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The biodiversity of marine microorganisms opens a promising potential for the discovery of new technical enzymes. During this study a characterization of marine microorganisms, isolated from Arctic or Antarctic ice, sea water or sediment from the ocean was performed based on a comprehensive strain collection at the Alfred-Wegener-Institut für Polar- und Meeresforschung. These marine psychrophilic bacteria indicated a wide spectrum of extracellular cold-active enzymes. 16S rRNA sequencing revealed that many of these psychrophilic bacteria represent new species. Characterization of selected isolates by means of transmission electron or raster electron microscopy showed remarkably pleomorphic cellular structures throughout their growth. The major part of this thesis focuses on a marine Antarctic, psychrophilic bacterium (strain ANT/505) isolated from sea ice covered surface water from the Southern Ocean, which was identified to express a very uncommon enzymatic activity for the marine environment, namely a pectinolytic activity. The sequencing of the 16S rRNA of isolate ANT/505 and biochemical tests indicated a taxonomical affiliation to the specie Pseudoalteromonas haloplanktis. The supernatant of this bacterial isolate showed after growth on citrus pectin three different pectinolytic activities. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames of 1671 and 1968 nt corresponding to proteins of 68 and 75 kDa. The deduced amino acid sequence of the two orfs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids length that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at a temperature of 30°C and a pH range of 9-10. The Km values of both lyases for pectate and citrus pectin were 1 g⋅l-1 and 5 g⋅l-1, respectively. Calcium was required for activity on pectic substrates, while the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two cold-adapted enzymes represent the first pectate lyases isolated and characterized from a marine bacterium. Further cloning and sequence analyses revealed that PelA from P. haloplanktis is an exceptionally big bifunctional enzyme featuring pectate lyase and pectin methylesterase activity. The deduced amino acid sequence of the pectin methylesterase domain showed homology to group I pectin methylesterases from Erwinia chrysanthemi and Erwinia carotovora. The pectin methylesterase domain of PelA was found to show highest homology to a potential pectin methylesterase from Saccharophagus degradans strain MD2-40. Maximum pectin methylesterase activity of PelA was detected at a pH of 7.5 and a maximum temperature of 30°C. This cold-adapted enzyme revealed high remaining pectin methylesterase activity at low temperatures around 5°C and was quickly unstabilized at temperatures above 45°C. The analysis of the localization of the two pectinolytic genes on the genome of P. haloplanktis ANT/505 revelaed that these pectinase genes are expressed from independent cistrons, which are not clustered but located at distant positions on chromosome I of the P. haloplanktis genome. It was found that the transcription of both pectinase genes is induced by the presence of pectin. By means of primer extension the promoter regions of both cistrons were detected.
Synthesis and evaluation of pseudosaccharin amine derivatives as potential elastase inhibitions
(2006)
Elastase is a serine protease which by definition is able to solubilize elastin by hydrolytic cleavage.Human Leukocyte Elastase, HLE (EC 3.4.21.37), is involved in deseases such as adult respiatory distress syndrome, pulmonary emphysema, smoking related chronic bronchitits, ischemic-reperfusion injury and rheumatoid arthritis. Hence, the elastase inhibitors have clinical utility in these diseases. Heterocyclic compounds are one of the most important classes of the elastase inhibitiors. In the present work different pseudosaccharin amine derivatives were synthesized and tested against the elastase. The synthesis of pseudosaccharin amine dervatives was carried out from the amines and(1,1-dioxobenzo[d]isothiazol-3-ylsulfanyl)acetonitrile in different solvents. Futhermore, the pseudosaccharin amines were obtained by refluxing the thiosaccarinates in absolute acetic acid. The reaction of 3-ethoxybenzo[d]isothiazole 1,1-dioxide with different amines in dioxane under reflux resulted into the desired pseudosaccharin amine derivatives in higher yields. Pseudosaccharin chloride was also used in the synthesis of these derivatives.A detail study of the synthesis of pseudosaccharin amine dervatives from the above differnt routes is described. Peptides were also synthesized by using the mixed anhydride method. The ester, acid, amide and peptide derivatives were tested against the Porcine Pancreatic Elastase (PPE) and Human Leukocyte Elastase (HLE). The esters were found to be the reversible inhibitors of HLE. The process of the PPE inhibion by cyanomethyl(2S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylbutanoate was studied. Michaelis-Menten curve and Lineweaver-Burk double reciprocal plot were constructed in order to study the kinetic of this reaction. The compounds showing high inhibition of HLE were further stuied for determination of their inhibitory constant(Ki). The esters were found to be the higly active compounds against HLE. The cyanomethyl(2S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylbutanoate and cyanomethyl(2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylpentanoate showed the competitive reversible inhibition of HLE.The cyanomethyl(2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylpentanoate is highly potent inhibitor of HLE. The possible mechanism of inhibition of elastase by these compounds is discussed. Molecular modelling of some of the ester derivatives is also discussed.
With the development of new functional genomics methods that can access the whole genome, transcriptome, proteome and metabolome more comprehensive insights in cellular processes are possible. Largely based on these advances, our knowledge about molecular constituents for many organisms is increasing at a tremendous rate. Until today, the genomes of several organisms including pathogenic bacteria are already sequenced and pave the way for metabolic network constructions. Interest in metabolomics, the global profiling of metabolites in a cell, tissue or organism, has been rapidly increased. A range of analytical techniques, including nuclear magnetic resonance (NMR) spectroscopy, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS), Fourier Transform mass spectrometry (FT–MS), high performance liquid chromatography (HPLC) are required in order to maximize the number of metabolites that can be identified in a matrix. With the help of microbial metabolomics (qualification and quantification of a huge variety of metabolites from a bacterium) deciphering of the bacterial metabolism is feasible. The metabolome pipeline or workflow encompasses the processes of (i) sample generation and preparation, (ii) establishment of analytical techniques (iii) collection of analytical data, raw data pre-processing, (iv) data analysis and (v) data integration into biological questions. The present work contributes to the above mentioned steps in a metabolomics workflow. A specific focus was set to the exo- and endometabolome analysis of Gram-positive bacteria
Summary Cyanobacteria are a diverse and ancient group of photosynthetic prokaryotic organisms that can inhabit a wide range of environments including extreme conditions such as hot springs, desert soils and the Antarctic. They are abundant producers of natural products well recognized for their bioactivity and utility in drug discovery and biotechnology applications. Novel intracellular and extracellular compounds from various cultured and field cyanobacteria with diverse biological activities and a wide range of chemical classes have considerable potential for development of pharmaceuticals and other biomedical applications. However, cyanobacteria are still viewed as unexplored source of potential drugs. Especially the collections of cyanobacterial strains from South East Asia where biodiversity is high are still largely unexplored. Thus, we investigated twelve soil cyanobacterial strains isolated from soil samples collected from rice, cotton, and coffee fields in Dak Lak province of Vietnam and one marine strain, Lyngbya majuscula collected from Khanh Hoa province of Vietnam for the search for new compounds with antimicrobial and cytotoxic activities. From the 12 soil cyanobacterial strains, 48 extracts prepared with n-hexane, methanol, and water for biomasses and ethyl acetate for growth media were screened for antibacterial activity against Gram-positive bacteria (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538) and Gram-negative bacteria (Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 27853). Of 48 extracts, 47.92% and 45.83% showed activity against Bacillus subtilis and Staphylococcus aureus, respectively, while 22.92% and 6.25% exhibited activity against Escherichia coli and Pseudomonas aeruginosa, respectively. All investigated cyanobacteria (12/12) showed antibacterial activity to at least one of the test organisms applied. Among the active extracts, extracts obtained from 5 cyanobacterial strains, Westiellopsis sp. VN, Calothrix javanica, Scytonema ocellatum, Anabaena sp. and Nostoc sp. showed the highest strength and range of antibacterial activity and therefore were selected for chemical investigation with an emphasis on the isolation and structure elucidation of antimicrobial compounds. Bioassay-guided fractionation of the methanol extract prepared from biomass of Westiellopsis sp. VN by silica gel chromatography, followed by sephadex LH-20 chromatography and reversed-phase HPLC led to isolation and identification of 6 compounds as ambiguine D isonitrile, ambiguine B isonitrile, dechloro-ambiguine B isonitrile, fischerellin A, hydroxy-eicosatetraenoic acid and methoxy-nonadecadienoic acid. Identification of these active compounds was established by direct comparison of our spectroscopic data, including 1H NMR and HR-ESI-MS with those reported in the literature. All these compounds showed biological activity. The identification of fatty acids and other volatile components by GS-MS in the active MeOH fraction obtained from EtOAc extract of growth medium was done before commencing further fractionation processes. Culture optimization of Westiellopsis sp.VN showed that NaNO3 deficiency increased accumulation of antimicrobial compounds. Biosynthesis of antimicrobial compounds increased over cultivation time resulting in increased diameter of inhibition zone of the methanol extract towards the end of the 7-to 8- week growth period, but the most clear inhibition zone of this extract was detected after cultivation time of 8 weeks. Bioassay-guided fractionation of the methanol extract prepared from biomass of either Calothrix javanica by C18 chromatography followed by reversed-phase HPLC or Scytonema ocellatum by C18 chromatography followed by silica gel chromatography and reversed-phase HPLC led to isolation and structure elucidation of new cyclic peptide named daklakapeptin. Structure of daklakapeptin was elucidated by exhaustive 1D (1H) and 2D (COSY, TOCSY, NOESY, HMQC, HMBC) NMR spectroscopy in combination with HR-ESI-MS. Daklakapeptin was found to have totally 12 residues including 6 proteinogenic amino acids (Pro, Tyr, Ile, Leu, Gln, Thr), 4 complexes (X,Y,T,Z) and the methyl derivative of Ile. The exact sequence of daklakapeptin is shown in following figure with X: (CH3)2CHCH2CH2CH(NH-)CH2CO-, Y:(CH3)2CHCH(OH)CH(NH-)CO-, T: HOCH2CH2CH(NH-)CO-, Z: HOCH2CHOHCH(NH-)CO- This new cyclic peptide exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 12.5 mm in concentration of 200 mg/disc. Further test for activity to other bacteria and for cytotoxic activity are in progress. Using reversed-phase HPLC to separate compounds in the crude ethyl acetate extract obtained from culture medium of Anabaena sp. led to isolation and structure elucidation of flourensadiol. The structure of flourensadiol was established using an extensive array of 1D (1H, 13C, DEPT-135) and 2D (HMQC, COSY, HMBC) NMR and HR-ESI-MS experiments. Flourensadiol was isolated previously from the common western shrub Flourensia cernua. However, only MS, IR, and proton NMR data but no reports on biological activity were available. In this study, we report the complete NMR data of flourensadiol for the first time. Flourensadiol was found to be very strong antibacterial active against Escherichia coli with diameter of inhibition zone of 20.0 mm in concentration of 200 mg/disc. Further test for activity to other bacteria and cytotoxic activity are in progress. Bioassay-guided fractionation of the methanol extract from biomass of Nostoc sp. by silica gel chromatography followed by C18 chromatography and reversed phase HPLC led to isolation of the active fraction NsF2 which exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 10.0 mm in concentration of 500 mg/disc. The low resolution ESI-MS of fraction NsF2 showed signal at m/z 426 [M+H]+. The NMR and MS characterization of compounds in fraction NsF2 is in progress. Bioassay-guided fractionation of the methanol extract prepared from biomass of marine cyanobacterium Lyngbya majuscula collected from Khanh Hoa province of Vietnam by various chromatographic methods (CC, PTLC, HPLC) afforded 3 cytotoxic compounds anhydrodebromoaplysiatoxin, debromoaplysiatoxin, and anhydroaplysiatoxin. Identification of these cytotoxic compounds was established by direct comparison of our spectroscopic data, including (1H, 13C) NMR and HR-ESI-MS with those reported in the literature. In our study, debromoaplysiatoxin and anhydroaplysiatoxin exhibited cytotoxic activity against bladder cancer cell line 5637 with IC50 of 86 ng/ml and 40 ng/ml, respectively but anhydrodebromoaplysiatoxin was not yet tested for cytotoxic activity. The identification of fatty acids by GS-MS technique in the n-hexane extract obtained from biomass of this marine cyanobacterium was undertaken before commencing further fractionation processes. The presented results prove that soil cyanobacteria are a promising source to yield chemical and pharmaceutical interesting compounds.
Bacillus licheniformis is one of the most important hosts used in the biotechnological industry for the production of technical enzymes, antibiotics and a number of biochemicals. Although this bacterium has been used for a long time as an expression host, only little information on expression systems of this host is available. An expression system could be controlled by a cell density signal, a specific chemical inducer or a thermal shift. A limiting substrate such as glucose or phosphate limitation is suggested to use as the signal for the induction of an expression system. When B. licheniformis cells are subjected to nutrient limitation conditions, numerous genes involved in the metabolism of alternative nutrient sources are induced in order to keep cell survival. Therefore, the main topic of this study was to identify and investigate the regulation of genes or operons which are strongly induced in B. licheniformis cells grown under nutrient limitation conditions in order to apply for the construction of potential new expression systems. The research includes studies on the regulation of genes which are responsible for the acetoin and 2,3-butanediol utilization in B. licheniformis cells grown under glucose limitation conditions. Furthermore, we also analyzed the regulation of phytase gene expression as well as investigated the function of a putative ribonuclease expressed in B. licheniformis under phosphate limitation conditions. From this study, it was shown that in B. licheniformis, the utilization of acetoin and 2,3-butanediol was mainly mediated by enzymes encoded by the acoABCL operon. The transcription of this operon was regulated by sigma L transcription factor and was induced by acetoin. The acuABC operon was suggested to play as an indirect regulatory role for the acetoin utilization in B. licheniformis. This operon was controlled by a typical sigma A dependent promoter, however, acetoin was not an inducer for its expression. Furthermore, the regulation of phytase gene expression was suggested to be controlled by PhoPR-two component systems. The results showed that phytate, which is the substrate of phytase enzyme, was not an inducer for the expression of phy gene. However, growth experiments revealed that phytate served as a good alternative phosphate source for the growth of B. licheniformis cells under these conditions. Finally, the inactivation of BLi03719 gene, coding for a putative ribonuclease, resulted in an increase of the total RNA concentration of B. licheniformis cells grown in phosphate limited medium. However, the mutation did not affect the expression of the heterologous reporter gene. Therefore, it could be speculated that the putative ribonuclease BLi03719 plays a role in ribosomal RNA degradation under these conditions.
In the search for bioactive compounds, 32 fungal strains were isolated from Indonesian marine habitats. Ethyl acetate extracts of their culture broth were tested for cytotoxic activity against a urinary bladder carcinoma cell line and for antifungal and antibacterial activities against fish and human pathogenic bacteria as well as against plant and human pathogenic fungi. Bioassay-guided fractionation led to the isolation of bioactive compounds. Altogether 14 compounds were isolated and further elucidated. The compounds were obtained from the ethyl acetate and dichloromethane extracts of six fungal strains. They included 9 polyketides, 2 terpenes, 1 alkaloid and 2 till now undefined structures.
Transition metal complexes play a crucial role in antitumor therapy. Complexes of platinum, ruthenium as well as lanthanum and gallium have been investigated in preclinical as well as in clinical studies. The best known platinum(II) agents approved worldwide, cisplatin or carboplatin, are used in nearly 50% of all cancer therapies. This work focused on the development of new metal-based drugs that could act against human cancer cells. It was motivated in part by previous work with Cu(II) complexes, reporting new coordination compounds of SOD mimicking and cytotoxic activities. On the basis of this work we chose several commercially available heterocyclic ligands to synthesize new metal ion complexes in search of their interesting biological activity. New as well as previously reported Cu(II), Co(II), Pt(II) and Zn(II) complexes were synthesized using various ligands (1-6). Almost all chelating 2:1 ligand-metal complexes were obtained generally in water at room temperature in the reaction of metal(II) chloride with corresponding aromatic nitrogen ligands bearing an O-carboxylate group ligand. The synthesized chelating complexes were characterized by the use of spectroscopic methods, elemental analyses and HPLC chromatography and some by X-ray crystallography. Such coordination compounds are easily formed by transition metals with free orbitals d that can accept the donor electron pairs. The coordination is through the heterocyclic nitrogen and carboxylate oxygen donor atoms, which was shown by analysis of the characteristic functional groups in the IR spectra. The d-d transitions and absorption of visible light in Cu(II) and Co(II) complexes make them highly colored, blue, green or green-blue, respectively. The configuration of the coordination center was established in some cases by X-ray crystallography. Most of the already published structures possess the trans configuration. This led to the assumption that other uncrystallized complexes were also trans configured. However, X-ray data of the Cu(II) complex of 5 showed quite unexpectedly the cis configuration. On the other hand, the LC/MS experiments with the Pt(II) complex of 5 indicated that this complex exists in two isomeric forms, i.e., cis and trans at the Pt(II) center. Through the use of density functional calculations we optimized the structures and calculated the energies and dipole moments. The differences in energy for all complexes were about 6 to 15-fold lower when compared to cis and transplatin. The DFT calculations confirmed that the trans-isomers are more stable than their cis-isomers. UV-Vis stability studies with most of the synthesized complexes as well as some other Cu(II) complexes were performed to study the spectral changes over 24 h in addition of glutathione, a tripeptide present in the cancer cells and ascorbate that were added to the incubations. The results indicated time-dependent changes and instability of the complexes in the cells and their possible decomposition to lose the ligand and release the metal ion. In the case of Cu(II) complexes, reduction of Cu(II) to Cu(I) may take place. New species such as GSSG could arise and the complexes may decarboxylate, but these structures were not elucidated. The synthesized coordination metal(II) complexes were tested for their potential antiproliferative activities by using the crystal violet staining method in a panel of human cancer cell lines. Out of all complexes, three Pt(II) complexes of 2, 5 and 6 showed satisfactory activity and for these complexes the IC50 values were additionally determined in new RT-4, DAN-G and MCF-7 cancer cell lines. Interestingly, the active complexes were the chelating trans complexes which is quite unexpected, based on the difference in activities between cis and transplatin. All of the complexes were tested for their potential antimicrobial activities in comparison to the standard antibiotics on such bacterial strains as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and yeast Candida maltosa. Co(II) complexes have been especially known to act against bacterial strains. The activity of the Co(II) complexes was indeed the highest of all metal(II) complexes. The ligand 2 (a nicotinic acid isomer) was also found active. This fact could explain why some antibacterial activity was found in the MIC assay. In addition to the complexes synthesized in this work, several novel heterocyclic metal(II) complexes of copper, ruthenium, platinum, gallium, osmium and lanthanum from other research groups were screened for their antiproliferative activity, some of which exhibited very potent activity in the cancer cell lines. In conclusion, Pt(II) complexes with bis-chelating heterocyclic carboxylate ligands represent a particularly interesting new class of compounds from the view point of their structural and biological properties.
