Cloning and heterologous expression of three type II PKS gene clusters from Streptomyces bottropensis

Mensacarcin is a potent cytotoxic agent isolated from Streptomyces bottropensis. It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of S. bottropensis. Screening a cosmid library of S. bottropensis with minimal PKS-gene-specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the msn cluster (mensacarcin biosynthesis), the rsl cluster (rishirilide biosynthesis), and the mec cluster (putative spore pigment biosynthesis). Interestingly, luciferase-like oxygenases, which are very rare in Streptomyces species, are enriched in both the msn cluster and the rsl cluster. Three cosmids, cos2 (containing the major part of the msn cluster), cos3 (harboring the mec cluster), and cos4 (spanning probably the whole rsl cluster) were introduced into the general heterologous host Streptomyces albus by intergeneric conjugation. Expression of cos2 and cos4 in S. albus led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope-feeding experiments in S. bottropensis, a putative biosynthesis pathway for mensacarcin is proposed.     

Effect of continuous and intermittent ultrasound on drying time and effective diffusivity during convective drying of apple and red bell pepper

A new approach of ultrasound-assisted convective drying was tested in this study using a screen as product support and sound transmitting surface. Red bell pepper and apple cubes were dried at 70 °C in a laboratory scale hot-air drying oven. In addition to continuous ultrasound used for both products, intermittent ultrasound was applied for apple drying reducing net sonication time to 50% and 10%. Significant improvements of drying characteristics were observed for continuous sonication and intermittent ultrasound at 50% net sonication time. Experimental data were fitted with eight models, among which the Midilli model resulted in the best fit with R² > 0.9988 and reduced χ² ? 0.0002. The influence of ultrasound on drying time to reach 20% and 30% residual moisture content as well as on effective moisture diffusivities and drying periods is discussed.     

The effect of NaCl-free processing and high pressure on the fate of Listeria monocytogenes and Salmonella on sliced smoked dry-cured ham

NaCl is an important multifunctional ingredient applied in dry-cured ham elaboration. However, its excessive intake has been linked to serious cardiovascular diseases causing a recent increase in the development of reduced salt products. In the present study Listeria monocytogenes and Salmonella, food-borne pathogens which can cross-contaminate post processed products, were spiked with < 100 CFU/g on slices of both standard (S) and NaCl-free processed (F) (elaborated with KCl + potassium lactate instead of NaCl) smoked dry-cured ham. Although L. monocytogenes and Salmonella counts decreased faster in S ham, pathogens were present in both types of non-pressure treated ham during the entire refrigerated storage period (112 days). Pressurisation at 600 MPa for 5 min caused the elimination of both pathogens in S ham after 14 days. In contrast, Salmonella and L. monocytogenes were present in F ham until days 28 and 56, respectively, indicating that the NaCl-free processed dry-cured ham had lower stability than standard smoked dry-cured ham.     

Switching azonaphthols containing a side chain with limited flexibility. Part 1. Synthesis and tautomeric properties

A series of azo dyes, possessing amide fragments with restricted flexibility tethered to 4-(phenyldiazenyl)naphthalen-1-ol, was obtained from 1-hydroxy-2-naphthoic acid by subsequent conversion to amides and diazo coupling. It was shown that the position of the tautomeric equilibrium in solution strongly depends on the solvent in both UV and NMR concentration scale. The compounds exist as pure enol forms in chloroform and hydrocarbons, while in polar solvents (acetone, acetonitrile, alcohols) a tautomeric mixture is observed. According to the quantum-chemical calculations the aggregation of the keto tautomer is the possible reason for this shift in the position of the tautomeric equilibrium. To support the theoretical predictions, it was found that from acetone the keto form crystallizes as a dimer with hydrogen bonding between N₁-H in the one molecule and amide CO in the other forming a three-dimensional structure. The importance of the side-chain nitrogen atom on the dimer formation was confirmed by solution and solid state study of 4-(phenyldiazenyl)-2-acetylnaphthalen-1-ol. The results indicate that the new azo-dyes obtained could be suitable candidates for switching and sensing applications in non-polar solvents.     

Pro-oxidative effects of melanoidin–copper complexes on isolated and cellular DNA

High molecular weight Maillard reaction products (melanoidins) are described to possess metal-chelating properties. Whereas in food systems, this ability contributes to antioxidant properties, the consequences on biological systems are not quite clear. The study was aimed to evaluate the implication of metal complexation by melanoidins on DNA damage. Melanoidins prepared with d-glucose and different l-amino acids under water-free reaction conditions were charged with cupric ions. The effect on isolated DNA was investigated by the PM2 assay and on cellular systems in the human colon carcinoma cell line HCT-116 by alkaline unwinding. Independent of the amino acid composition, pure melanoidins (MW >14 kDa) did not cause significant DNA damage. By charging melanoidins with Cu²⁺ ions, a considerable DNA strand breaking activity was detectable, which was again amplified in an oxidative milieu (addition of hydrogen peroxide). Since Cu²⁺ normally does not provoke the formation of reactive oxygen species (ROS) via Fenton-type reaction, the results obtained have to be attributed to reducing properties of melanoidins. Thus, in melanoidin–copper complexes redox cycling may take place leading to Cu⁺ which subsequently undergoes Fenton-type and Haber–Weiss reactions. As a consequence, ROS are formed, which may explain the generation of DNA strand breaks.     

