. Tissue from twelve human subjects was used to determine adhesion using a tensile strength method. Thawing of tissue frozen at −20 °C resulted in a significantly higher work of adhesion (p = 0.0005) when a low contact force was applied for one minute, whereas the maximum detachment force was not affected. When the contact force and time were increased, no differences were found for thawed tissue compared to fresh tissue. No change in adhesion was observed depending on the sampling location. Initial results from a comparison of adhesion to porcine and human mucosa suggest that the tissues are equivalent.
Humans consume snail flesh as part of their diet. To assess its nutritional value and toxicity, chemical analyses were conducted to confirm the presence of protein, total and reduced carbohydrates, fat, fatty acid composition and mineral components. Furthermore, an acute toxicity study was carried out to determine the safety of Helix aspersa Müller snail flesh. H. aspersa Müller snail flesh exhibits a high nutritional content, a good ω3/ω6 ratio and higher levels of unsaturated fatty acids. Various minerals have been found in the flesh of H. aspersa Müller. Around 76.91 kcal, or 3.84% of the energy of a daily meal of 2000 kcal, are present in 100 g of this flesh. The evaluation of the antioxidant capacity indicated that the flesh’s extracts contained a large quantity of antioxidant biomolecules. Administration of the aqueous extract of H. aspersa Müller flesh didn’t cause death in laboratory rats, indicating that the lethal dose 50 is greater than 2000 mg·kg−1 body weight. The consumption of the flesh of H. aspersa Müller is highly recommended for human consumption due to its high concentration of nutrients and essential elements, as well as unsaturated fats, and due to its safety.
The microbiome of the colon is characterized by its great diversity. This varies not only intra- but also interindividually and is influenced by endogenous and exogenous factors, such as dietary and lifestyle factors. The aim of this work was to investigate the extent to which the degradation of the drug sulfasalazine is influenced by different microbiota. Therefore, the in vitro model MimiCol3 was used, which represents the physiological conditions of the ascending colon. In addition to a representative physiological volume, the pH value, redox potential and an anaerobic atmosphere are important to provide the bacteria with the best possible growth conditions. Stool samples were taken from three healthy subjects, comparing omnivorous, vegetarian and meat-rich diets, and cultured for 24 h. However, the nutrient medium used for cultivation led to the alignment of the bacterial composition of the microbiota. The previously observed differences between the diets could not be maintained. Nevertheless, the similar degradation of sulfasalazine was observed in all microbiota studied in MimiCol3. This makes MimiCol3 a suitable in vitro model for metabolism studies in the gut microbiome.
Development of Test Programs for the Biorelevant Characterization of Esophageal-Applied Dosage Forms
(2023)
In the local treatment of the esophageal mucosa, the retention time of the different dosage forms, such as tablets, films or liquids, is of high relevance for the effective treatment of diseases. Unfortunately, there are only few in vitro models describing the esophageal route of administration. To predict the behaviour of an esophageal-applied dosage form, it is necessary to simulate the site of application in a biorelevant way. The aim of this work was to develop two test setups for an esophageal peristalsis model which was described in a previous study. Different parameters such as flow rate, peristalsis, angle of inclination or mucous membrane were varied or introduced into the model. A stimulated and unstimulated modus were developed and tested with two different dosage forms. The time until the dosage form was cleared from the in vitro model was shorter with the stimulated than with the unstimulated modus. Also, esophageal-applied films had a prolonged transit time compared to a viscous syrup. The modification of the simulated esophageal surface made it possible to estimate the retention time of the dosage forms. It could be demonstrated that the residence time of a dosage form depends on different parameters affecting each other.