Acute pancreatitis is a common clinical inflammatory disease with variable severity from mild, self-limiting attacks to a severe lethal attack with a high mortality. In most of the cases, acute pancreatitis is either caused by gallstone obstruction or excessive alcohol consumption. Clinical symptoms include elevated levels (minimum 3 times than normal) of pancreatic enzymes such as amylase or lipase in serum. It is generally believed that earliest event in acute pancreatitis occur in acinar cells which includes premature protease activation and cytoplasmic vacuole formation. Premature trypsinogen activation has been considered as chief culprit as it can activate other proteases in a cascade like manner in acinar cells. Trypsin activity takes place in a biphasic curve with elevated levels at 1 h and 8 h in the initial stages up to 24 h in caerulein induced pancreatitis in mice. It has been shown that cytoplasmic vacuoles observed in pancreatitis are of autophagic nature. The role of autophagy for the disease onset and its role in trypsinogen is much of a debate. Hence, we studied the relation between autophagosome formation and trypsinogen activation in first 12h of pancreatitis. Although autophagosomes were found to be co-localised with trypsin in vivo, this was found to be a late event occuring only by 4 h. Substrate specific trypsin activity and western blotting from both sub-cellular fractions over the time course of pancreatitis and multiple fractions prepared from 1 h caerulein induced pancreatic tissue revealed that trypsin activity observed at 1 h occured in a zymogen enriched fraction. In line simultaneous confocal imaging of trypsin activity and autophagosome formation in hyperstimulated acini isolated from GFP-LC3 mice showed that both processes are independent and take place in parallel. Furthermore, protease inhibition by gabexate mesilate did not prevent autophagosome formation indicating that trypsinogen activation is not a prerequisite for vacuole formation. Even though, autophagosomes and active trypsin were found to be co-localised around 30 minutes to some degree upon cholecystokinin hyperstimulation, the earliest trypsin activation started to appear by 15 minutes and was independent of autophagosomes. The earliest active trypsin was found to be co-localised along with the cis-Golgi complex suggesting that the Golgi apparatus and its pre-condensed zymogen granules are the compartment responsible for the trypsinogen activation. 2) Protease activation in pancreatic acinar cells considered as the early hallmark event in the acute pancreatitis. However, the disease is aggravated by the infiltration of the leukocytes. Activated proteases mediate acinar cell injury and hereby cause the release of chemokines, which in turn attract inflammatory cells. Transmigrated inflammatory cells cause systemic damage that deteriorates the condition of the disease. Neutrophil elastase has been reported to be involved in the dissociation of cell-cell contact at adherens junctions by the extracellular cleavage of E-cadherin. This subsequently leads to transmigration of leukocytes into the epithelial tissue during the initial phase of experimental pancreatitis and aggravates the disease condition. On the other hand, pancreatic elastase substantially contributes to acinar cell necrosis. In this study, ZD0892, an orally bioavailable dual inhibitor against both elastases was tested for its efficacy to ameliorate severity in acute pancreatitis. ZD0892 orally fed mice showed increased survival compared to the control group in the taurocholate model of severe pancreatitis. In the initial stages of pancreatitis up to 24 h, the severity markers were found to be significantly lower in the inhibitor treated group. Treatment of mice with ZD0892 did not impede the defensive property of the leukocytes such as phagocytosis or oxidative burst. In caerulein induced pancreatitis, a mild form of acute pancreatitis, in rats, the local damage measured as serum amylase and lipase, wet dry ratio, and pancreatic myeloperoxidase levels were significantly lower in the inhibitor group. Systemic inflammatory parameters such as myeloperoxidase activity in lung was found to be significantly lower in the inhibitor fed rats. Inhibitor feeding resulted in lesser elastolytic activity compared to control group indicating that extracellular matrix was less damaged. Prophylactic treatment of pancreatitis with an orally available inhibitor with a dual specificity against pancreatic elastase and PMN-elastase was shown to ameliorate both local and systemic damage. Hence, in overall, ZD0892 treatment is proved to be beneficial to the mice and rats in experimental pancreatitis and should be considered for treatment in humans as the substance has been already studied in phase I and II trails for other indications.
The biological decontamination and sterilization is a crucial processing step in producing and reprocessing of medical devices. Since polymer-based materials are increasingly used for the production of medical devices, the application of conventional sterilization processes are restricted to a certain extent. Conventional sterilization techniques on the basis of high temperatures, toxic gases, or ionizing radiation can be detrimental to the functionality and performance of polymeric materials. For this reason, alternative, gentle, and efficient decontamination processes are required. One possible approach is the use of non-thermal physical plasmas. Especially atmospheric pressure plasma is receiving great interest due to the absence of vacuum systems which is highly attractive for the practical applicability. Its mechanisms of action enable the efficient killing and inactivation of micro-organisms which are attributed to the interaction of plasma-generated reactive oxygen and nitrogen species (ROS, RNS) as well as plasma-emitted (V)UV radiation. Owing to the moderate gas temperatures (near or at room temperature) so-called cold plasmas are well-suitable for the treatment of heat-sensitive materials, such as polymers, without affecting their bulk properties. The present work focuses on the investigation of atmospheric pressure plasma processes for the biological decontamination of polymers. The objective is to help elucidate on the one hand the impact of varied plasma process parameters on the inactivation of micro-organisms and on the other hand the influence of plasma on the surface properties of the substrate. The investigations were performed by means of a high-frequency driven plasma jet (from the product line kINPen) operated with argon and argon-oxygen mixtures. Three main aspects were analyzed: 1. The effect of plasma on the viability of micro-organisms dependent on working gas, treatment time, and the sample distance (distance between the jet nozzle and the substrate). 2. The plasma-based removal of microbial biofilms. 3. The effects of the plasma treatment on the surface properties of selected polymers. Additionally to the capability of the applied plasma jet in killing microbes the efficacy of this plasma jet for the removal of complex biological systems (e.g. biofilms) is shown. To model cell constituents of bacteria different synthetic polymers were chosen to gain insight into the decomposition process responsible for biofilm degradation. By investigating the impact of atmospheric pressure plasma on physico-chemical surface properties of various synthetic aliphatic and aromatic polymers the interaction mechanisms between plasma and plasma-exposed material are discussed. These studies are accompanied by applying different optical plasma diagnostic techniques (optical emission spectroscopy and two-photon absorption laser induced fluorescence spectroscopy) to obtain information on the plasma gas phase which contributes to the elucidation of the reaction mechanisms occurring during plasma exposure. Moreover, it is presented to which extent the plasma treatment influences the surface properties of polymers during the plasma-based bio-decontamination process and further, the benefits of surface-functionalized polymers for biomedical application is discussed.
Bacteria are an integral part of modern biotechnology. They are used to make a variety of products, such as foods, drugs, as well as a multitude of chemicals. In order to increase their production rates molecular biotechnology offers many tuning points, starting from the selection of an applicable host, over its geno- and phenotypical characterization, followed by genetic manipulations for an optimized metabolism and stabilisation of production processes. This work comprises the optimization of Bacillus subtilis as an expression system. It describes the steps taken for selection and genomic characterization of the B. subtilis wild type strain ATCC 6051, the subsequent optimizations of the strain in respect to growth and productivity, as well as the characterization of its behaviour in a variety of cultivation conditions. The B. subtilis strain most commonly found in laboratories around the world is the first sequenced Gram-positive organism B. subtilis 168. Zeigler et al. showed that strain 168 is not a real wild type. Instead it was created through random mutagenesis with X-rays and selected for transformability. This strain has been used as the basis for popular B. subtilis strains in heterologous gene expression such as the extracellular protease deficient WB strains. Growth experiments showed the real wild type strain ATCC 6051 to be superior to its mutated ancestor 168, making it a solid basis for the construction of an optimized B. subtilis expression system. In order to gain a full understanding of the genomic and corresponding physiological differences between the two systems, B. subtilis ATCC 6051 was sequenced and compared to the genome of B. Subtilis 168. Several variations on geno- and phenotypic level could be revealed, that resulted in particular from genes involved in natural competency, the metabolism of amino acids and chemotaxis. This genomically well characterized B. subtilis ATCC 6051 was improved in respect to its application as an expression host. Improvements were achieved through the inactivation of both sporulation and reduction of autolysis, leading to a more robust behaviour during the overproduction and secretion of a reporter enzyme. A positive effect on the activity of an acetoin induced promoter by the addition of second copies for its transcription factors SigmaL and AcoR could be observed. Anaerobic zones and areas with excess glucose caused by insufficient mixing are common conditions in large scale bioprocesses and lead to oscillating conditions for the cells. In turn, this oscillation provokes an excretion of so called overflow metabolites, which can negatively affect the bacterial productivity. Detailed scientific characterizations of industrial scale processes under such oscillating conditions are scarce due to the high costs and logistics involved. A B. Subtilis sporulation mutant was thus examined in respect to its extra- and intracellular metabolites in a scale-down, two-compartment reactor giving hints about conditions the host is exposed to and how it reacts. To improve tolerance thresholds and utilization capacity for such metabolites in B. subtilis, the glyoxylate cycle was transferred from its close relative Bacillus licheniformis into the genome of B. subtilis. This feature enabled our B. subtilis ACE mutant to grow on acetate. The improved strain showed higher tolerance towards excess glucose in a fed-batch as well as higher productivity during the expression of a reporter enzyme in comparison to the wild type. The ACE strain and B. licheniformis showed an increased formation of glycolate during growth with the glyoxylate cycle. This with regard to bacteria undescribed metabolite seems to play a role as a by-product of the glyoxylate cycle. Summarizing, this thesis deals with the characterization and optimization of B. subtilis for growth on overflow metabolites, enhancements of the acoA-expression system and the influence of sporulation and lysis mutants on its activity. Complementary, the host was begun to be characterized in respect to its behaviour in industrial scale processes.
There is a growing interest in the application of non-thermal atmospheric pressure plasma for the treatment of wounds. Due to the generation of various ROS and RNS, UV radiation and electric fields plasma is a very promising tool which can stimulate skin and immune cells. However, not much is known about the mammalian cell responses after plasma treatments on a molecular level. The present work focusses on the impact of plasma on cell signaling in the human keratinocyte cell line HaCaT by using the methods DNA microarray, qPCR, ELISA and flow cytometry. Here, cell signaling mediators such as cytokines and growth factors which could promote wound healing by enhancing angiogenesis, reepithelization, migration and proliferation were of major interest. Additionally, the crosstalk between keratinocytes and monocytes was studied using a co-culture. For the first time extensive investigations on the impact of plasma on cell signaling in human keratinocytes were conducted. The most prominent cytokines and growth factors which were regulated by plasma at gene and protein level were VEGF-A, GM-CSF, HB-EGF, IL-8, and IL-6. The latter was not activated due to the JAK/STAT-pathway but probably by a combined activation of MAPK- and PI3K/Akt-pathways. By the use of conditioned medium it was found out that ROS and RNS generated directly after plasma treatment induced larger effects on cell signaling in keratinocytes than the subsequently secreted growth factors and cytokines. Furthermore, monocytes and keratinocytes hardly altered their secretion profiles in co-culture. From these results it is deduced that the plasma generated reactive species are the main actors during cell signaling. In order to differentiate the impact of ROS and RNS on the cellular response the ambience of the plasma effluent was controlled, varying the ambient gas composition from pure nitrogen to pure oxygen. Thereby a first step towards the attribution of the cellular response to specific plasma generated reactive species was achieved. While IL-6 expression correlated with ROS generated by the plasma source, the cell signaling mediators VEGF-A, GM-CSF and HB-EGF were significantly changed by RONS. Above all hydrogen peroxide was found to play a dominant role for observed cell responses. In summary, plasma activates wound healing related cell signaling mediators as cytokines and growth factors in keratinocytes. It was also shown that the generated reactive species mainly induced cell signaling. For the first time cell responses can be correlated to ROS and RONS in plasma treated cells. These results underline the potential of non-thermal atmospheric pressure plasma sources for their applications in wound treatment.
Rich knowledge about global nutrient cycles and functional interactions can be gained from the perspective of complex microbial proteomes. In this thesis, the application of environmental proteomics allowed for a direct in situ analysis of habitat-specific proteomes expressed by respective microbial communities from two different marine ecosystems. In the first part of this thesis, unculturable symbiont populations from tubeworms that colonize hydrothermal vents of the Pacific deep sea became accessible by use of community proteomics. This branch of environmental proteomics is generally employed to ascertain simple microbial assemblages derived from in situ samples. The proteome study was aimed at analyzing adaptations of seemingly monospecific symbionts to different hosts, the tubeworms Tevnia jerichonana und Riftia pachyptila. A comparison of the newly sequenced genomes of symbiont populations from both hosts confirmed that both symbioses involve the same bacterial species. Also the proteome analysis by 2D-PAGE showed a high physiological homogeneity for symbionts from both worm species, although the hosts are exposed to different geochemical conditions. Thus, the hosts provide their symbionts with a relatively stable internal environment by attenuation of external influences. Only minor variations in the symbionts proteomes reflected the differential environmental conditions outside the worms. Hence, the symbionts were able to fine-tune major metabolic pathways and oxidative stress in response to only minor chemical changes within their hosts. Moreover, new components of important physiological processes of the bacterial symbionts, like the sulfide oxidation and carbon fixation, were identified by in-depth proteomics of the Riftia symbiosis model system. The in situ protein samples showed as well that, in contrast to an earlier hypothesis, nitrate is used as an alternative electron acceptor. In the second part of this thesis, another branch of environmental proteomics called metaproteomics was applied to investigate the response of a bacterioplankton community to a spring phytoplankton bloom in the North Sea. Recurrent plankton blooms are a common phenomen of coastal areas, which however has only been investigated with limited resolution in biodiversity. Based on large-scale proteomic data sets it was found that specialized populations of Bacteroidetes, Gammaproteobacteria and Alphaproteobacteria exhibited differential protein expression patterns. These involved oligomer transporters, glycoside hydrolases and phosphate acquisition proteins. A successive utilization of algal organic matter by microbes indicated a series of ecological niches occupied by the heterotrophic picoplankton. Key proteins, identified by metaproteomics, were further investigated by studying a model bacterium to define their specificities regarding the utilization of algal glycans. By isotope labeling of proteins, quantitative proteomics of the North Sea isolate Gramella forsetii KT0803, a Bacteroidetes representative could be conducted. The adaptation to the algal polysaccharides alginate and laminarin in comparison with glucose was analyzed. G. forsetii proved to be a specialist for the chosen algal polymers, in particular for glucans like laminarin. Primarily comprehensive clusters, the so-called polysaccharide utilization loci (PULs) were activated. The results of this model study complemented the basic concepts obtained by the metaproteomic approach about carbon cycling in coastal systems. The accessibility of numerous unculturable marine microbes by environmental proteomics allows to improve our understanding of interactions that drive symbioses or complex communities. Adaptations to environmental parameters, such as the abundance of substrates, can be analyzed and associated with respective populations. Thus statements can be made for functional groups of microorganisms, their ability for the creation of niches and their flexibility to respond to varying environmental impacts. The increasing number of marine model bacteria enables targeted analysis of specificities and adaptations and hence to support the environmental proteomics approach.
In many industrial sectors biotechnological production processes have replaced pure chemical methods and allowed new, ecologically friendly and enzyme-based processes. Microorganisms, such as modified Bacillus strains are used in particular for the industrial enzyme synthesis. The two organisms Bacillus licheniformis and Bacillus pumilus are of great industrial importance. B. licheniformis is able to secrete proteins in large amounts, while B. pumilus shows high resistance to oxidative stress. During production processes different conditions can occur that affect the physiology of the production hosts and may result in a quantitative, but also a qualitative impairment of the products. This influence is based on e.g. chemical processes, the setting of temperature, pH, or oxygen availability and can lead to various stress situations for the bacteria. Cells respond to changes in their environment by sensing stressors and initiate a response to the stress, which is usually implemented by an induction or derepression of various regulons. In order to conduct an optimal production process, the metabolism and stress responses of the utilized bacteria should be known exactly. The aim of this study was to analyze of the stress response of B. licheniformis to heat and salt stress, and the stress response of B. licheniformis and B. pumilus to oxidative stress. These analyses were performed at the level of transcriptomics using cDNA microarrays, which is the most direct and global method for the analysis of changes in the physiology of a cell. The identification of stress specific markers genes and their differentiation from the SigB regulated general stress response has been another purpose of this work. Knowledge of these marker genes enables a prompt analysis of the fermentation conditions and thus a possible optimization of the process. The transcriptome analyses of this work show that B. licheniformis responds to heat stress by the induction of heat shock genes belonging to different regulons. These include the htpG gene, the HrcA regulon or the CtsR regulon, encoding chaperones and proteases, which mainly contribute to the protein quality control. The heat stress response of B. licheniformis revealed no fundamental differences to the heat stress response of the Gram-positive model organism Bacillus subtilis. The general stress response (SigB regulon), which is activated by heat stress, could be analyzed in more detail by the study of a ΔsigB mutant of B. licheniformis. Salt stress also provokes a strong induction of the general stress response in B. licheniformis. Genes for the transport and synthesis of compatible solutes were strongly induced, as well as several genes for transport systems with more or less known functions. The synthesis of the osmoprotective metabolites proline and glycine betaine could be verified in more detail by a metabolomics approach. The response to oxidative stress showed differences between both B. licheniformis and B. pumilus, and also to the oxidative stress response of B. subtilis. In B. licheniformis, the genes of the glyoxylate cycle are induced during oxidative stress. An activation of the glyoxylate bypass under oxidative conditions could be confirmed by a metabolome analysis of B. licheniformis. In addition, the PerR regulon of B. licheniformis is extended to include another two genes compared to B. subtilis. In contrast, several genes of the PerR regulon lack in the genome of B. pumilus, such as katA (vegetative catalase) or ahpCF (alkyl hydroperoxide reductase). However, other genes were induced in B. pumilus that were upregulated under oxidative stress conditions neither in B. subtilis nor in B. licheniformis. In addition, known regulons, regulated by e.g. Spx, CtsR or SOS were induced in both organisms. In summary, this dissertation transcriptionally analyzes the stress responses of B. licheniformis to heat, salt and oxidative stress, and in addition the oxidative stress response of B. pumilus. Several stress-specific regulons were identified in both, B. pumilus and B. licheniformis, which also correspond to the stress response of B. subtilis. However, it was possible to additionally assign genes to the stress specific responses of both organisms and to find differences, such as the absence of parts of the PerR regulon of B. pumilus, or the activation of the glyoxylate pathway in B. licheniformis during oxidative stress.