Antibacterial Surface Coatings from Zinc Oxide Nanoparticles Embedded in Poly(N‐isopropylacrylamide) Hydrogel Surface Layers

Despite multiple research approaches to prevent bacterial colonization on surfaces, device‐associated infections are currently responsible for about 50% of nosocomial infections in Europe and significantly increase health care costs, which demands development of advanced antibacterial surface coatings. Here, novel antimicrobial composite materials incorporating zinc oxide nanoparticles (ZnO NP) into biocompatible poly(N‐isopropylacrylamide) (PNIPAAm) hydrogel layers are prepared by mixing the PNIPAAm prepolymer with ZnO NP, followed by spin‐coating and photocrosslinking. Scanning electron microscopy (SEM) characterization of the composite film morphology reveals a homogeneous distribution of the ZnO NP throughout the film for every applied NP/polymer ratio. The optical properties of the embedded NP are not affected by the matrix as confirmed by UV‐vis spectroscopy. The nanocomposite films exhibit bactericidal behavior towards Escherichia coli (E. coli) for a ZnO concentration as low as ≈0.74 μg cm^?2 (1.33 mmol cm^?3), which is determined by inductively coupled plasma optical emission spectrometry. In contrast, the coatings are found to be non‐cytotoxic towards a mammalian cell line (NIH/3T3) at bactericidal loadings of ZnO over an extended period of seven days. The differential toxicity of the ZnO/hydrogel nanocomposite thin films between bacterial and cellular species qualifies them as promising candidates for novel biomedical device coatings.     

Modular Ligation of Thioamide Functional Peptides onto Solid Cellulose Substrates

Hetero Diels‐Alder (HDA) cycloaddition – as an effective modular conjugation approach – is employed to graft thioamide endfunctional oligopeptides onto solid cyclopentadienyl (Cp) functional cellulose substrates generating cellulose‐peptide hybrid materials. The highly reactive Cp moieties serve as diene functionality in the consecutive HDA reaction on the biosubstrate surface. Oligopeptides (i.e., the model peptide Gly‐Gly‐Arg‐Phe‐Pro‐Trp‐Trp‐Gly and the antimicrobial peptide tritrpticin) are functionalized at their N‐termini employing strongly electron deficient thiocarbonyl thio compounds resulting in biomacromolecules bearing a thioamide endgroup. The dienophile‐ functional peptides readily undergo HDA reactions at ambient temperature and under mild conditions in solution with synthetic polymers as well as on solid (bio)substrates. An in‐depth investigation is provided of the influence of the temperature, the Lewis acid catalysis and the side group exchange of thioamide functional oligopeptides reacting with Cp terminated poly(methyl methacrylate) (M_n = 2100 g·mol^?1, PDI = 1.1) in homogenous solution as well as Cp functionalized cellulose in a heterogeneous system. To assess the success of the grafting reaction, the soluble samples were subjected to characterization methods such as size exclusion chromatography (SEC) and SEC‐electrospray ionization mass spectrometry (SEC‐ESI‐MS). The heterogeneous “grafting‐to” reactions were monitored using high resolution attenuated total reflection (ATR) Fourier transform infrared microscopy (HR‐FTIRM) imaging, X‐ray photoelectron spectroscopy (XPS) and elemental analysis. Evaluation via elemental analysis leads to quantitative peptide cellulose surface loading capacities.     

Strong influence of polymer architecture on the microstructural evolution of hafnium-alkoxide-modified silazanes upon ceramization

The present study focuses on the synthesis and ceramization of novel hafnium-alkoxide-modified silazanes as well as on their microstructure evolution at high temperatures. The synthesis of hafnia-modified polymer-derived SiCN ceramic nanocomposites is performed via chemical modification of a polysilazane and of a cyclotrisilazane, followed by cross-linking and pyrolysis in argon atmosphere. Spectroscopic investigation (i.e., NMR, FTIR, and Raman) shows that the hafnium alkoxide reacts with the N-H groups of the cyclotrisilazane; in the case of polysilazane, reactions of N-H as well as Si-H groups with the alkoxide are observed. Consequently, scanning and transmission electron microscopy studies reveal that the ceramic nanocomposites obtained from cyclotrisilazane and polysilazane exhibited markedly different microstructures, which is a result of the different reaction pathways of the hafnium alkoxide with cyclotrisilazane and with polysilazane. Furthermore, the two prepared ceramic nanocomposites are unexpectedly found to exhibit extremely different high-temperature behavior with respect to decomposition and crystallization; this essential difference is found to be related to the different distribution of hafnium throughout the ceramic network in the two samples. Thus, the homogeneous distribution of hafnium observed in the polysilazane-derived ceramic leads to an enhanced thermal stability with respect to decomposition, whereas the local enrichment of hafnium within the matrix of the cyclotrisilazane-based sample induces a pronounced decomposition upon annealing at high temperatures. The results indicate that the chemistry and architecture of the precursor has a crucial effect on the microstructure of the resulting ceramic material and consequently on its high-temperature behavior.