The investigated bacterial strain 64G3 was isolated from an offshore oil reservoir in Vung Tau, Vietnam. By means of 16S rDNA sequence alignment and DNA-DNA hybridization with Petrotoga mexicana DSM 14811, the isolate was identified as Petrotoga mexicana species. Morphologically, the 64G3 cells were rod-shaped and cell sizes varied widely from 1.0 µm up to 60 µm in length and from 0.6 to 1.2 µm in width. The cells appeared single, pairwise or in chains within a sheath-like structure (a typical characteristic of the order Thermotogales) that ballooned over the cell ends. Cells were immobile and no flagella were observed. Strain 64G3 grew anaerobically at temperatures ranging from 30 to 65°C and within the pH range of 5.0 to 8.5 with optimum growth at 55°C and the pH 7.0. Elemental sulfur and thiosulfate served as alternative electron acceptors whereas sulfate did not. Cellular extract of strain 64G3 grown in a basal medium containing soluble starch displayed hydrolytic activity towards soluble starch. The amylase system includes at least two individual enzymes. Amylase activity of the cell extract was detected in a wide temperature range (30-80°C), with optimal enzyme activity at 75°C. By using degenerate primer for PCR amplification of GH13 enzyme coding regions in combination with other molecular methods, a full amylase coding gene containing four conserved regions of α-amylase was obtained. The deduced sequence showed low identities (up to 40%) to other known amylases. This 1992 bp coding gene was heterologously expressed in E. coli and its product (amylase) was characterized. Under common expression conditions, the 77 kDa amylase (rAmyA) was predominantly produced as inclusion bodies (insoluble protein). The minor amount of soluble active amylase was used for purification and characterization of the enzyme. rAmyA was active on starch at temperatures between 30-55°C, with an optimum at 45oC. It is not thermostable because it was completely inactive after incubation at 65°C for 15 min. The enzyme was active over a pH range from 4.5-8.0, with an optimum at pH 6.5. Beside starch, rAmyA also hydrolysed glycogen, amylose, amylopectin and other oligosaccharides. Pullulan and cyclodextrins were not the substrates for this amylase. The enzyme hydrolyzed starch in an endo-acting manner, releasing maltose and maltotriose as major products and a lesser amount of glucose. On the basis of the primary structure, the substrate specificities and the hydrolysis pattern, rAmyA was classified as an endo-acting α-amylase (EC. 3.2.1.1). The cpn10/60 operon from psychrophilic O. antarctica was cloned and expressed in B. subtilis using a multi-copy plasmid. The amounts of soluble 60 kDa Cpn60 and 10 kDa Cpn10 produced at temperature ranging from 10 - 30°C were high and stable during cell growth. To investigate the impact of psychrophilic chaperonin on cold adaptation, cells with (cpn+) and without (cpn-) cpn10/60 operon were grown at 10 and 15°C. Growth comparison between two strains revealed that psychrophilic chaperonin did not support cold adaptation of B. subtilis at 10 and 15°C as it did in E. coli. A single copy of O. antarctica cpn10/60 operon was integrated into the amyE locus of the B. subtilis chromosome. The yeast α-glucosidase, a theoretic protein substrate for this chaperonin, was heterologously produced in B. subtilis at temperatures ranging from 15-30°C. Within this temperature range, the major amount of this protein appeared as inclusion bodies. Co-expression of O. antarctica cpn10/60 operon at 15°C, however, did not result in a higher activity of glucosidase. Moreover, SDS-PAGE analysis of cellular insoluble fractions revealed that the amount of insoluble enzyme produced in cpn+ cells did not decrease in comparison with that produced in cpn- cells, indicating that the recombinant chaperonin had no impact on recovery of active α-glucosidase from the inclusion bodies.
Chemistry and biology of Phenolics isolated from Myricaria germanica (L.) Desv. (Tamaricaceae)
(2014)
In accordance with the recent worldwide interest in plant phenolics, which emerges from their broad range of biological activities, particular emphasis has been focused, in the present thesis, on the constitutive phenolics of the extract of Myricaria germanica (L.) Desv. (Tamaricaceae). During the current thesis twenty phenolics (1 – 20) were isolated and identified from the aqueous/ethanol extract of the whole Myricaria germanica plant. The isolates include four hitherto unknown natural phenolics (2, 10, 12 and 20). Also, the cytotoxic activities of M. germanica extract, column fractions, and one new natural isolate against three different solid tumor cell lines, namely, breast cancer (MCF-7), prostate (PC-3), and liver (Huh-7) cancer cell using SRB viability assay have been investigated and first insights into mode of action have been obtained.
In the search for new antifungal agents, this study dealt with the antimicrobial screening, extraction, isolation, structural elucidation as well as selective biological investigations of the isolated compounds. In addition, the impact of the culture conditions on growth and on biosynthesis of bioactive compounds was also studied. Besides, selective cyanobacteria were axenized and the taxonomy as well as the genetic relationship of axenic cyanobacteria that produced bioactive compounds with some other cyanobacteria was identified basing on the 16S rRNA gene sequences. 22 Vietnamese and 6 German cyanobacterial strains were screened for their antifungal activity using the agar diffusion assay. Among them, the MeOH/water extract from the biomass obtained from a laboratory culture of strain Bio 33, isolated from the Baltic Sea near Rügen Island, exhibited a specific antifungal activity against Candida maltosa and others human pathogenous fungi such as Candida albicans, Candida krusei, Aspergillus fumigatus, Microsporum gypseum, Trichophyton rubrum and Mucor sp. Besides, it was very impressed that extracts of strain Bio 33 showed no antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The taxonomy basing on 16S rRNA gene sequence of the axenic Bio 33 identified this strain as Anabaena cylindrica species. As a result of the bioassay-guided fractionation of the crude MeOH/water extract, four new lipopeptides, named balticidins A – D, were isolated. These lipopeptides represent a new structural type with the co-occurrence of a glycosylated cyclic peptide, a fatty acid containing chlorine and a disaccharide moiety. The main active fraction isolated from the MeOH/water extract of the biomass of Bio 33 which contains the four lipopeptides exhibited only marginal cytotoxic activity against the human bladder carcinoma cell line 5637 (IC50 = 93 μg/ml). The weak cytotoxic activity and the absence of antibacterial effects in the used in vitro test systems opens a promising future for further investigations to clarify the antifungal mechanism and for in vivo applications of the new lipopeptides. Different media, temperature, light intensity and period of irradiance, the depletion of nitrate and the trace element cobalt were investigated to figure out conditions at which Bio 33 produces maximum of balticidins under laboratory conditions. Temperature was the most apparent factor influencing the growth of Bio 33 and the production of balticidins. Bio 33 grew best in BG 11 medium plus 0.5% NaCl at 26°C, under white fluorescent continuous light and a light intensity of 20 μmol photons m-2 s-1. Nevertheless, under the same conditions, 22.5°C was the best temperature for the production of balticidins. Besides, harvesting of Bio 33 during the logarithmic growth phase, particularly at 20th day, should supply approximately maximum quantity of balticidins. At 22.5°C and 20 μmol photons m-2 s-1 under 24 h continuous irradiance, the depletion of nitrate had no negative effect on the growth and concentration of balticidin A but increased balticidin B and decreased balticidin C; the absence of cobalt slightly decreased the growth but had no clear effect on the production of balticidins. On the other hand, extracts of the culture medium of the Vietnamese cyanobacterium TVN40, exhibited antifungal activity against Candida maltosa and weak antibacterial activity. Extraction of the culture medium with XAD-16 and elution of the XAD-bounded compounds by different solvents resulted in five fractions (water, 80% MeOH, 100% MeOH, acetone, dichloromethan). Four compounds have been isolated from the 80% MeOH fraction and one was identified as a dioxindole derivative. Structural elucidation of the other three compounds is still in progress. TVN40 was formerly identified as an Anabaena sp. according to the morphological properties, but the 16S rRNA gene sequence confirms that the strain belongs to the genus Nostoc. Microscopic examination of TVN40 revealed that the filamentous strain was not a unialgal but a mixed culture with strange round cells (SRCs) - a unicellular cyanobacterium belonging to the order Chroococcales. Laboratory cultures of the pure filamentous strain TVN40, the isolated SRCs and the mixed culture of both strains were established. Both TVN40 and SRC culture media were responsible for the antibacterial activity against B. subtilis, S. aureus and E. coli. However, only the extract of the culture medium of TVN40 was active against C. maltosa. The supplement of cobalt enhanced the antimicrobial activity of the culture medium. Pure strains showed higher activity in comparison to the mixed culture of TVN40 and SRC.
Non-thermal atmospheric pressure plasma has drawn more and more attention to the field of wound healing research during the last two decades. It is characterized by a unique composition, which includes amongst others free radicals, ions and electrons. Furthermore, non-thermal plasma exhibits temperatures that are below those inducing thermal cell damage. Next to its well-established anti-bacterial properties, plasma can have lethal as well as stimulating effects on mammalian cells. Therefore, the medical application of non-thermal plasma on chronic wounds seems to be a promising tool to enable healing processes. However, less is known about the plasma-mediated induction of intracellular signaling pathways in human immune cells, which play a leading part in the process of wound recovery and removal of pathogens. Therefore, this thesis examined the cellular effects of a non-thermal atmospheric pressure plasma treatment on human immune cells using the argon plasma jet kinpen 09. Here, the CD4+ T helper cell line Jurkat, the monocyte cell line THP-1 as well as the corresponding primary cells were investigated. First, cell survival and apoptosis induction was assessed in response to non-thermal plasma treatment by growth curves and flow cytometric assays. On the one hand it could be shown that primary cells are more susceptible to plasma treatment than the respective cell lines. On the other hand, monocytes responded less sensitive to plasma exposure than lymphocytes. Furthermore, this thesis outlined the impact of non-thermal plasma treatment on the gene expression level of immune cells. Therefore, DNA microarray analysis was performed with the cell lines Jurkat and THP-1. It became obvious that plasma exposure modulated the expression of several genes in both cell types. Differential expression of distinct target genes was further validated by quantitative PCR in the immune cell lines. Here, elevated gene expression levels of JUN and FOS in Jurkat cells and increased transcription of JUND in THP-1 cells in response to plasma treatment were made visible. JUN, FOS and JUND are components of the transcription factor AP-1, which is involved amongst others in gene expression of IL-8 and HMOX-1. Consequently, transcriptional induction of the inflammatory cytokine IL-8 as well as the enzymes HMOX-1 and GSR was detected in plasma-treated THP-1 cells. In addition, alterations in the protein activation levels were analyzed in plasma-treated Jurkat, THP-1 cells and primary monocytes. Since some of the identified target genes are known to be associated with the MAPK pathways, the regulation of these cascades was further investigated by western blot analysis. In all investigated cell types the pro-proliferative signaling molecules ERK 1/2 and MEK 1/2 as well as the pro-apoptotic signaling proteins p38 MAPK and JNK 1/2 were activated in a plasma treatment time dependent manner. In contrast to Jurkat and primary monocytes, the anti-apoptotic HSP27 was only induced in THP-1 cells in response to plasma exposure. Moreover, modulation of cytokine production and secretion was examined in the different immune cell types and co-cultured THP-1 and HaCaT keratinocytes by ELISA or flow cytometry. While Jurkat cells showed no plasma-mediated regulation of cytokine expression, THP-1 cells revealed an increased IL-8 secretion after long plasma time duration (360 s). Additionally, the intracellular expression levels of IL-6 and IL-8 were modulated in primary monocytes by plasma exposure. While short plasma treatment caused no alteration of the number of cells expressing IL-8 an up-regulation of the intracellular IL-6 level occurred after 30 s of plasma treatment. Long plasma treatment times resulted in a significant decrease of the intracellular IL-8 and IL-6 production levels. Furthermore, co-cultured THP-1 and HaCaT cells as well as mono-cultured THP-1 and HaCaT cells were examined regarding their cytokine secretion profile. Here, cells treated with plasma (180 s) as well as LPS and plasma (180 s and LPS) were compared with untreated cells. IL-6, IL-8 and GM-CSF secretion was induced by both plasma and plasma combined with LPS treatment in mono-cultivated HaCaT cells and co-cultured cells. Though, the highest cytokine secretion levels were reached in the plasma and LPS exposed co-culture. In contrast, mono-cultivated THP-1 cells only showed an increased secretion of IL-6, IL-8 and TNFa after incubation with plasma together with LPS exposed medium. In conclusion, this study revealed for the first time the non-thermal plasma-modulated expression of numerous genes and cytokines and the activation state of various signaling cascades in human immune cells. Thus, it contributes to gain a better understanding of the immune-modulatory impacts of plasma that might promote the wound healing process.
Accelerated drug release tests are essential for quality control (QC) of long acting (non-oral) controlled release formulations. Real-time release experiments are usually required for product development, to understand the mechanism of release and to establish a correlation with in vivo release. Ideally, the accelerated test should maintain the biorelevant aspect of the in vitro method and the mechanism of release should not change under accelerated test conditions. At the same time adequate discriminatory ability is a prerequisite as the accelerated test should be able to discriminate between batches with respect to manufacturing variables that can impact on bioavailability. The objective of this thesis was to develop accelerated drug release tests for intravaginal rings (IVRs) and to gain a mechanistic understanding of the principles that facilitate in vitro drug release under accelerated test conditions. A detailed evaluation of the in vitro release characteristics of the formulations under real-time test conditions was considered as a prerequisite for developing predictive accelerated tests. Two formulations were subject of this study, in which the mechanism of release is primarily governed by drug diffusion. One formulation was the commercially available Nuvaring®, a combined hormonal contraceptive IVR that releases etonogestrel and ethinylestradiol with a constant rate over a duration of 3 weeks. The second formulation was a prototype of an investigational IVR that is supposed to be bioequivalent to the marketed formulation. The Nuvaring® provides an example of a reservoir system in which a membrane mediates diffusion, resulting in release rates that are almost constant with time, whereas the investigational IVR is a matrix-type IVR. In these devices drug release is driven by Fickian diffusion through a homogeneous matrix and decays with time. Both IVRs are based on different grades of polyethylene vinyl acetate (PEVA). Accelerated drug release tests were performed at elevated temperature and in hydro-organic solvents since these two parameters were expected to increase drug diffusion through the semicrystalline EVA copolymer. Release experiments with IVRs or endcapped segments were performed in an incubator shaker. The devices were placed in stoppered flasks containing an adequate release medium that was continuously shaken and completely replaced at predetermined time points. Release experiments with endcapped segments were also performed in a small volume version of UPS apparatus 7 (the Reciprocating Holder). Results from release experiments in these two setups were in general comparable when the release from segments was standardized to release per ring with respect to the mass ratio (segment/IVR). Real-time drug release in an aqueous release medium at a temperature of 37 °C from the Nuvaring® was slightly affected by variations in the in vitro test conditions, i.e. media volume and composition (addition of solubility enhancing agents). These variations, however, did not affect the release kinetics that continued to be zero-order with exception of the initial burst. In contrast, real-time drug release from the matrix IVR was affected by the steroid solubility in the release medium, increased with increasing media volume and reached a maximum in release media containing solubility enhancing agents, resulting in distinct release kinetics. Interestingly the steroid solubility had a distinct influence on the release rate under conditions that are commonly assumed to provide sink conditions. Even under experimental conditions that provided minimum drug solubility, the concentration of ethinylestradiol in the receptor medium never exceeded 3 % of the saturation solubility. Accelerated drug release from both IVRs could be observed after exposure to elevated temperature and/or hydro-organic release media. Overall, increased drug release in different hydro-organic media correlated with polymer swelling. The higher swelling capacity of the investigational IVR, when compared with the Nuvaring®, was accounted for a stronger degree of acceleration in different hydro-organic release media. These observations were in agreement with literature sources that report that swelling as well as diffusivity in EVA copolymers increases with increasing VA content, which is lower in the rate-controlling membrane of the Nuvaring®. For the investigational IVR a good correlation between accelerated and real-time release profiles could be obtained if changes in steroid solubility under accelerated conditions were taken into consideration. For example, a good correlation could be observed between accelerated release profiles in hydro-organic media and real-time release profiles in media containing surfactants that provide maximum drug solubility and thus eliminate boundary layer effects. This observation appears reasonable since hydro-alcoholic release media also enhance steroid solubility in the receptor compartment. In case of the Nuvaring® variations in the in vitro test parameters under real-time test conditions did not affect the release kinetics. For this IVR, the mechanism of release was maintained in hydro-organic release media and at elevated temperature. The quantitative relationship between the zero-order release constants and the test temperature could be described by the Arrhenius equation, indicating that accelerated release is governed by an increase in drug diffusion. Validation of the accelerated method with a prototype of the investigational IVR with a different drug load demonstrated that the accelerated methods were able to detect formulation changes with similar discriminatory ability as the real-time test. However, the temperature-controlled accelerated method was less sensitive to detect changes in the release characteristics of a Nuvaring® that have been induced by preliminary heat-treatment, indicating that the accelerated method may be less sensitive to detect changes in IVRs that are a result of physical aging. When the aim is to develop an accelerated method for batch release it is therefore crucial to validate the accelerated method with appropriate samples from non-conforming batches that are out of specification under real-time test conditions and have been obtained by small but deliberate variations in the critical process parameters. In both formulations the degree of acceleration could be further increased by combining the effect of hydro-organic release media with an increase in temperature. Under these test conditions the ability to differentiate between the different prototypes of the investigational IVR was maintained. Moreover, in both IVRs the mechanism of release was not affected by an additional increase in temperature when compared with release profiles in hydro-organic solvents. In conclusion, the results of this study indicate that both temperature and hydro-organic release media are valid parameters to accelerate drug release from delivery systems in which the mechanism of release is primarily governed by diffusion through dense (inert) polymer matrices (i.e. inserts, implants). A correlation between real time and accelerated release will be facilitated if drug release under real-time test conditions is independent of the test parameters. To assure the outcome of the test with respect to quality and safety it is crucial to validate the accelerated method with appropriate batches.
Oral drug delivery is the preferred route of administration for the majority of drugs. Solid dosage forms arewell-accepted because of ease of administration, accurate dosing and high degree of patient compliance. The orodispersible technology platform has attracted increasing interest. Fast disintegrating in the mouth before swallowing, orodispersible dosage forms like orodispersible tablets (ODTs) address the need for patient-compliant medicines. ODTs represent a convenient alternative to conventional tablets or capsules. ODTs are an interesting approach when a rapid onset of therapeutic action is important. So far, ODTs have often been considered as an innovative variant of conventional oral solid dosage forms. Still, the development of ODT formulations is typically assisted by compendial in vitro test methods. However, the techniques described in international pharmacopoeias are non-specific for ODTs. After administration, the dispersion of an ODT in the mouth may provide effects which might influence the absorption of the drug. The performance of ODTs is more comparable to solutions/suspensions than to traditional tablets. To better guide the development of a new ODT formulation, this lack needs to be addressed. It is the aim of this work to design more specific in vitro test methods helping to improve understanding ODT formulations. To reflect the physiological conditions experienced by an ODT after administration, particular attention was given to the mouth where the ODT disperses and releases the drug before swallowing. In vitro biorelevant test setups simulating in vivo conditions were designed. An electronic tongue system was used to assess taste properties of ODTs. These test methods were applied in different stages of the ODT formulation development. Diclofenac being a poorly soluble and weakly acidic NSAID which is a standard medication for acute painful inflammatory conditions was used as a drug model. Three forms, i.e. the free acid and its sodium/potassium salt, were investigated for the formulation of palatable and fast acting ODTs. In Chapter 1, the development of biorelevant test setup reflecting the physiological conditions experienced by ODTs is described in detail. The newly-designed in vitro models successfully discriminated the different diclofenac forms in successive in vitro compartments simulating the mouth, the stomach and the small intestine. It was possible to identify peculiar dissolution profiles with diclofenac salts. Characterizing in-depth the diclofenac free acid and salt particles provided a better understanding of the peculiar dissolution profiles. Critical behaviors of diclofenac salts on their way from the mouth to the stomach and passing different pH conditions were extensively evaluated. Reasons for pH-dependent API precipitation and particle agglomeration were studied in detail. In pre-formulation studies, the proposed biorelevant test setups succeeded in helping to early identify critical pharmaceutical properties for diclofenac salts and to select diclofenac free acid as the most appropriate drug form providing the most stable in vitro performance. In Chapter 2, the electronic tongue method as an in vitro taste assessment tool for ODTs is proposed. Using the TS-5000Z taste sensing system (Insent Inc., Japan), the method was able to differentiate between the taste/aftertaste qualities and intensities of the three diclofenac candidates. The electronic tongue was also successfully used to differentiate different ODT formulations. The results obtained proved that valuable information can be gained. By this means, the taste perception of the diclofenac drug candidates were classified and rank against each other. For manufacturing taste-masked ODTs, diclofenac free acid, could be selected easily. The electronic tongue found out to be a precious tool in assisting the development of a new ODT product and finding the most appropriate multi-component formulation. Both proposed methods successfully showed their discriminative ability and also their utility in pre-formulation studies of ODTs. In the previous chapters, it was indeed possible to early select diclofenac free acid as the most suitable drug candidate for the targeted product profile. In Chapter 3, said methods were further used to guide the development of the taste masked diclofenac ODT formulation. This study highlights the importance of considering in vitro the physiological aspects which may have an impact on the in vivo performance of ODT dosage forms. The contact of ODTs with the mouth should be simulated in vitro for a better understanding of the in vivo behavior. With feasible biorelevant in vitro dissolution methods, an optimized correlation of in vitro and in vivo results may be achieved. The proposed in vitro test methods may provide data of predictive value and may support the rational development of ODT formulations.
Heparin is an anticoagulant drug. It is important in the treatment of deep vein thrombosis,pulmonary embolism and during surgeries. Heparin-induced thrombocytopenia (HIT) is a severe adverse reaction caused by the formation of ultralarge complexes of platelet factor 4 (PF4) with unfractionated heparin (UFH). It can lead to limb loss or fatal events like stroke, myocardial infarction or pulmonary embolism. HIT has an incidence of about 3% in patients receiving anticoagulative heparin treatment. PF4 is a tetrameric protein, released from the α-granules of platelets upon activation. PF4 is known to form antigenic complexes with UFH accompanied by structural changes of PF4. In this thesis, the size and size distribution of PF4 and PF4/heparin complexes were analyzed using asymmetrical flow field-flow-fractionation (AF4), photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). PF4 tends to form auto-aggregates and to adsorb to different surfaces, including regenerated cellulose, polyethersulfone, quartz and glass. The aggregates are less pronounced in solutions at isotonic NaCl concentration. Arginine and Tween 20 were identified as possible ingredients to hinder the auto-aggregation of PF4. Also, it is shown by combining circular dichroism (CD) spectroscopy, atomic force microscopy (AFM) and isothermal titration calorimetry (ITC) with UFH and defined chain length (16-, 8-, 6-, 5-mer) heparins that structural changes (i.e., increase in β-sheets) alone are not sufficient to induce antigenicity. While UFH, 16-, 8-, and 6-mer heparins all induced an increase in the antiparallel β-sheet content to > 30% (as determined by CD spectroscopy), complex antigenicity as measured by anti-PF4/heparin antibody binding in an enzyme-linked immunosorbent assay (EIA) was only induced by UFH and 16-mer heparin. Fondaparinux (5-mer heparin), which forms in vitro non-antigenic complexes with PF4, did not induce structural changes of PF4. Interestingly, the structural changes induced by antigenic UFH and 16-mer heparin but not by non-antigenic shorter heparins were reversible at higher heparin concentrations. Furthermore, the complexes formed by PF4 with longer heparins were larger than those formed with shorter heparins as shown by atomic force microscopy (AFM). UFH, HO16 and HO08 are able to form ultralarge multimolecular complexes with PF4. ITC data indicated strong electrostatic interactions and energetically unfavorable conformational changes of PF4 with longer heparins, while for the short heparins, favorable conformational changes in the structure of PF4 are induced. This explains the reversibility of the structural changes seen for UFH and HO16 upon addition of an over-saturating amount of heparin. Finally, using differential scanning calorimetry (DSC) the thermal stability of PF4 and PF4/heparin complexes was assessed. Despite its tendency to form auto-aggregates, PF4 is a heat-stable protein. This stability is, length dependently, even increased in complex with heparins. This work shows important differences in the binding between PF4 and heparins of different chain length and might be relevant for the understanding of other biological functions of heparins (e.g., involvement in allergic and inflammatory reactions).
HPMC (Hydroxypropylmethylcellulose) based hydrophilic gel matrix tablets are one of the most commonly used monolithic extended release dosage forms used in the pharmaceutical industry. Drug release from the hydrated HPMC matrix is generally controlled by either diffusion or erosion, or a combination of both. Several studies have shown that for HPMC-based matrices with a high amount of poorly water-soluble additives, erosion is the predominant release mechanism. Erosion rates of these formulations vary significantly with changes in the matrix composition. Depending on the erosion rate, the drug delivery might occur over a shorter or longer time span and thus to different sites of action that are proximal or distal gastrointestinal tract (GIT). Erosion rates of HPMC-based matrices can be modulated by changing the amount and molecular weight of the HPMC. In the present study, four different HPMC-based hydrophilic matrix formulations developed by AstraZeneca R&D, Sweden, were investigated for in vitro as well as in vivo erosion behavior. Formulations F1, F2, and F3 consist of 40% HPMC, which is a mixture of two different HPMC viscosity grades (Methocel K100LV and Methocel K4M). Formulations F1, F2, and F3 contained 23%, 10%, and 0% of Methocel K4M, respectively, while formulation F4 was composed of 20% Methocel K100LV. Calcium hydrogen phosphate dihydrate (a poorly water-soluble compound) was used as the filling excipient. The in vitro HPMC release from the matrices was investigated using a USP dissolution apparatus II equipped with a stationary basket in a phosphate buffer (PB) pH 6.8 and simulated gastric fluid without pepsin (SGFsp) pH 1.2 at various rotation speeds. The HPMC concentration in the dissolution samples were analyzed using size exclusion chromatography coupled with multiangle light scattering and refractive index detectors (SEC-MALS/RI). In order to establish a correlation function between the magnetic moment and HPMC release, the formulations were tested in a magnetic moment dissolution tester (MMDT), a modified in vitro dissolution apparatus equipped with a magnetometer. The in vivo gastrointestinal imaging and erosion behavior of the tablets were investigated by magnetic marker monitoring (MMM) using a superconducting quantum interference devices (SQUIDs) sensor system in five healthy male volunteers at Physikalisch-Technische Bundesanstalt (PTB), Berlin. All formulations were administered after an overnight fast of at least 10 hours. However, formulations 3 and 4 were also administered 30 minutes after a standard FDA breakfast. The in vivo HPMC release was calculated using the correlation function from the recorded in vivo magnetic moment data. A linear correlation function was not observed, since the decrease of the magnetic signal was driven by both erosion and diffusion. The in vitro and in vivo erosion-time profiles show that erosion was strongly dependent on the composition of the formulation. The formulations containing a larger proportion of high molecular weight HPMC, or a higher content of HPMC, exhibited relatively slower erosion rates and vice versa. However, unlike in vitro erosion rates, the in vivo erosion rates for different formulations did not always significantly differ from each other. In vivo erosion rates of the investigated formulations were significantly higher under postprandial administration than under fasted state administration. No rapid disintegration of any of the formulations (that is, formulation failure that can potentially cause dose dumping) was observed. A good linear (point-to-point) correlation between the in vitro HPMC release at 50 rpm in PB pH 6.8 and the in vivo HPMC release was observed for all formulations in the individual volunteers for both administration conditions. The predictability of the in vivo HPMC release for all formulations in fasting as well as postprandial administrations was better with phosphate buffer pH 6.8 at 50 rpm in comparison to SGFsp pH 1.2 or higher stirring rate in phosphate buffer pH 6.8. In postprandial administrations, the gastric emptying time was significantly delayed compared to fasting administrations. For postprandial administrations, the localized erosion rate in the distal stomach was significantly higher than in the proximal stomach. The in vivo HPMC release of the investigated formulations under both intake conditions was not dependent on the motility of the tablet in the gastrointestinal tract. The in vivo HPMC release for all the investigated formulations when administered under fasting conditions was underestimated, while under postprandial conditions, the HPMC release was overestimated by the in vitro dissolution method in PB pH 6.8 at 50 rpm.
Pharmaceutical residues are found in increasing concentrations in the environment and in potable water where they have verifiable effects on aquatic life. Conventional methods for water treatment are not able to sufficiently abate these generally stable compounds. It was found that physical plasma generated directly in water can degrade several of these recalcitrant organic pollutants. Studies on the basic plasma chemical processes for the model system of phenol showed that the degradation is primarily caused by hydroxyl radicals. This was confirmed by reaction chemistry and spin trap enhanced electron paramagnetic resonance spectroscopy (EPR). The degradation of diclofenac and its by-products were investigated in detail to perform a first risk-assessment of the new technology. Findings are not limited to the application of plasma but applicable to other advanced oxidation processes (AOP) that are based on the generation of hydroxyl radicals as well. Additionally, pulsed corona plasma and pulsed electric fields were assessed for their capacity to kill Legionella pneumophila in water. Whereas it was possible to kill L. Pneumophila with both methods, plasma treatment resulted in an enhanced bacterial killing. Therefore, advanced oxidation processes (AOP) and plasma treatment in particular are some of the few feasible approaches to decompose recalcitrant compounds in water.
Introduction: Ketamine (KET) is widely used as anaesthetic drug. Beside its pronounced an-aesthetic effects as caused by antagonism of NMDA receptors, ketamine also causes potent analgesia. Moreover, There are ample new evidences, firstly, that 2R,6R/2S,6S-enantiomers of hydroxynorketamine (HNK), exert neuro-modulating effects by AMPA-receptor activation and, secondly, that the plasma levels of norketamine (n-KET) after oral dosing are higher than after intravenous administration. From the physicochemical point of view ketamine is expected to be a substrate of drug transporters. Thus, it was the aim of this study to separate and quantify KET and its metabolites in human serum, urine and feces; investigate the role of transporter proteins in the intestinal absorption, distribution and elimination of ketamine; and evaluate pharmacokinetics and metabolism of a newly developed prolonged-release keta-mine dosage form to confirm its suitability for chronic treatment of CNS-diseases (e.g. de-pression) according to the new “ketamine metabolite paradigm”. Materials and methods: Quantification of ketamine was done by a LC-MS/MS-based quantifi-cation method on the QTRAP4000 instrument. Samples were extracted by methyl tert-butyl ether after addition of sodium carbonate to liberate the free base; Single transfected MDCKII cells overexpressing OCT1, OCT2, OCT3, and MATE1 or MATE2K, and HEK293 cells over-expressing OATP2B1 were used to study the cellular uptake of ketamine. Inside-out lipovesi-cles were used to determine the affinity of ketamine to the efflux transporter P-glycoprotein (P-gp). Uptake into cells or vesicles was determined by liquid scintillation counting. Func-tionality of all in vitro systems was assured by using in each case appropriate probe sub-strates; The dose-escalation study was performed in five consecutive periods (7 days wash-out) in 15 healthy subjects (5 females and 10 males. 20-35 years, BMI 19.4-27.6 kg/m2). Results: We introduce for the first time the separation and quantification of the active me-tabolites 2R,6R/2S,6S-HNK; Ketamine was shown to be taken up significantly in a time- and concentration-dependent manner by OCT1-3. The affinity to OCT transporters at pH=6.5 was several fold higher than that at pH=7.4. ), ketamine showed a significant but low affinity to P-gp. In contrast to this, we could not detect any transport of ketamine by MATE1 / 2K or OACPT2B1; and PR-KET was safe and well tolerated with higher metabolites productivity, different pharmacokinetic properties and longer T1/2 when compared to IV-KET or IR-KET. Conclusion: the uptake transporters OCT1 & 3 and the efflux transporter P-gp may play a role in the intestinal absorption of the drug. On the other side, P-gp, MATE1 / 2K and OCT are not expected to contribute significantly to tissue (brain) distribution or renal excretion of ketamine; Moreover, the prolonged-release ketamine undergoes dose-dependent “first-pass” metabolism which generates substantially increased plasma exposure of downstream me-tabolites with potential neuro-modulating effects compared to ketamine after intravenous administration.
Chemosymbiosis in marine bivalves – unravelling host-symbiont interactions and symbiotic adaptions
(2018)
Symbiosis essentially forms the cornerstone of complex life on earth. Spearheading
symbiosis research in the last few decades include the exploration of diverse mutualistic
animal-bacterial associations from marine habitats. Yet, many facets of symbiotic
associations remain under-examined. Here we investigated marine bivalves of the genera
Bathymodiolus and Codakia, inhabiting hydrothermal vents and shallow water
ecosystems, respectively, and their bacterial symbionts. The symbionts reside
intracellularly within gill epithelia and supply their host with chemoautotrophically fixed
carbon. They oxidize reduced substrates like sulfide (thiotrophic symbionts) and methane
(methanotrophic symbionts) from surrounding fluids for energy generation. The nature of
interactions between host and symbiont at the metabolic and physical level, as well as
between the holobiont and its environment remain poorly understood. In vitro cultivations
of both symbiont and host are difficult till date, hampering the feasibility of targeted
molecular investigations.
We bypassed culture-based experiments by proteogenomically investigating physically
separated fractions of host and symbiont cell components for the bivalves Bathymodiolus
azoricus, Bathymodiolus thermophilus and Codakia orbicularis. Using these
enrichments, we sequenced the symbionts’ genomes and established semi-quantitative
host-symbiont (meta-) proteomic profiles. This combined approach enabled us to resolve
symbiosis-relevant metabolic pathways and adaptations, detect molecular factors
mediating physical interactions amongst partners and to understand the association of
symbiotic traits with the environmental factors prevailing within habitats of the respective
bivalve.
Our results revealed intricate metabolic interdependence between the symbiotic partners.
In Bathymodiolus, these metabolic interactions included (1) the concentration of essential
substrates like CO2 and thiosulfate by the host for the thiotrophic symbiont, and (2) the
host’s replenishment of essential TCA cycle intermediates for the thiotroph that lacks
biosynthetic enzymes for these metabolites. In exchange (3), the thiotroph compensates
the host’s putative deficiency in amino acid and cofactor biosynthesis by cycling aminoacids
derived from imported precursors back to the host. In case of Codakia orbicularis,
the symbionts may metabolically supplement their host with N-compounds derived from
fixation of molecular nitrogen, a trait that was hitherto unknown in chemosynthetic
thiotrophic symbionts.
Individual proteogenomic investigations of the bivalves Bathymodiolus azoricus and
Bathymodiolus thermophilus showed that their symbionts are able to exploit a multitude
of energy sources like sulfide, thiosulfate, methane and hydrogen to fuel chemosynthesis.
The bivalves and their thiotrophic symbionts, however, are particularly adapted to
thiosulfate-utilization, as indicated by mitochondrial production and concentration of
thiosulfate by host and dominant expression of thiosulfate oxidation enzymes in the
symbiont. This may be advantageous, because thiosulfate is less toxic to the host than
sulfide. The central metabolic pathways for energy generation, carbon and nitrogen
assimilation and amino acid biosynthesis in thiotrophic symbionts of both Bathymodiolus
host species are highly conserved. Expression levels of these pathways do, however, vary
between symbionts of both species, indicating differential regulation of enzyme synthesis,
possibly to accommodate differences in host morphology and environmental factors.
Systematic comparison of symbiont-containing and symbiont-free sample types within
and between B. azoricus and B. thermophilus revealed the presence of ‘symbiosisspecific’
features allowing direct host-symbiont physical interactions. Host proteins
engaged in symbiosis-specific functions include 1) a large repertoire of host digestive
enzymes predominant in the gill, possibly facilitating symbiont population control and
carbon acquisition via direct enzymatic digestion of symbiont cells and 2) a set of host
pattern-recognition receptors, which may enable the host to selectively recognize
pathogens or even symbionts “ripe” for consumption. Symbiont proteins engaged in
symbiosis-specific interactions included 3) an enormous set of adhesins and toxins,
putatively involved in symbiont colonization, persistence and host-feeding.
Bathymodiolus symbionts also possess repertoires of CRISPR-Cas and restrictionmodification
genes for phage defense that are unusually large for intracellular symbionts.
Genomic and proteomic comparisons of thiotrophic symbionts of distinct Bathymodiolus
host species from different vent sites revealed a conserved core genome but divergent
accessory genomes. The B. thermophilus thiotroph’s accessory genome was notably more
enriched in genes encoding adhesins, toxins and phage defense proteins than that of other
Bathymodiolus symbionts. Phylogenetic analyses suggest that this enrichment possibly
resulted from horizontal gene acquisition followed by multiple internal gene duplication
events. In others symbionts, these gene functions may be substituted by alternate
mechanisms or may not be required at all: The methanotrophic symbionts of B. azoricus,
for example, has the genetic potential to supplement phage defense functions. Thus, the
accessory genomes of Bathymodiolus symbionts are species- or habitat-associated,
possibly facilitating adaptation of the bivalves to their respective micro- and macroenvironments.
In support of this, we show that symbiont biomass in B. thermophilus,
which hosts only one thiotrophic symbiont phylotype, is considerably higher than in B.
azoricus that hosts thiotrophic and methanotrophic symbionts. This suggests that different
symbiont compositions in each species produce distinct microenvironments within the
holobiont.
Our study presents an exhaustive assessment of the genes and proteins involved in this
bivalve-microbe interaction, hinting at intimate host-symbiont interdependencies and
symbiotic crosstalk between partners. The findings open novel prospects for
microbiologists with regard to mechanisms of host-symbiont interplay within highly
specialized niches, origin and distribution of prokaryote-eukaryote interaction factors
across both mutualistic and pathogenic associations.
Profiling the activity and hepatotoxicity of flupirtine through medicinal chemistry approaches
(2019)
Drug induced liver injury (DILI) and tissue discoloration led to the recent discontinuation of the therapeutic use of the closely related drugs flupirtine and retigabine, respectively. Experience gained with these drugs strongly suggests that heterodimer, voltage‐gated potassium channels 2 and 3 (KV2/3) are valid targets for effective treatment of pain and epilepsy. Because the adverse effects are not related to the mechanism of action, it appears promising to investigate chemical modifications of these clinically validated, drug‐like leads. In the present retro metabolic drug design study, a series of 44 compounds were
synthesized and characterized with regards to KV7.2/3 opening activity and efficacy. The most active compounds displays excellent potency (EC50 = 4 nM) and efficacy (154%) as an Kv7.2/3 opener. Limited aqeous solubility hampered toxicity testing at concentrations higher than 63 μM, but this concentration was nontoxic to two hepatocellular cell ilnes (HEP‐G2 and TAMH) in culture.
The aim of the present dissertation was to investigate the biological and chemical potential of two European mushroom species: Fomitopsis betulina and Calvatia gigantea. For this purpose, different extracts of both fungi were tested for: antimicrobial, antifungal, cytotoxic, in vitro wound healing, and anti-adhesive properties. Bioassay-guided fractionation led to the isolation of bioactive compounds, altogether 20 compounds were isolated and identified. The compounds were obtained from the ethyl acetate extracts, they included triterpenes, sterols and aromatic compounds. The separated substances from both fungi were proved for biological activities, some of them showed antimicrobial and cytotoxic activities.
Microalgae are aquatic, unicellular, eukaryotic organisms, which perform photosynthesis. They have gained interest within the last decades not only for biofuel production due to their high amount of lipids, but also for pharmaceutical and for nutraceutical purposes. Interesting compounds are proteins, carbohydrates, or pigments, such as carotenoids. However, microalgae possess strong and rigid cell walls, which hinder a sufficient and yet, gentle extraction of those valuable compounds. Although standard extraction techniques are available, several shortcomings occur, e.g. high energy demand, use of environmentally harmful solvents or alteration of compounds due to heat or chemicals. Therefore, an alternative method is needed, which is able to address these disadvantages. Physical plasmas were thus studied to answer the question whether they are able to disintegrate the cell walls of microalgae effectively and yet, without degradation of the extractives.
First step of the thesis was to find a suitable plasma source that has an effect on the cell walls because plasma effects, such as electric fields, shockwaves, UV light emission, and the generation of reactive species can be tailored with the respective setup. It was found that spark discharges are most effective for the extraction of Chlorella vulgaris, which was chosen as model organism. All extraction yields were compared to reference methods, whereat microwave radiation was found to be the most effective reference method and were hence, applied for comparative studies.
For the next step, proteins were selected as targets to answer the question, which differences can be determined between plasms-treated and microwave-radiated proteins are observable although the extraction yields were equal. Furthermore, plasma effects, especially the effects of reactive species on the extracted proteins had to be studied. Findings indicate that heat sensitive proteins, such as photosystem-related proteins, or histones are better extractable with spark discharges than with microwave exposure and the effect of reactive species is only minor.
The last step was to determine, which plasma effect is responsible for the observed cell wall disintegration. Therefore, the tensile strength of Chlorella vulgaris was determined and compared to the shockwave pressure, which is generated from the spark channel. It was proven that the shockwave pressure exceeds by far the tensile strength of the microalgae an can be thus held responsible for mechanism for cell wall rupture.
In this thesis, it was found that spark discharges are a promising alternative for the extraction of valuable compounds from microalgae. The discharges are not only effective, but also gentle enough for sensitive compounds, such as proteins or pigments.
From a biopharmaceutical point of view, poor oral bioavailability of a drug is one of the greatest challenges for formulation scientists. The majority of new chemical entities (NCEs) are weakly basic drugs. Consequently, these drugs exhibit pH-dependent solubility, being higher under acidic conditions in the fasted stomach and lower under neutral conditions in the small intestine, the main site of drug absorption. For theses compounds, pH-dependent precipitation testing represents a key parameter during early development stages. In this development phase, the amount of drug available is limited, and fast and detailed investigations of simulated drug solubility are desired. Therefore, an automated small-scale in vitro transfer model, simulating drug transfer from a donor (stomach; simulated gastric fluid, SGF pH 2.0) to an acceptor (small intestine; fasted state simulated intestinal fluid, FaSSIF-phosphate pH 6.5) compartment, has been developed. In contrast to the originally published transfer model, this model allowed a detailed investigation of drug supersaturation and precipitation in a small-scale, feasible for pre-formulation purposes, through miniaturization and automation in an in-line analytical set-up. In-line drug concentration analysis in turbid samples, due to pH-dependent drug precipitation, was achieved by a pre-filtration step, the use of flow-through cuvettes and the application of UV derivative spectroscopy. Compared to the common procedure of manual sampling followed by HPLC-UV analysis for concentration determination, the supersaturation and precipitation of the model drug ketoconazole was more accurately captured by the newly developed in-line analytical set-up. In addition, the newly developed small-scale model was compared to a USP II-based transfer model, representing an established scale of the transfer model. Using a physiologically relevant simulated gastric emptying rate of 5 min half-time, supersaturation and precipitation of the model drugs ketoconazole and a new chemical entity from the research laboratories of Merck Healthcare KGaA, MSC-A, were observed to be highly comparable. Following miniaturization and automation, the developed small-scale model was used to establish eight physiologically relevant test-sets. These test-sets were used to assess the impact of gastrointestinal (GI) variability, i.e. gastric pH, gastric emptying, and GI fluid volumes, on supersaturation and precipitation of two weakly basic model compounds, ketoconazole and MSC-A. The experiments revealed that variations in all GI parameters investigated affected the in vitro supersaturation and precipitation of ketoconazole. For example, faster gastric emptying yielded higher supersaturation and faster precipitation of ketoconazole. In contrast, MSC-A supersaturation and precipitation was only affected by variability in gastric pH. Consequently, the effect of varying GI parameters was found to be drug-specific. Elevated gastric pH, as it can result from co-medication with acid-reducing drugs, resulted in lower degrees of supersaturation for both substances. For ketoconazole, this result is in agreement with the observation that the oral bioavailability of ketoconazole is lowered when proton pump inhibitors are co-administered. In addition to the physiological considerations, the small-scale model developed herein was used to establish an in vitro screening assay for precipitation inhibitors (PIs). The use of PIs represents one option of reducing the process of pH-dependent drug precipitation during simulated GI transfer. For this purpose, ketoconazole and five orally administered kinase inhibitors (i.e. pazopanib, gefitinib, lapatinib, vemurafenib, and MSC-A) were analyzed with and without the polymeric PIs HPMC, HPMCAS, PVPK17 and K30, PEG6000, and Soluplus® in the small-scale transfer model. This screening revealed that at least one effective PI could be identified for each model drug. Moreover, HPMCAS and Soluplus® were the most effective PIs. Another outcome of these studies was that gefitinib expressed highly variable amorphous precipitation which was confirmed by powder X-ray diffraction (PXRD). During the transfer model experiments, the intermediate amorphous and supersaturated state of gefitinib was stabilized using HPMCAS and Soluplus®. After the polymer investigations, the impact of the buffer species in the simulated intestinal medium on drug supersaturation and precipitation was assessed. Since luminal fluids are mainly buffered by hydrogen carbonate ions, a USP II-based transfer model equipped with the pHysio-grad® device was proposed. This allowed the use of a complex bicarbonate buffer for the preparation of FaSSIF-bicarbonate in an in vitro transfer model. Results of transfer model experiments using standard phosphate-based FaSSIF and a more physiologically relevant bicarbonate-based FaSSIF were compared. Therefore, ketoconazole, pazopanib, and lapatinib were analyzed with and without the precipitation inhibitor HPMCAS. While HPMCAS was found to be an effective precipitation inhibitor for all drugs in FaSSIF-phosphate, the effect in FaSSIF-bicarbonate was much less pronounced. Additionally, performed rat PK studies revealed that HPMCAS did not increase the exposure of any of the model compounds significantly, indicating that the transfer model employing bicarbonate-buffered FaSSIF was more predictive compared to the model using phosphate-buffered FaSSIF. The in vitro and in vivo results of these studies demonstrated that the supersaturation precipitation of poorly soluble weakly basic drugs can be significantly affected by GI variability. Furthermore, the use of the automated small-scale transfer model enabled the identification of effective precipitation inhibitors for the model drugs involved in these studies. At the same time the buffer species has been observed to be especially important to reliably predict the in vivo solubility/dissolution behavior of HPMCAS and the weakly basic model drugs.
Research on the science and the fiction of supercritical fluid chromatography (SFC) has been ongoing for more than five decades. Today, packed column SFC promises speedy solutions to chiral and semi-preparative separation problems, but academia has been reluctant to incorporate SFC into its curriculum, as doubts linger concerning its practicability. This work sought to explore the merits of SFC in hyphenation with electrospray ionization--single quadrupole mass spectrometry (ESI-MS) and supercritical fluid extraction (SFE) in various aspects of medicinal chemistry and bioanalysis within an academic setting.
SFC was investigated for its usefulness in assessing the purity and the stability of synthesis products, and the quantification of chiral and achiral metabolites - domains conventionally occupied by high performance liquid chromatography (HPLC).
Confronted with analytes prone to hydrolysis (cyclic polysulfides) and UV-induced configurational changes (aza-stilbenes), fast elution by water-free SFC-MS proved complementary to traditional chromatographic techniques.
The quantification of antidepressant ketamine metabolites presented an opportunity to assess supercritical fluid techniques within a bioanalytical context. While SFC hyphenated to single quadrupole MS did not reach the sensitivity levels of HPLC coupled to triple quadrupole MS/MS, exploitation of supercritical CO2 reduced analysis times more than six-fold (60 minutes by HPLC vs 10 minutes by SFC). When coopted for both extraction and analysis, SFE-SFC-MS simplified sample preparation and promoted the transition from off- to on-line bioanalysis. Similar results were obtained when SFC was applied to acidic and basic metabolites of the controversial anodyne flupirtine. Again, SFC featured shorter run times but also expanded the target metabolite spectrum covered within one run.
Finally, a tiered approach to validation demonstrated the reliability achievable by SFC. Critical applications such as quantification of the newly approved antidepressant ketamine or the recently withdrawn analgesic flupirtine were comprehensively validated according to guidelines on bioanalytical method validation by the European Medicines Agency. Notably, this included the first fully validated chromatographic methods for the putative antidepressant (2R,6R)-6-hydroxynorketamine, and the first report of EMA-conforming quantification by on-line SFE-SFC-MS from urine.
Separation scientists find themselves confronted with diverse problems and tools. Although parsing only a microscopic subsection of the available chemical and analytical space, the results obtained here suggest SFC to be a fast and versatile addition to conventional chromatographic methods employed at the intersection of medicinal chemistry and bioanalysis.
Polysaccharide is a major constituent of the total organic carbon that is generated by photosynthetic eukaryotes. In the marine realm, where approximately half of annual global carbon fixation occurs, algae can produce large amounts of polysaccharide during bloom events. Phytoplankton blooms are frequently seasonal phenomena, and spring blooms in particular have been a focus of study as they are predictable in space and time. This makes them much more amenable model systems in which to explore the processes that occur as organic carbon is recycled.
It is assumed that the bulk of the polysaccharides algae produce serve one of two primary functions - namely acting as an energy storage molecule, or they serve as structural polymers in the cell walls. Other polysaccharides may also have protective functions as exudates. Regardless of function in algae, the polysaccharides are a valuable energy source for heterotrophic bacteria. The combination of abundance and predictable or semi-predictable structure of the polysaccharides has led to proliferation of variations on a particular sequestration and degradation strategy among the Bacteroidetes and Gammaproteobacteria that is frequently characterised as being ‘selfish’. The strategy is based on uptake of poly- and especially oligosaccharides into the periplasm via the use of TonB-dependent transporters. Once in the periplasmic space, oligomers can be further degraded to monomers that can then be transported into the cytosol. This mechanism is beneficial to the cell as it needn’t then lose the nutritive benefit of the polysaccharide to other cells, which may or may not have manufactured their own degradative carbohydrate active enzymes (CAZymes).
The research articles that make up this thesis are thus based around attempts to find and elucidate the polysaccharide preferences of heterotrophic bacteria that become abundant following phytoplankton blooms.The first article is a study into the abundance of TonB-dependent transporter proteins in metaproteomes and metagenomes across a single spring phytoplankton bloom at the long term research station at Helgoland. This investigation identifies transporters for laminarin and alpha-glucans, the two most abundant glucose-based storage polysaccharides, are the most abundant predicted polysaccharide transporting TonB-dependent transporters during the bloom. However, as the bloom progressed, and particularly following a doubling of bacterial cell numbers, the proportion of predicted polysaccharide transporters dedicated to laminarin and alpha-glucan transport declined relative to transporters for less readily degraded mannose-, xylose-, and fucose-containing polysaccharides. The inference is that this change is an active response to the availability of the different polysaccharides, or their relative attractiveness as growth substrates during the period.
The second article is an in-depth look at one of the most abundant Bacteroidetes clades, which was previously unnamed, and has not to date been cultivated. The most abundant species in this clade grows rapidly and often peaks earlier than other heterotrophic clades. It was found to be limited in predicted polysaccharide consumption capability, having only PULs for predicted laminarin degradation. It is also detectable in many locations at higher latitudes where phytoplankton blooms are expected to occur, indicating this is a globally successful consumer of algal organic matter, and may have an outsize significance for global laminarin degradation given its high abundance.
The third article is a more holistic study of phytoplankton bloom associated Gammaproteobacteria, which have otherwise been rather ignored compared to the Bacteroidetes. Gammaproteobacteria overlap with Bacteroidetes to some extent in being clear consumers of laminarin, but fewer of them are clearly capable of consuming the more complex cell-wall derived polysaccharides. Some may, however, be producers of alginate, an otherwise mysteriously popular polysaccharide with Bacteroidetes, given that it is not known to be produced by bloom forming microalgae.
The fourth article then goes into detail on the PUL content of Bacteroidetes, based on metagenomic data. It finds five substrates, alpha- and beta-glucans, xylose and mannose rich polysaccharides, and alginate, are the most frequent predicted polysaccharide substrates for Bacteroidetes PULs among populations responding to the Helgoland spring blooms.
This thesis thus summarises multiple metagenomic and metaproteomic investigations into the polysaccharide consumption capabilities of marine heterotrophic bacteria. These bacteria have a profound impact on the overall carbon cycle in coastal regions, and are critical for understanding how changes in atmospheric carbon concentrations impact carbon turnover and storage in the world's oceans.
Abstract
Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide‐mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub‐micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long‐chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo‐based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell‐based activity assay.
Symbiotic interactions are a key element of biological systems. One powerful strategy to gain insight into these interactions, and into biological systems in general, is the analysis of proteins expressed in situ using metaproteomics. In this thesis, host-microbe interactions in two mutualistic associations between chemosynthetic sulfur-oxidizing endosymbionts and marine invertebrates, the deep-sea tubeworm Riftia pachyptila and the shallow-water clam Codakia orbicularis, were studied by adapted and optimized metaproteomics methods.
The Riftia symbiosis, which inhabits hydrothermal vents in the deep sea, and in which the host completely depends on its symbiont for nutrition, has fascinated researchers for about four decades. Yet, the interaction mechanisms between both partners have been understudied so far. Additionally, while different aspects of the host’s biology have been described, a comprehensive analysis has been lacking. Moreover, although only one symbiont 16S rRNA phylotype is present in Riftia, the symbiont population of the same host expresses proteins of various redundant or opposed metabolic pathways at the same time. As the symbionts also exhibit a wide variety in size and shape, symbionts of different size might have dissimilar physiological functions, which remained as of now to be elucidated. In this thesis, we addressed both, the host-symbiont interaction mechanisms, and physiological roles of symbiont subpopulations. A comprehensive Riftia host and symbiont protein database was generated as prerequisite for metaproteomics studies by de novo sequencing the host’s transcriptome and combining it with existing symbiont protein databases. This database was then used for metaproteomics comparisons of symbiont-containing and symbiont-free Riftia tissues, to gain insights into host-symbiont interactions on the protein level. The impact of energy availability on host-symbiont interactions was studied by comparing specimens with stored sulfur (i.e., high energy availability) with specimens in which sulfur storages were depleted. We employed optimized liquid chromatography peptide separation to increase metaproteome coverage. With this analysis, we identified proteins and mechanisms likely involved in maintaining the symbiosis, under varying environmental conditions. We unraveled key interaction mechanisms, i.e.: (i) the host likely digests its symbionts using abundant digestive enzymes, and, at the same time, (ii) a considerable part of the worm’s proteome is involved in creating stable internal conditions, thus maintaining the symbiont population. Furthermore, (iii) the symbionts probably employ eukaryote-like proteins to communicate with the host. (iv) Under conditions of restricted energy availability, the host apparently increases digestion pressure on the symbiotic population to sustain itself.
Riftia symbionts of different size apparently have dissimilar metabolic roles, as revealed in this thesis. We enriched symbionts of different sizes using gradient centrifugation. These enrichments were subjected to protein extraction using a protocol optimized for the small sample amount available. Metaproteomics analysis included a gel-based workflow and evaluation of the complex dataset with machine learning techniques. Based on our metaproteomics study, we propose that Riftia symbionts of different cell size correspond to dissimilar physiological differentiation stages. Smaller cells are apparently engaged in cell differentiation and host interactions. Larger cells, on the other hand, seem to be more involved in synthesis of various organic compounds. Supposedly, in large symbionts endoreduplication cycles lead to polyploidy. Our results indicate that the Riftia symbiont employs a large part of its metabolic repertoire at the same time in the stable host environment.
The symbiont of the shallow-water clam Codakia orbicularis, which, like the Riftia symbiont, relies on reduced sulfur compounds as energy source and fixes inorganic carbon, is, unexpectedly, also able to fix atmospheric nitrogen, as shown by metaproteomic, genomic and biochemical analysis. Potentially, this benefits the host, as Codakia digests its symbiont and might thus supplement its diet with organic nitrogen fixed by the symbionts in addition to organic carbon in its nitrogen-poor seagrass habitat.
Infections with Helicobacter pylori are a global challenge that affects both developed and developing countries. This infection is currently treated using multiple antimicrobials that are mostly absorbed after oral administration and subsequently secreted into the gastric lumen. The eradication rates from the different therapeutic regimens, however, are declining nowadays, primarily due to high antibiotic resistance and possibly the mode of drug delivery. H. pylori is commonly found adhering to epithelial cells, and therefore, intragastric drug delivery may be a more direct treatment option. In this work, we developed a new strategy for the local eradication of H. pylori within the stomach.
Initial in vitro experiments revealed that penicillin G shows promising antibiotic activity against resistant strains of H. pylori with MIC values of 0.125 µg/mL. To provide luminal concentrations above the MIC for an extended time, we decided to follow two different formulation strategies: effervescent granules and HPMC-based hydrogel matrix tablets. Among the granule formulations, only one batch was stable and demonstrated excellent performance with respect to drug content, effervescent action, and drug release. It was therefore selected for further in vitro studies. All matrix tablets showed the desired tablet quality requirements and drug release was scalable in vitro by the HPMC concentration.
In order to quantify PGS in various formulations and media, an HPLC method was developed and validated. Due to the stability concerns, the degradation behavior of PGS was studied at different pH. PGS was found to be unstable at acidic pH values, but its stability was higher at more neutral pH values. Sufficient stability was exhibited at pH values above pH 4.5. Due to the instability of PGS in acidic media, alkalizers were added to the matrix tablets to prevent the degradation of the drug within the tablet. Among the alkalizers tested, NaHCO3 showed the most promising results as it significantly enhanced the stability within the matrix and also the concentration of PGS in the dissolution media. The stabilizing effect was caused mainly by the modulation of the microenvironmental pH rather than a pH change in the dissolution media. As a result, these matrix tablets were selected for further in vitro characterization.
In order to guide formulation development, a flow-through model (FTM), which was able to simulate various physiological conditions of the gastric environment, was developed and applied. In contrast to compendial dissolution methods, the FTM allowed studying the effect of gastric secretion, mixing and emptying on the gastric concentration of the drug in vitro. It could be shown that the granules generated a high initial concentration, which decreased over time. On the contrary, the matrix tablets did not provide such a profile due to the absence of pressure events in the model. Further investigations of the matrix tablets in a dissolution stress test device revealed faster drug release if pressure events of physiological relevance are simulated.
In the last part of this thesis, the two formulation concepts were compared in vivo by using the salivary tracer technique. For this purpose, caffeine was used as a model drug. The in vivo investigations suggested that granules administered in a fed state demonstrated longer gastric retention than in a fasted state. In a fed state, effervescent granules provided longer gastric retention of caffeine in comparison to the matrix tablets. Interestingly, the administration of the granules together with 240 mL of tap water provided an even better gastric retention of caffeine than the smaller volume (20 mL). Additional MRI investigations after 4 h of tablets’ intake revealed that the matrix tablets were already disintegrated in vivo.
In conclusion, effervescent granules dosed after food are expected to better maintain intragastric drug concentration over an extended period compared to matrix tablets. Moreover, the carbon dioxide generated after disintegration supports the mixing of the drug with the chyme and thus, provides a uniform distribution of the drug. By this, bacterial sanctuary sites within the stomach can be avoided. The major challenge could be the stability of PGS in acidic media. This problem could be addressed via concomitant administration of PPIs. H2 blockers could also be recommended to address nocturnal acid-breakthrough during the mid-night. In combination with an acid-reducing agent, PGS granule formulations alone or part of the treatment regimens could enable the local eradication of H. pylori directly within the stomach.
Oral administration of drugs is the most common, convenient, safest and economical route of drug administration. There is lack of established tools to study the function of transporters in the intestinal absorption of drugs. Because of its favorable physico-chemical, pharmacokinetic and pharmacodynamic characteristics, trospium could be potentially used as a probe substrate to study the function of drug transporters. Therefore, this study was conducted to examine the suitability of trospium chloride as a probe drug to study the function of multidrug transporters in the human body. To this end, two randomized, controlled, four-period, cross-over pharmacokinetic drug interaction studies of oral and intravenous trospium with co-medication of oral clarithromycin or ranitidine were performed in 24 healthy subjects to mechanistically characterize the role of P-gp, OATP1A2, OCT1, OCT2, MATE1 and MATE2-K in the absorption and disposition of trospium. The contribution of the drug transporters in the absorption and disposition of trospium were examined in isolated systems using in vitro uptake and inhibition assays in transporter transfected human cell lines.
OCT1 (Vmax = 0.8 ± 0.1 nmol/min × mg) is a high capacity transporter of trospium compared to OCT2 (Vmax = 0.04 ± 0.01 nmol/min × mg). But the OCT2 (Km = 0.5 ± 0.1 µM) transporter demonstrated a high affinity in the transport of trospium compared to OCT1 (Km = 17.4 ± 2.1 µM). OCT1 genetic alleles *2, *3, *4 and *7 resulted in significant loss of activity and the alleles *5 and *6 caused complete loss of uptake of trospium. The common OCT2 genetic allele Ser270 caused slight but significant increase in activity of OCT2.
Ranitidine inhibits OCT1 (IC50 = 186 ± 25 µM), MATE1 (IC50 = 134 ± 37 µM) and MATE2-K (IC50 = 35 ± 11 µM)-mediated uptake of trospium in vitro. But it is a weak inhibitor of OCT2 transporter (IC50 = 482 ± 105 µM). Using FDA and EMA in vitro to in vivo extrapolation models, ranitidine was predicted to have a potential inhibition effect on intestinal OCT1 ([I]2/IC50 ~40), renal MATE1 ([I]1/IC50 ~0.02) and MATE2-K ([I]1/IC50 ~0.1) transporters in vivo. Clarithromycin was predicted to cause DDI by inhibiting P-gp-mediated efflux of trospium at the intestine ([I]2/IC50 of ~310) and hepatocytes ([I]3/IC50 ~1). Therefore, co-medication of oral clarithromycin was expected to result in an increase in oral absorption and hepatic clearance of trospium but not changes in distribution volume.
In healthy subjects, oral trospium is slowly (MAT ~10 h) and poorly (F ~10 %) absorbed from the jejunum and cecum/ascending colon, widely distributed into the body (Vss = 5 - 6 l/kg) and slowly eliminated (t1/2 = 9 - 10 h) majorly via renal glomerular filtration and tubular secretion (CLR ~500 ml/min). After co-medication of clarithromycin (inhibitor of P-gp), on the contrary to our IVIVE prediction, we found a non-expected but significant expansion of the shallow and deep distribution spaces for trospium by ~27 %. A single dose administration of trospium with co-medication of ranitidine (inhibitor of OCT1) resulted in no effect on the intestinal absorption of trospium. But the renal clearance of trospium decreased slightly (15 %) but significantly.
Intravenously administered trospium (2 mg TC) might be a suitable probe drug to evaluate the effects of a P-gp inhibitor on distribution of a drug. Oral trospium chloride can be selected for DDI studies with new chemical entities (NCE) with predicted inhibitory potential on OCT1 and P-gp and which are available after oral absorption along the small intestine and in the cecum/ascending colon. Another kind of application of trospium chloride might be pharmacogenomics studies in subjects with functionally relevant polymorphisms of P-gp and OCT1 or in patients with suspected transport failure due to intestinal diseases. The function of the efflux transporters MATE1 and MATE2-K in the PTC of the kidneys can be well assessed with the probe drug trospium by measuring its renal clearance.
Background
In addition to the broad dissemination of pathogenic extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli in human and veterinary medicine and the community, their occurrence in wildlife and the environment is a growing concern. Wild birds in particular often carry clinically relevant ESBL-producing E. coli.
Objectives
We analyzed ESBL-producing and non-ESBL-producing E. coli obtained from wild birds in Mongolia to identify phylogenetic and functional characteristics that would explain the predominance of a particular E. coli clonal lineage in this area.
Methods
We investigated ESBL-producing E. coli using whole-genome sequencing and phylogenetics to describe the population structure, resistance and virulence features and performed phenotypic experiments like biofilm formation and adhesion to epithelial cells. We compared the phenotypic characteristics to non-ESBL-producing E. coli from the same background (Mongolian wild birds) and genomic results to publicly available genomes.
Results and Conclusion
We found ESBL-producing E. coli sequence type (ST) 1159 among wild birds in Mongolia. This clonal lineage carried virulence features typical for extra-intestinal pathogenic or enterotoxigenic E. coli. Comparative functional experiments suggested no burden of resistance in the ST1159 isolates, which is despite their carriage of ESBL-plasmids. Wild birds will likely disseminate these antibiotic-resistant pathogens further during migration.
The role of glutathione peroxidases (GPx) in cancer and their influence on tumor prognosisand the development of anticancer drug resistance has been extensively and controversially discussed.The aim of this study was to evaluate the influence of GPx1 expression on anticancer drug cytotoxicity.For this purpose, a GPx1 knockout of the near-haploid human cancer cell line HAP-1 was generatedand compared to the native cell line with regards to morphology, growth and metabolic rates,and oxidative stress defenses. Furthermore, the IC50values of two peroxides and 16 widely usedanticancer drugs were determined in both cell lines. Here we report that the knockout of GPx1 in HAP-1cells has no significant effect on cell size, viability, growth and metabolic rates. Significant increasesin the cytotoxic potency of hydrogen peroxide andtert-butylhydroperoxide, the anticancer drugscisplatin and carboplatin as well as the alkylating agents lomustine and temozolomide were found.While a concentration dependent increases in intracellular reactive oxygen species (ROS) levelswere observed for both HAP-1 cell lines treated with either cisplatin, lomustine or temozolamide,no significant enhancement in ROS levels was observed in the GPx1 knockout compared to the nativecell line except at the highest concentration of temozolamide. On the other hand, a ca. 50% decreasein glutathione levels was noted in the GPx1 knockout relative to the native line, suggesting thatfactors other than ROS levels alone play a role in the increased cytotoxic activity of these drugs in theGPx1 knockout cells.
Multidrug-resistant gram-negative (MRGN) bacteria are a serious threat to global health. We used genomics tostudy MRGN obtained from houseflies in a tertiary Rwandan hospital. Our analysis revealed a high abundance ofdifferent MRGN includingE. colipathogenic lineage ST131 suggesting the important role of flies in disseminatinghighly virulent pathogens in clinical settings and beyond
Natural products comprise a rich reservoir for innovative drug leads and are a constant
source of bioactive compounds. To find pharmacological targets for new or already known
natural products using modern computer-aided methods is a current endeavor in drug discovery.
Nature’s treasures, however, could be used more effectively. Yet, reliable pipelines for the
large-scale target prediction of natural products are still rare. We developed an in silico workflow
Int. J. Mol. Sci. 2020, 21, 7102; doi:10.3390/ijms21197102 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020, 21, 7102 2 of 18
consisting of four independent, stand-alone target prediction tools and evaluated its performance
on dihydrochalcones (DHCs)—a well-known class of natural products. Thereby, we revealed
four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1,
17β-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a
thorough strategy on how to perform computational target predictions and guidance on using the
respective tools.
Analysis and Reduction of Cellular Heterogeneity in Strain Optimization of Bacillus licheniformis
(2021)
Bacillus species invest substantial resources in inherent cellular processes for pre-adaptation to environmental changes, many of which are dispensable in the controlled environment of industrial bioprocesses. The underlying physiological mechanisms are well characterized in B. subtilis, but only little is known about these processes in the closely related B. licheniformis. Moreover, experimental conditions in previous studies differ from industrial settings in most parameters, foremost in batch cultures or plate-based analysis over fed-batch processes. In this thesis, cellular heterogeneity was analyzed in B. licheniformis in optimized, nutrient-rich media in batch and fed-batch cultivations. Systematic inactivation of genes involved in biofilm formation and synthesis of the flagellar apparatus or global regulators thereof resulted in higher protein production and provided new insights into biofilm formation and cellular heterogeneity in this strain.
The investigation of complex molecular systems by molecular dynamics simulations has been successfully established and proven as a standard method during the last decades. The use of highly optimized algorithms and steadily increasing, generally available computing resources enables even larger and longer simulations. However, the dynamics of the system itself is not accelerated, and it can be trapped in low energy minima that can only be overcome slowly. A number of methods have therefore been developed to address this problem.
Within the context of this dissertation, a novel algorithm based on replica exchange was developed to solve problems with existing methods, which can now be used for large molecular systems with a low resource consumption. Parameter dependence was systematically evaluated and optimized to define guidelines for correct application. This algorithm was successfully applied to various pharmaceutical and biochemical problems, such as protein folding or protein-protein interactions.
Crab Spa, is a stable diffuse-flow hydrothermal vent site located at the 9°N hydrothermal vent field on the East Pacific Rise (EPR). Remarkably, the physicochemical conditions at Crab Spa have remained largely constant since its discovery in 2007 providing a uniquely stable environment in which a well-adapted and stable microbial community has evolved. This microbial community is dominated by the class Campylobacteria, accounting for up to 90% of the community. Little is known, however, about the metabolic pathways that allow the Campylobacteria to dominate the bacterial community at Crab Spa. To address this fundamental question, a two-pronged approach was taken consisting of first determining the dominant metabolic pathways in situ, and second to study those same metabolic pathways and their controls in more detail under defined conditions in vitro in the model campylobacterium Sulfurimonas denitrificans.
Metagenomic analysis of two environmental samples provided the blueprint to determine the metaproteomic profile of the Crab Spa microbial community. This allowed to identify the dominant organisms and their major metabolic pathways sustaining the microbial community at Crab Spa. About 90% of the genes for transcription and protein synthesis of the metagenome sequences belonged to just three genera of Campylobacteria: Sulfurimonas, Sulfurovum and Arcobacter. The metaproteomic analyses confirmed that the active microbial community was dominated by Campylobacteria, carrying out carbon fixation via the reductive TCA cycle predominantly fueled by the oxidation of sulfide and sulfur with nitrate and oxygen. The analysis further revealed that pathways might be partioned between different members of the bacterial community. Proteins involved in electron acceptor–related pathways, in particular denitrification, accounted for up to 20% of the whole metaproteome, which could be seen as an adaptation to the scarcity of electron acceptors at Crab Spa. Conversely, proteins related to electron donor–associated metabolic pathways accounted for less than 0.1% of the metaproteome, possibly in response to the high concentration of the electron donor. To follow up on this hypothesis, chemostat experiments with S. denitrificans were performed under either electron-acceptor or -donor limitation. These experiments confirmed that electron-acceptor limitation lead to the elevated expression of electron-acceptor proteins. However, a higher expression of electron-donor proteins was not observed under electron-donor limitation. Besides hydrogen sulfide, elemental sulfur has the potential to serve as an important electron donor at Crab Spa. However, up to know no information was available on how Campylobacteria might be able to utilize elemental sulfur. For this, S. denitrificans grew with either thiosulfate or cyclooctasulfur (S8) as sole electron donors and its transcriptome and proteome was compared. The results revealed a differential expression of the SOX sulfur oxidation pathway (soxCDYZ and soxABXYZ) in response to the two different sulfur compounds. Based on these findings, a model for the oxidation of cylcooctasulfur was proposed that also applies to other sulfur-oxidizing Campylobacteria and helps in the interpretation of environmental metatranscriptomic and –proteomic data (Götz, Pjevac, et al., 2018; Lahme et al., 2020). The presented results help to better understand the microbial processes at hydrothermal vents.
Marine bacteria represent the most diverse organisms in the marine environment. The majority of these microbes is unknown and unculturable. Algae represent the main nutrient source for bacteria. Macro- and microalgae can consist to 70% of polysaccharides. The metabolic degradation of marine polysaccharides is underexplored and thus these mechanisms have to be investigated. These mechanisms are of high importance to generate defined oligosaccharides for the medical and pharmaceutical applications. The specific structure of marine poly- and oligosaccharides show antiviral activities, e.g. carrageenans from red algae are used for the inhibition of human papillomavirus. Another alginate derived marine polysaccharide show inhibition of the replication of the human immunodeficiency virus (HIV). The degradation mechanisms of marine CAZymes and the structure of marine polysaccharides should be further investigated for their high potential of antiviral activities and the creation of new marine drugs.
Many marine bacteria produce membrane extension like membrane vesicles or appendages but the function of these is poorly understood. In order to investigate their function, especially concerning polysaccharide utilization, proteomic analyses of subcellular compartments were performed. Microscopy analyses revealed that, beside MV, P. distincta forms different appendages, vesicle chains (VC) and thin filaments which were dedicated to extracellular polymeric substance. The formation of MV and VC was independent of growth phase or carbon source. The proteomic data showed that transporters end enzymes for the initial degradation of pectin and alginate were highly abundant in these membrane extensions and that there could be a kind of sorting for proteins in the membrane extensions. Additionally, two PUL encoded alkaline phosphatases and other phosphate acquiring enzymes were abundant in the MV and VC fractions. This indicates, that P. distincta constitutively produces enzymes for phosphate uptake, which would be necessary in the phosphate-limiting environment of the Southern Ocean. On the one hand marine bacteria produce membrane extensions in order to create a larger surface in the nutrient limiting marine environment for an increased chance to get in contact to nutrients and on the other hand the results indicate an accumulation of enzymes responsible for uptake and degradation of carbohydrates and phosphates in the MV and VC. Therefore, the membrane extensions act as nutrient traps and this might be beneficial for the bacteria in the diffuse aquatic environment.
The microbial community structure and the metabolism of bacteria in the Southern Ocean are very poorly investigated. The SO is a harsh environment for all organism but nevertheless, the SO is of high importance for the climate in the world due to the high carbon dioxide uptake. In this study water samples from two different sampling sites (S1 and S2) in the SO were investigated. With a metagenomic and metaproteomic approach the key players and the metabolic activity were analyzed. Additionally, the surface water was inoculated with pectin and incubated for several days in order to analyze polysaccharide utilization loci for pectin degradation and to isolate new pectin degraders. 16S-rDNA analyses revealed the bacterial community from the genomic data. Bacteria were separated in particle-associated and free-living bacteria. The overall particle associated bacterial community at both sampling sites was comparable, with Bacteroidetes and Gammaproteobacteria as the abundant phylum. Within the Gammaproteobacteria the Alteromonadaceae and Colwelliaceae were more abundant at S2 than at S1. The free-living bacteria at S1 were dominated by the Alphaproteobacteria, especially the SAR11 clade I. Metagenomic analyses showed that both sampling sites had comparable PUL composition, but taxonomical classification of PULs was differently. The metaproteome data revealed that PUL encoded enzymes were not highly abundant. Only few CAZymes were found, mostly TonB-dependent transporters belonged to the detected PUL proteins. Taxonomical classification of proteins showed differences between the sampling sites. At S2 the genus Colwellia and Arcobacter were highly increased compared to S1. At this location Candidatus Pelagibacter, Planktomarina and Polaribacter were the abundant taxa. The functional classification at both sampling sites was comparable. The only difference was the high abundance of Epsilonproteobacteria at S2 referable to the Arcobacter species. Nevertheless, the notably taxonomical differences could not be explained by the proteomic data and the functional classification, because no specific metabolic function could be highly addressed to these bacteria. These results assumed that different abundance of the key players could be explained by different environmental conditions. The pectin enriched cultured at both sampling sites were investigated for the functional potential of pectin degrading enzymes. No metaproteomic approach could be performed due to less sampling material. Only one PUL for the degradation of rhamnogalacturonan, a component of pectin, was found at S1. In contrast, bacteria grown on pectin could be isolated from these samples. Genome sequencing of five isolates showed that functional potential of pectin degradation is available. Due to the limitations of sequence alignments, it was not possible to detect a PUL responsible for pectin utilization in the metagenomic data. The results show that the polysaccharide degradation mechanism in the Southern Ocean has to be more investigated to get knowledge about the bacterial activity in the ocean’s surface and the carbon turnover in this underexplored environment.
Within the last decades cancer treatment improved by the availability of more specifically
acting drugs that address molecular target structures in cancer cells. However, those target-sensitive
drugs suffer from ongoing resistances resulting from mutations and moreover they are affected
by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be
treated with the common drugs, so that there have been long efforts to develop drugs to combat
that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in
cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective
efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those
inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4
we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a
MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition
of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active
compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant
anticancer drug as proof-of-principle to encourage for further preclinical studie
The objectives of this study were to ascertain the fecal ESBL/AmpC-E. coli prevalence and to detect risk factors for their occurrence in young pre-weaned calves and their dams on large dairy farms in Germany. From 2018–2019 we investigated 2816 individual fecal samples from pre-weaned dairy calves and their dams, representing seventy-two farms (mean = 667 milking cows) from eight German federal states. To assess possible risk factors associated with ESBL/AmpC-E. coli prevalence in calves and dams, a questionnaire was performed, collecting management data. We observed an ESBL/AmpC-E. coli prevalence of 63.5% (95% CI: 57.4–69.5) among the sampled calves and 18.0% (95% CI: 12.5–23.5) among the dams. On all farms, at least one positive sample was obtained. To date, this is the highest ESBL/AmpC-E. coli prevalence observed in dairy herds in Europe. Feeding with waste milk was identified as a significant risk factor for a high prevalence of ESBL/AmpC-E. coli in calves. Many calves at large dairies in Germany are fed with waste milk due to the large amounts generated as a result of antibiotic dry-off routines and mastitis treatment with antibiotics. Other notable risk factors for high ESBL/AmpC-E. coli in calves were the general fitness/health of dams and calves, and the quality of farm hygiene. Taken together, these findings suggest that new or improved approaches to animal health management, for example, antibiotic dry cow management (selective dry cow therapy) and mastitis treatment (high self-recovery), as well as farm hygiene, should be researched and implemented.
Multi-drug resistant (MDR), gram-negative Enterobacteriaceae, such as Escherichia coli (E. coli) limit therapeutic options and increase morbidity, mortality, and treatment costs worldwide. They pose a serious burden on healthcare systems, especially in developing countries like Rwanda. Several studies have shown the effects caused by the global spread of extended-spectrum beta-lactamase (ESBL)-producing E. coli. However, limited data is available on transmission dynamics of these pathogens and the mobile elements they carry in the context of clinical and community locations in Sub-Saharan Africa. Here, we examined 120 ESBL-producing E. coli strains from patients hospitalized in the University Teaching Hospital of Butare (Rwanda), their attending caregivers as well as associated community members and livestock. Based on whole-genome analysis, the genetic diversification and phylogenetics were assessed. Moreover, the content of carried plasmids was characterized and investigated for putative transmission among strains, and for their potential role as drivers for the spread of antibiotic resistance. We show that among the 30 different sequence types (ST) detected were the pandemic clonal lineages ST131, ST648 and ST410, which combine high-level antimicrobial resistance with virulence. In addition to the frequently found resistance genes blaCTX–M–15, tet(34), and aph(6)-Id, we identified csg genes, which are required for curli fiber synthesis and thus biofilm formation. Numerous strains harbored multiple virulence-associated genes (VAGs) including pap (P fimbriae adhesion cluster), fim (type I fimbriae) and chu (Chu heme uptake system). Furthermore, we found phylogenetic relationships among strains from patients and their caregivers or related community members and animals, which indicates transmission of pathogens. Also, we demonstrated the presence and potential transfer of identical/similar ESBL-plasmids in different strains from the Rwandan setting and when compared to an external plasmid. This study highlights the circulation of clinically relevant, pathogenic ESBL-producing E. coli among patients, caregivers and the community in Rwanda. Combining antimicrobial resistance with virulence in addition to the putative exchange of mobile genetic elements among bacterial pathogens poses a significant risk around the world.
Previous research identified veterinary clinics as hotspots with respect to accumulation and spread of multidrug resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (EC). Therefore, promoting the prudent use of antibiotics to decrease selective pressure in that particular clinical environment is preferable to enhance biosecurity for animal patients and hospital staff. Accordingly, this study comparatively investigated the impact of two distinct perioperative antibiotic prophylaxis (PAP) regimens (short-term versus prolonged) on ESBL-EC carriage of horses subjected to colic surgery. While all horses received a combination of penicillin/gentamicin (P/G) as PAP, they were assigned to either the “single-shot group” (SSG) or the conventional “5-day group” (5DG). Fecal samples collected on arrival (t0), on the 3rd (t1) and on the 10th day after surgery (t2) were screened for ESBL-EC. All isolates were further investigated using whole genome sequences. In total, 81 of 98 horses met the inclusion criteria for this study. ESBL-EC identified in samples available at t0, t1 and t2 were 4.8% (SSG) and 9.7% (5DG), 37% (SSG) and 47.2% (5DG) as well as 55.6% (SSG) and 56.8% (5DG), respectively. Regardless of the P/G PAP regimen, horses were 9.12 times (95% CI 2.79–29.7) more likely to carry ESBL-EC at t1 compared to t0 (p < 0.001) and 15.64 times (95% CI 4.57–53.55) more likely to carry ESBL-EC at t2 compared to t0 (p < 0.001). ESBL-EC belonging to sequence type (ST) 10, ST86, ST641, and ST410 were the most prevalent lineages, with blaCTX–M–1 (60%) being the dominant ESBL gene. A close spatio-temporal relationship between isolates sharing a particular ST was revealed by genome analysis, strongly indicating local spread. Consequently, hospitalization itself has a strong impact on ESBL-EC isolation rates in horses, possibly masking differences between distinct PAP regimens. The results of this study reveal accumulation and spread of multi-drug resistant ESBL-EC among horses subjected to colic surgery with different P/G PAP regimens, challenging the local hygiene management system and work-place safety of veterinary staff. Moreover, the predominance of particular ESBL-EC lineages in clinics providing health care for horses needs further investigation.
Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis was able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.
Abstract
Neutrophils are the most abundant leukocytes in circulation playing a key role in acute inflammation during microbial infections. Phagocytosis, one of the crucial defence mechanisms of neutrophils against pathogens, is amplified by chemotactic leukotriene (LT)B4, which is biosynthesized via 5‐lipoxygenase (5‐LOX). However, extensive liberation of LTB4 can be destructive by over‐intensifying the inflammatory process. While enzymatic biosynthesis of LTB4 is well characterized, less is known about molecular mechanisms that activate 5‐LOX and lead to LTB4 formation during host–pathogen interactions. Here, we investigated the ability of the common opportunistic fungal pathogen Candida albicans to induce LTB4 formation in neutrophils, and elucidated pathogen‐mediated drivers and cellular processes that activate this pathway. We revealed that C. albicans‐induced LTB4 biosynthesis requires both the morphological transition from yeast cells to hyphae and the expression of hyphae‐associated genes, as exclusively viable hyphae or yeast‐locked mutant cells expressing hyphae‐associated genes stimulated 5‐LOX by [Ca2+]i mobilization and p38 MAPK activation. LTB4 biosynthesis was orchestrated by synergistic activation of dectin‐1 and Toll‐like receptor 2, and corresponding signaling via SYK and MYD88, respectively. Conclusively, we report hyphae‐specific induction of LTB4 biosynthesis in human neutrophils. This highlights an expanding role of neutrophils during inflammatory processes in the response to C. albicans infections.
The EyeFlowCell: Development of a 3D-Printed Dissolution Test Setup for Intravitreal Dosage Forms
(2021)
An in vitro dissolution model, the so-called EyeFlowCell (EFC), was developed to test intravitreal dosage forms, simulating parameters such as the gel-like consistency of the vitreous body. The developed model consists of a stereolithography 3D-printed flow-through cell with a polyacrylamide (PAA) gel as its core. This gel needed to be coated with an agarose sheath because of its low viscosity. Drug release from hydroxypropyl methylcellulose-based implants containing either triamcinolone acetonide or fluorescein sodium was studied in the EFC using a schematic eye movement by the EyeMovementSystem (EyeMoS). For comparison, studies were performed in USP apparatus 4 and USP apparatus 7. Significantly slower drug release was observed in the PAA gel for both model drugs compared with the compendial methods. Drug release from fluorescein sodium-containing model implants was completed after 40 min in USP apparatus 4, whereas drug release in the gel-based EFC lasted 72 h. Drug release from triamcinolone acetonide-containing model implants was completed after 35 min in USP apparatus 4 and after 150 min in USP apparatus 7, whereas this was delayed until 96 h in the EFC. These results suggest that compendial release methods may overestimate the drug release rate in the human vitreous body. Using a gel-based in vitro release system such as the EFC may better predict drug release.
Controlling the time point and site of the release of active ingredients within the gastrointestinal tract after administration of oral delivery systems is still a challenge. In this study, the effect of the combination of small capsules (size 3) and large capsules (size 00) on the disintegration site and time was investigated using magnetic resonance imaging (MRI) in combination with a salivary tracer technique. As capsule shells, Vcaps® HPMC capsules, Vcaps® Plus HPMC capsules, gelatin and DRcaps® designed release capsules were used. The three HPMC-based capsules (Vcaps®, Vcaps® Plus and DRcaps® capsules) were tested as single capsules; furthermore, seven DUOCAP® capsule-in-capsule combinations were tested in a 10-way crossover open-label study in six healthy volunteers. The capsules contained iron oxide and hibiscus tea powder as tracers for visualization in MRI, and two different caffeine species (natural caffeine and 13C3) to follow caffeine release and absorption as measured by salivary levels. Results showed that the timing and location of disintegration in the gastrointestinal tract can be measured and differed when using different combinations of capsule shells. Increased variability among the six subjects was observed in most of the capsule combinations. The lowest variability in gastrointestinal localization of disintegration was observed for the DUOCAP® capsule-in-capsule configuration using a DRcaps® designed release capsule within a DRcaps® designed release outer capsule. In this combination, the inner DRcaps® designed release capsule always opened reliably after reaching the ileum. Thus, this combination enables targeted delivery to the distal small intestine. Among the single capsules tested, Vcaps® Plus HPMC capsules showed the fastest and most consistent disintegration.
Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations
(2021)
Transcription factors play a crucial role in regulating biological processes such as cell
growth, differentiation, organ development and cellular signaling. Within this group, proteins
equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding
transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a
variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary
glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which
serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown
to be critically important for efficient regulation of the BCL11B target genes and could therefore
represent a promising target for novel drug therapies. Here, we report the structural basis for
BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure
prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy
transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of
the single ZF domain and directly involved in forming the dimer interface. These findings not only
provide deep insight into how BCL11B acquires its active structure but also represent an important
step towards rational design or selection of potential inhibitors.
Abstract
Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single‐quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13C‐ and 32S‐labeled internal standards with external standard calibration confirmed the superiority of stable isotope‐labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2 = 0.945). In contrast to off‐line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2‐based extraction and separation techniques for potentially infective biomatrices.
Summary
Outer membrane extensions are common in many marine bacteria. However, the function of these surface enlargements or extracellular compartments is poorly understood. Using a combined approach of microscopy and subproteome analyses, we therefore examined Pseudoalteromonas distincta ANT/505, an Antarctic polysaccharide degrading gamma‐proteobacterium. P. distincta produced outer membrane vesicles (MV) and vesicle chains (VC) on polysaccharide and non‐polysaccharide carbon sources during the exponential and stationary growth phase. Surface structures of carbohydrate‐grown cells were equipped with increased levels of highly substrate‐specific proteins. At the same time, proteins encoded in all other polysaccharide degradation‐related genomic regions were also detected in MV and VC samples under all growth conditions, indicating a basal expression. In addition, two alkaline phosphatases were highly abundant under non‐limiting phosphate conditions. Surface structures may thus allow rapid sensing and fast responses in nutritionally deprived environments. It may also facilitate efficient carbohydrate processing and reduce loss of substrates and enzymes by diffusion as important adaptions to the aquatic ecosystem.
Pentathiepins are cyclic polysulfides that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and potently inhibit GPx1. These properties render this class of compounds promising candidates for the development of anticancer drugs. However, the biological effects and how they intertwine to promote high cytotoxicity have not been systematically assessed throughout a panel of cancer cell lines from distinct tissues of origin. In this thesis, six novel pentathiepins were analyzed and constitute the second generation of compounds with additional properties such as fluorescence or improved water solubility to facilitate cellular testing. All compounds underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigations of the inhibitory potential with regards to GPx1 and cell proliferation, examined the cytotoxicity in human cancer cell lines, as well as the induction of oxidative stress and DNA strand breaks. Furthermore, selected hallmarks of apoptosis, ferroptosis, and autophagy were studied. Experimental approaches regarding these cellular mechanisms included observing morphological changes, detecting phosphatidyl serine exposure and caspase activity, and quantifying cleaved PARP1 and levels of LC3B II. In addition, the analysis of the cell cycle aimed to identify aberrations or arrests in cell division.
Five of the six tested pentathiepins proved to be potent inhibitors of the GPx1, while all six exerted high cytotoxic and antiproliferative activity, although to different extents. There was a clear connection observed between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks both extra- and intracellularly. Furthermore, various experiments supported apoptosis but not ferroptosis as the mechanism of cell death in four different cell lines. In particular, the externalization of PS, the detection of activated caspases, and the cleavage of PARP1 corroborated this conclusion. Additionally, indications for autophagy were found, but more investigations are required to verify the current data. The findings of this dissertation are mainly in line with the postulated mechanism of action proposed for pentathiepins and a previous publication from our group that described their biological activity. However, the influence of modulators such as oxygen and GSH on the biological effects was ambiguous and dependent on the compound. The expression profile of the cell lines concerning GPx1 and CAT did not influence the cellular response toward the treatment, whereas the cell doubling time correlated with the cytotoxicity.
As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for future optimization of the anticancer activity of pentathiepins. An analysis of the structure-activity relationships revealed that the piperazine scaffold was associated with superior biological activity compared to the pyrrolo-pyrazine backbone. Furthermore, substituents with electron-withdrawing properties or those providing a free electron pair, such as fluorine or morpholine, were advantageous. These findings should help design and synthesize the next generation of pentathiepins, thereby expanding the library of compounds, allowing for the further deduction of structure-activity relationships and an improved understanding of their mechanism of action.
The poor aqueous solubility of many drug substances has been addressed using different solubility enhancement approaches in the pharmaceutical technology field over the last decades. In this context, advanced drug delivery systems based on lipids referred to as SNEDDS were used to overcome solubility limitations of drugs, that are often associated with a low bioavailability after oral administration. There are numerous examples in the literature for the development of L-SNEDDS, which have led to some pharmaceutical products available on the market. As L-SNEDDS development using conventional methods requires a lot of time and experimental effort, a streamlining of this procedure was aimed in the first part of the presented work.
Starting with the development of L-SNEDDS formulations for solubility enhancement of poorly-water soluble drugs, extensive solubility studies with different BCS Class II drugs were performed in various excipients to determine drugs with high solubilities in these excipients as well as to evaluate multiple excipients for their suitability to be used in L-SNEDDS formulations. Celecoxib, efavirenz and fenofibrate were selected as model drugs and a pre-selection of excipients for further development was made. In a next step, a novel screening approach for L-SNEDDS formulation development based on a customized mapping method in a special triangular mixture design was established. This customized tool for L-SNEDDS development comprised the systematic analysis of results obtained with different in vitro characterization methods such as droplet size analysis and distribution, transmittance measurement and emulsification performance assessment. Furthermore, the novel approach streamlined the procedure for L-SNEDDS development as a reduction of experimental effort and time compared to conventional methods was achieved. The most promising L-SNEDDS formulations determined via the customized screening tool approach showed high drug release of celecoxib, efavirenz as well as fenofibrate, and clearly indicated that this method was suitable for efficiently designing stable and rapidly releasing L-SNEDDS formulations incorporating poorly water-soluble drugs.
After the successful development of L-SNEDDS formulations with different drug substances using the novel screening approach, a further aspect of this work dealt with conversion of L-SNEDDS into S-SNEDDS, since a limited storage stability has been reported for many L-SNEDDS formulations. The conversion into S-SNEDDS required the determination of appropriate solid carriers with different material properties depending on the manufacturing process. As a first technological approach, adsorption to a solid carrier was investigated by adding a carrier to drug-loaded L-SNEDDS applying a defined mixing ratio resulting in a solid, particulate formulation. When performing drug release studies, S-SNEDDS based on different commercial
carrier materials revealed major limitations due to incomplete drug release. Thus, a tailor-made microparticulate carrier material based on cellulose was developed for the purpose of adsorbing L-SNEDDS and presented with superior performance compared to conventional adsorbents based on cellulose or silica. Based on the obtained results, this novel cellulose-based microparticle prepared with gum arabic as a binder was determined to be the most promising material amongst all adsorptive carriers that were investigated.
In addition to the technology approach of adsorption, another manufacturing process was considered in the course of the present work, which focused on the preparation of S-SNEDDS by means of HME. As a successful conversion of L-SNEDDS into S-SNEDDS using HME processing requires at least one additional polymeric component, a selection of marketed (co-)polymers that were frequently used in the field of solubility enhancement were evaluated for their suitability in this context. Critical process parameters and target properties of the (co-)polymers were determined, ultimately leading to the idea of developing a novel, customized polymer in order to perform the conversion step via HME in a more suitable and effective manner. In this context, a new copolymer referred to as ModE, as it disclosed a structural association with the commercially available copolymer EUDRAGIT® E PO, was developed. The novel copolymer ModE was evaluated for its suitability for different formulation technologies and showed promising results when used for S-SNEDDS and ASD formulations prepared by the HME process. Different variants of ModE in terms of Mw, Tg and PDI were synthesized via radical polymerization and it was found that the modification of Mw, Tg and PDI of the novel aminomethacrylate-based copolymer had significant effects on drug release as well as storage stability of S-SNEDDS and ASDs. The ModE copolymer type with a Mw of 173 kDa turned out to be the most suitable candidate for S-SNEDDS development using HME technology. In addition, drug-loaded S-SNEDDS based on the ModE variant 173 kDa were storage stable and presented with the highest drug release among all S-SNEDDS formulations tested.
In conclusion, a novel screening tool approach for efficient L-SNEDDS development was established in order to streamline the process for obtaining stable and rapidly releasing L-SNEDDS formulations which improved the solubility of poorly water-soluble drugs. Apart from the L-SNEDDS development process, the conversion from L-SNEDDS into S-SNEDDS was successfully performed using the technology approaches of adsorption to a solid carrier and HME processing. An improved storage stability compared to L-SNEDDS as well as high drug release were achieved for several S-SNEDDS formulations, especially for those prepared with tailor-made materials. Based on the results obtained for S-SNEDDS formulations produced via adsorption, especially in terms of drug release performance, the new cellulose-based
microparticle carriers (M-GA and M-MC) turned out to be the most suitable materials. S-SNEDDS that were manufactured via HME presented with a superior performance regardless of the incorporated drug when comparing the results of S-SNEDDS with those of the corresponding ASDs regarding drug release performance, amorphicity/crystallinity and storage stability. In this context, among all S-SNEDDS formulations prepared via HME, S-SNEDDS based on the ModE variant 173 kDa showed the best results, especially when using the drug substances celecoxib and efavirenz. Although the S-SNEDDS formulation approach is still largely unexplored, based on the research results generated in the present work, it represents a promising technology platform that should definitely be further developed in future experiments.
Klebsiella pneumoniae is a common member of the intestinal flora of vertebrates. In addition to opportunistic representatives, hypervirulent (hvKp) and antibiotic-resistant K. pneumoniae (ABR-Kp) occur. While ABR-Kp isolates often cause difficult-to-treat diseases due to limited therapeutic options, hvKp is a pathotype that can infect healthy individuals often leading to recurrent infection. Here, we investigated the clinical K. pneumoniae isolate PBIO3459 obtained from a blood sample, which showed an unusual colony morphology. By combining whole-genome and RNA sequencing with multiple in vitro and in vivo virulence-associated assays, we aimed to define the respective Klebsiella subtype and explore the unusual phenotypic appearance. We demonstrate that PBIO3459 belongs to sequence type (ST)20 and carries no acquired resistance genes, consistent with phenotypic susceptibility tests. In addition, the isolate showed low-level virulence, both at genetic and phenotypic levels. We thus suggest that PBIO3459 is an opportunistic (commensal) K. pneumoniae isolate. Genomic comparison of PBIO3459 with closely related ABR-Kp ST20 isolates revealed that they differed only in resistance genes. Finally, the unusual colony morphology was mainly associated with carbohydrate and amino acid transport and metabolism. In conclusion, our study reveals the characteristics of a Klebsiella sepsis isolate and suggests that opportunistic representatives likely acquire and accumulate antibiotic resistances that subsequently enable their emergence as ABR-Kp pathogens.
Essential Oils as Multicomponent Mixtures and Their Potential for Human Health and Well-Being
(2022)
Essential oils (EOs) and their individual volatile organic constituents have been an inherent part of our civilization for thousands of years. They are widely used as fragrances in perfumes and cosmetics and contribute to a healthy diet, but also act as active ingredients of pharmaceutical products. Their antibacterial, antiviral, and anti-inflammatory properties have qualified EOs early on for both, the causal and symptomatic therapy of a number of diseases, but also for prevention. Obtained from natural, mostly plant materials, EOs constitute a typical example of a multicomponent mixture (more than one constituent substances, MOCS) with up to several hundreds of individual compounds, which in a sophisticated composition make up the property of a particular complete EO. The integrative use of EOs as MOCS will play a major role in human and veterinary medicine now and in the future and is already widely used in some cases, e.g., in aromatherapy for the treatment of psychosomatic complaints, for inhalation in the treatment of respiratory diseases, or topically administered to manage adverse skin diseases. The diversity of molecules with different functionalities exhibits a broad range of multiple physical and chemical properties, which are the base of their multi-target activity as opposed to single isolated compounds. Whether and how such a broad-spectrum effect is reflected in natural mixtures and which kind of pharmacological potential they provide will be considered in the context of ONE Health in more detail in this review.
The present study covers the synthesis, purification and evaluation of a novel aminomethacrylate-based copolymer in terms of its suitability for improving the solubility and in vitro release of poorly water-soluble drug compounds. The new copolymer was synthesized by solvent polymerization with radical initiation and by use of a chain transfer agent. Based on its composition, it can be considered as a modified type of dimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylate “EUDRAGIT® E PO” (ModE). ModE was specifically developed to provide a copolymer with processing and application properties that exceed those of commercially available (co-)polymers in solubility enhancement technologies where possible. By varying the concentration of the chain transfer agent in the radical polymerization process, the molecular weight of ModE was varied in a range of 173–305 kDa. To evaluate the solubility-enhancing properties of ModE, a series of drug-loaded extrudates were prepared by hot melt extrusion using the novel—as well as several commercially available—(co-)polymers. These extrudates were then subjected to comparative tests for amorphousness, solubility-enhancing properties, storage stability, and drug release. Celecoxib, efavirenz, and fenofibrate were used as model drugs in all experiments. Of all the (co-)polymers included in the study, ModE with a molecular weight of 173 kDa showed the best performance in terms of desired properties and was shown to be particularly suitable for preparing amorphous solid dispersions (ASDs) of the three model drugs, which in a first set of dissolution experiments showed better release behavior under pH conditions of the fasting stomach than higher molecular weight ModE types, as well as a variety of commercially available (co-)polymers. Therefore, the results demonstrate the successful synthesis of a new copolymer, which in future studies will be investigated in more detail for universal application in the field of solubility enhancement.
Antimicrobial resistance is an increasing global problem and complicates successful treatments of bacterial infections in animals and humans. We conducted a longitudinal study in Mecklenburg-Western Pomerania to compare the occurrence of ESBL-producing Escherichia (E.) coli in three conventional and four organic pig farms. ESBL-positive E. coli, especially of the CTX-M type, were found in all fattening farms, confirming that antimicrobial resistance is widespread in pig fattening and affects both conventional and organic farms. The percentage of ESBL-positive pens was significantly higher on conventional (55.2%) than on organic farms (44.8%) with similar proportions of ESBL-positive pens on conventional farms (54.3–61.9%) and a wide variation (7.7–84.2%) on organic farms. Metadata suggest that the farms of origin, from which weaner pigs were purchased, had a major influence on the occurrence of ESBL-producing E. coli in the fattening farms. Resistance screening showed that the proportion of pens with multidrug-resistant E. coli was similar on conventional (28.6%) and organic (31.5%) farms. The study shows that ESBL-positive E. coli play a major role in pig production and that urgent action is needed to prevent their spread.
In the search for alternative treatment options for infections with multi-resistant germs, traditionally used medicinal plants are currently being examined more intensively. In this study, the antimicrobial and anti-biofilm activities of 14 herbal drugs were investigated. Nine of the tested drugs were traditionally used in Europe for treatment of local infections. For comparison, another five drugs monographed in the European Pharmacopoeia were used. Additionally, the total tannin and flavonoid contents of all tested drugs were analyzed. HPLC fingerprints were recorded to obtain further insights into the components of the extracts. The aim of the study was to identify herbal drugs that might be useable for treatment of infectious diseases, even with multidrug resistant E. coli, and to correlate the antimicrobial activity with the total content of tannins and flavonoids. The agar diffusion test and anti-biofilm assay were used to evaluate the antimicrobial potential of different extracts from the plants. Colorimetric methods (from European Pharmacopeia) were used for determination of total tannins and flavonoids. The direct antimicrobial activity of most of the tested extracts was low to moderate. The anti-biofilm activity was found to be down to 10 µg mL−1 for some extracts. Tannin contents between 2.2% and 10.4% of dry weight and total flavonoid contents between 0.1% and 1.6% were found. Correlation analysis indicates that the antimicrobial and the anti-biofilm activity is significantly (p < 0.05) dependent on tannin content, but not on flavonoid content. The data analysis revealed that tannin-rich herbal drugs inhibit pathogens in different ways. Thus, some of the tested herbal drugs might be useable for local infections with multi-resistant biofilm-forming pathogens. For some of the tested drugs, this is the first report about anti-biofilm activity, as well as total tannin and flavonoid content.
In the search for alternative treatment options for infections with multi-resistant germs,
traditionally used medicinal plants are currently being examined more intensively. In this study,
the antimicrobial and anti-biofilm activities of 14 herbal drugs were investigated. Nine of the tested
drugs were traditionally used in Europe for treatment of local infections. For comparison, another
five drugs monographed in the European Pharmacopoeia were used. Additionally, the total tannin
and flavonoid contents of all tested drugs were analyzed. HPLC fingerprints were recorded to ob-
tain further insights into the components of the extracts. The aim of the study was to identify herbal
drugs that might be useable for treatment of infectious diseases, even with multidrug resistant E.
coli, and to correlate the antimicrobial activity with the total content of tannins and flavonoids. The
agar diffusion test and anti-biofilm assay were used to evaluate the antimicrobial potential of dif-
ferent extracts from the plants. Colorimetric methods (from European Pharmacopeia) were used for
determination of total tannins and flavonoids. The direct antimicrobial activity of most of the tested
extracts was low to moderate. The anti-biofilm activity was found to be down to 10 µg mL −1 for
some extracts. Tannin contents between 2.2% and 10.4% of dry weight and total flavonoid contents
between 0.1% and 1.6% were found. Correlation analysis indicates that the antimicrobial and the
anti-biofilm activity is significantly (p < 0.05) dependent on tannin content, but not on flavonoid
content. The data analysis revealed that tannin-rich herbal drugs inhibit pathogens in different
ways. Thus, some of the tested herbal drugs might be useable for local infections with multi-re-
sistant biofilm-forming pathogens. For some of the tested drugs, this is the first report about anti-
biofilm activity, as well as total tannin and flavonoid content.
Fibers and yarns are part of everyday life. So far, fibers that are also used pharmaceutically have mainly been produced by electrospinning. The common use of spinning oils and the excipients they contain, in connection with production by melt extrusion, poses a regulatory challenge for pharmaceutically usable fibers. In this publication, a newly developed small-scale direct-spinning melt extrusion system is described, and the pharmaceutically useful polyvinyl filaments produced with it are characterized. The major parts of the system were newly developed or extensively modified and manufactured cost-effectively within a short time using rapid prototyping (3D printing) from various materials. For example, a stainless-steel spinneret was developed in a splice design for a table-top melt extrusion system that can be used in the pharmaceutical industry. The direct processing of the extruded fibers was made possible by a spinning system developed called Spinning-Rosi, which operates continuously and directly in the extrusion process and eliminates the need for spinning oils. In order to prevent instabilities in the product, further modifications were also made to the process, such as a the moisture encapsulation of the melt extrusion line at certain points, which resulted in a bubble-free extrudate with high tensile strength, even in a melt extrusion line without built-in venting.
The study aimed to examine the influence of a rotating magnetic field (RMF) of two different frequencies (5 and 50 Hz) on the expression of regulatory (agrA, hld, rot) and staphylococcal enterotoxin (SE—sea, sec, sel) genes as well as the production of SEs (SEA, SEC, SEL) by the Staphylococcus aureus FRI913 strain cultured on a medium supplemented with a subinhibitory concentration of trans-anethole (TA). Furthermore, a theoretical model of interactions between the bacterial medium and bacterial cells exposed to RMF was proposed. Gene expression and SEs production were measured using quantitative real-time PCR and ELISA techniques, respectively. Based on the obtained results, it was found that there were no significant differences in the expression of regulatory and SE genes in bacteria simultaneously cultured on a medium supplemented with TA and exposed to RMF at the same time in comparison to the control (unexposed to TA and RMF). In contrast, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF or when they were exposed to RMF of 50 Hz (but not to TA), a significant increase in agrA and sea transcripts as compared to the unexposed control was found. Moreover, the decreased level of sec transcripts in bacteria cultured without TA but exposed to RMF of 50 Hz was also revealed. In turn, a significant increase in SEA and decrease in SEC and SEL production was observed in bacteria cultured on a medium supplemented with TA and simultaneously exposed to RMFs. It can be concluded, that depending on SE and regulatory genes expression as well as production of SEs, the effect exerted by the RMF and TA may be positive (i.e., manifests as the increase in SEs and/or regulatory gene expression of SEs production) or negative (i.e., manifests as the reduction in both aforementioned features) or none.
Highly Virulent and Multidrug-Resistant Escherichia coli Sequence Type 58 from a Sausage in Germany
(2022)
Studies have previously described the occurrence of multidrug-resistant (MDR) Escherichia coli in human and veterinary medical settings, livestock, and, to a lesser extent, in the environment and food. While they mostly analyzed foodborne E. coli regarding phenotypic and sometimes genotypic antibiotic resistance and basic phylogenetic classification, we have limited understanding of the in vitro and in vivo virulence characteristics and global phylogenetic contexts of these bacteria. Here, we investigated in-depth an E. coli strain (PBIO3502) isolated from a pork sausage in Germany in 2021. Whole-genome sequence analysis revealed sequence type (ST)58, which has an internationally emerging high-risk clonal lineage. In addition to its MDR phenotype that mostly matched the genotype, PBIO3502 demonstrated pronounced virulence features, including in vitro biofilm formation, siderophore secretion, serum resilience, and in vivo mortality in Galleria mellonella larvae. Along with the genomic analysis indicating close phylogenetic relatedness of our strain with publicly available, clinically relevant representatives of the same ST, these results suggest the zoonotic and pathogenic character of PBIO3502 with the potential to cause infection in humans and animals. Additionally, our study highlights the necessity of the One Health approach while integrating human, animal, and environmental health, as well as the role of meat products and food chains in the putative transmission of MDR pathogens.
Antimicrobial resistance (AMR) is a serious global health threat and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are a major contributor. This study aimed to gain a deeper insight into the AMR burden of wild animals. In total, 1595 fecal samples were collected by two systematic searches in Mecklenburg-Western Pomerania, north-east Germany. Samples were screened for ESBL-carrying Escherichia (E.) coli and isolates found were further analyzed using antimicrobial susceptibility testing and whole-genome sequencing. We found an estimated prevalence of 1.2% ESBL-producing E. coli in wild boar and 1.1% in wild ruminants. CTX-M-1 was the most abundant CTX-M type. We also examined fecal samples from wild boar and wild ruminants using shotgun metagenomics to gain insight into the resistome in wild animals. The latter revealed significantly lower normalized counts for AMR genes in wildlife samples compared to farm animals. The AMR gene levels were lower in wild ruminants than in wild boar. In conclusion, our study revealed a low prevalence of ESBL-producing E. coli and a low overall AMR gene burden in wild boar and wild ruminants, probably due to the secluded location of the search area.
Characterization, Chemical Compounds and Biological Activities of Marrubium vulgare L. Essential Oil
(2022)
As consumer trends shift towards more natural and ecological consumption patterns, industrialists are actively working towards substituting synthetic chemicals with natural and vegan products that contain bioactive properties. Thus, considering the shifts in customer demand and the growing concern around vegetable sourced productions, this work aims to contribute to the valorization of aromatic and medicinal Moroccan plants. By focusing on the Marrubium vulgare L. species, our objective is to carry out a physicochemical characterization to determine its chemical composition and biological activities. The volatile fraction collected by hydrodistillation (0.61%) and analyzed by GC-MS (gas chromatography coupled to mass spectrometry) contains five main compounds: 3-Thujanone, Eugenol, Topanol, Menthone and Piperitone. The antioxidant activity has been estimated by applying the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test and the ferric reducing antioxidant power (FRAP). The values of inhibitory concentration prove that our oil is a good antioxidant, with values of IC50 = 1.136 mg/mL and IC50 = 2.998 mg/mL, respectively, for the DPPH and FRAP tests. The results of the antifungal activity indicate a significant inhibition of mycelial growth for both tested molds, as well as a total inhibition of spore production at a concentration of 0.25 µL/mL.
In recent years, the colon has become a hot topic in biopharmaceutical research as several in vitro models of the human colon have been presented. A major focus is on the characterization of the microbiota and its capabilities. The aim of the present study was to further develop the MimiCol, preserving its properties and accelerating data acquisition. Emphasis was placed on the simplicity of its design and easy scalability. To prove the viability of the concept, degradation of sulfasalazine was investigated, and the bacterial composition during the experiment was assessed by 16S rRNA sequencing. The transfer of the experimental conditions to the new model was successful. Commercially available components were implemented in the setup. The model MimiCol3 represented the colon ascendens satisfactorily in its properties regarding volume, pH value, and redox potential. 16S rRNA sequencing led to further insights into the bacterial composition in the vessels. Degradation of sulfasalazine was in good agreement with in vivo data. The new model of the colon ascendens MimiCol3 enabled us to collect more reliable data, as three experiments were conducted simultaneously under the same conditions.
Transmucosal drug delivery systems can be an attractive alternative to conventional oral dosage forms such as tablets. There are numerous in vitro methods to estimate the behavior of mucoadhesive dosage forms in vivo. In this work, a tensile test system was used to measure the mucoadhesion of polyvinyl alcohol films. An in vitro screening of potential influencing variables was performed on biomimetic agar/mucin gels. Among the test device-specific factors, contact time and withdrawal speed were identified as influencing parameters. In addition, influencing factors such as the sample area, which showed a linear relationship in relation to the resulting work, and the liquid addition, which led to an abrupt decrease in adhesion, could be identified. The influence of tissue preparation was investigated in ex vivo experiments on porcine small intestinal tissue. It was found that lower values of Fmax and Wad were obtained on processed and fresh tissue than on processed and thawed tissue. Film adhesion on fresh, unprocessed tissue was lowest in most of the animals tested. Comparison of ex vivo measurements on porcine small intestinal tissue with in vitro measurements on agar/mucin gels illustrates the inter- and intra-individual variability of biological tissue.
Species of the genus Drosera, known for carnivorous plants, such as sundew, have been traditionally used for centuries as medicinal plants. Efficacy-determining compounds are naphthoquinones and flavonoids. Flavonoids possess a broad spectrum of bioactive properties, including biofilm inhibitory activity. Biofilms render antibiotics ineffective, contributing to the current rise in antimicrobial resistance. In this study, the biofilm inhibitory activity of two European sundew species (Drosera rotundifolia and Drosera intermedia) grown agriculturally in Germany and four commercial sundew products (declared as Drosera longifolia, Drosera sp. and Drosera planta trit.) against three multidrug-resistant Escherichia coli strains was tested. The aim of the study was to comparatively investigate the biofilm inhibitory potential of sundew species extracts grown locally in northern Germany and commercial sundew products. The minimum biofilm inhibitory concentration of the European sundew species was approx. 35 µg mL−1. In comparison, commercial sundew products ranged in concentration from 75 to 140 µg mL−1. Additionally, individual compounds isolated from European sundew were tested. Among these compounds, biofilm inhibitory activity was determined for four of the eight substances, with 2″-O-galloyl hyperoside standing out for its activity (38 µg mL−1). The whole plant extracts of Drosera rotundifolia and Drosera intermedia proved to be more effective than the commercial products and the single compounds in its biofilm inhibition activity against Escherichia coli strains. Sundew extracts may serve as a potential therapeutic approach for targeting biofilm production.
Abstract
Aim
To verify synergistic effects, we investigated the antimicrobial activity of seven phenolic phytochemicals (gallic acid; epicatechin; epigallocatechin gallate; daidzein; genistein; myricetin; 3‐hydroxy‐6‐methoxyflavone) in combination with six antibiotics against multidrug‐resistant isolates from the ESKAPE group.
Methods and Results
To investigate single phytochemicals and combinations, initial microdilution and checkerboard assays were used, followed by time‐kill assays to evaluate the obtained results. The research revealed that phenolic compounds on their own resulted in little or no inhibitory effects. During preliminary tests, most of the combinations resulted in indifference (134 [71.3%]). In all, 30 combinations led to antagonism (15.9%); however, 24 showed synergistic effects (12.8%). The main tests resulted in nine synergistic combinations for the treatment of four different bacteria strains, including two substances (3‐hydroxy‐6‐methoxyflavone, genistein) never tested before in such setup. Time‐kill curves for combinations with possible synergistic effects confirmed the results against Acinetobacter baumannii as the one with the greatest need for research.
Conclusions
The results highlight the potential use of antibiotic–phytocompound combinations for combating infections with multi‐resistant pathogens. Synergistic combinations could downregulate the resistance mechanisms of bacteria.
Significance and Impact of the Study
The aim of this study is to demonstrate the potential use of phenolic natural compounds in combination with conventional antibiotics against multidrug‐resistant bacteria of the ESKAPE group. Due to synergistic effects of natural phenolic compounds combined with antibiotics, pathogens that are already resistant to antibiotics could be resensitized as we were able to reduce their MICs back to sensitive. In addition, combination therapies could prevent the development of resistance by reducing the dose of antibiotics. This approach opens up the basis for future development of antimicrobial therapy strategies, which are so urgently needed in the age of multidrug‐resistant pathogens.
Livestock animals, especially poultry, are a known reservoir for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli). They may enter the pen either via positive day-old chicks or via the environment. We developed a mathematical model to illustrate the entry and dissemination of resistant bacteria in a broiler pen during one fattening period in order to investigate the effectiveness of intervention measures on this infection process. Different management measures, such as varying amounts of litter, a slow-growing breed or lower stocking densities, were tested for their effects on broiler colonization. We also calculated the impact of products that may influence the microbiota in the chicks’ digestive tract, such as pre- or probiotics, feed supplements or competitive exclusion products. Our model outcomes show that a contaminated pen or positive chicks at the beginning of the fattening period can infect the entire flock. Increasing the amount of litter and decreasing the stocking density were shown to be effective in our model. Differences in the route of entry were found: if the chicks are already positive, the litter quantity must be increased to at least six times the standard of 1000 g/m2, whereas, if the pen is contaminated on the first day, three times the litter quantity is sufficient. A reduced stocking density of 20 kg/m2 had a significant effect on the incidence of infection only in a previously contaminated pen. Combinations of two or three measures were effective in both scenarios; similarly, feed additives may be beneficial in reducing the growth rate of ESBL-producing E. coli. This model is a valuable tool for evaluating interventions to reduce the transmission and spread of resistant bacteria in broiler houses. However, data are still needed to optimize the model, such as growth rates or survival data of ESBL-producing E. coli in different environments.
The present study focused on a new formulation approach to improving the solubility of drugs with poor aqueous solubility. A hot melt extrusion (HME) process was applied to prepare drug-loaded solid self-nanoemulsifying drug delivery systems (S-SNEDDS) by co-extrusion of liquid SNEDDS (L-SNEDDS) and different polymeric carriers. Experiments were performed with L-SNEDDS formulations containing celecoxib, efavirenz or fenofibrate as model drugs. A major objective was to identify a polymeric carrier and process parameters that would enable the preparation of stable S-SNEDDS without impairing the release behavior and storage stability of the L-SNEDDS used and, if possible, even improving them further. In addition to commercially available (co)polymers already used in the field of HME, a particular focus was on the evaluation of different variants of a recently developed aminomethacrylate-based copolymer (ModE) that differed in Mw. Immediately after preparation, the L-SNEDDS and S-SNEDDS formulations were tested for amorphicity by differential scanning calorimetry. Furthermore, solubility and dissolution tests were performed. In addition, the storage stability was investigated at 30 °C/65% RH over a period of three and six months, respectively. In all cases, amorphous formulations were obtained and, especially for the model drug celecoxib, S-SNEDDS were developed that maintained the rapid and complete drug release of the underlying L-SNEDDS even over an extended storage period. Overall, the data obtained in this study suggest that the presented S-SNEDDS approach is very promising, provided that drug-loaded L-SNEDDS are co-processed with a suitable polymeric carrier. In the case of celecoxib, the E-173 variant of the novel ModE copolymer proved to be a novel polymeric carrier with great potential for application in S-SNEDDS. The presented approach will, therefore, be pursued in future studies to establish S-SNEDDS as an alternative formulation to other amorphous systems.