Protonation patterns in tetracycline:tet repressor recognition: simulations and experiments

Resistance to the antibiotic tetracycline (Tc) is regulated by its binding as a Tc:Mg2+ complex to the Tet Repressor protein (TetR). Tc:TetR recognition is a complex problem, with the protein and ligand each having several possible conformations and protonation states, which are difficult to elucidate by experiment alone. We used a combination of free-energy simulations and crystallographic analysis to investigate the electrostatic interactions between protein and ligand and the possible role of induced fit in Tc binding. Tc in solution was described quantum mechanically, while Tc:TetR interactions were described by a recent, high-quality molecular-mechanics model. The orientations of the amide and imidazole groups were determined experimentally by a careful analysis of Debye-Waller factors in alternate crystallographic models. The agreement with experiment for these orientations suggested that the simulations and their more detailed, thermodynamic predictions were reliable. We found that the ligand prefers an extended, zwitterionic state both in solution and in complexation with the protein. Tc is thus preorganized for binding, while the protein combines lock-and-key behavior for regions close to the ligand's amide, enolate, and ammonium groups, with an induced fit for regions close to the Mg2+ ion. These insights and the modeling techniques employed should be of interest for engineering improved TetR ligands and improved TetR proteins for gene regulation, as well as for drug design.     

Effect of the Extent of Conversion and Retrogradation on the Digestibility of Potato Starch

The effect of starch conversion on the susceptibility of potato granules to α‐amylase was studied by direct sampling at different pasting times corresponding to different points on the RVA profile of a 6.4% (w/w) suspension of starch in distilled water. Native granules showed high resistance to α‐amylase with 8.6 ± 0.4% digestibility for a 6 h incubation period with the enzyme. When the suspension was heated to 60 °C, the digestibility increased to 53.5 ± 0.7% although, at this temperature, there was still no noticeable increase in the measured viscosity (≤0.040 Pa · s). The material sampled after a pasting time corresponding to the RVA peak viscosity showed a digestibility of 88.4 ± 0.5%. This suggested, owing to the expected retrogradation of amylose on cooling, the quasi‐total susceptibility of amylopectin to enzymatic digestion at this pasting stage. The effect of ions on the swelling of potato starch was used to assess whether the decrease of the swelling of the granules in the presence of NaCl was paralleled by an increase in resistance to α‐amylase. A small (∼6.1%) but significant decrease in the digestibility of pasted starch was observed in the presence of salt. Finally, the effect of the retrogradation of the amylopectin fraction on its digestibility was assessed in extruded potato starch ribbons containing 35% (w/w) water and stored at different temperatures. After 14 days of storage, the digestibility decreased from 77.0 ± 0.9% in the freshly extruded samples to between 28.0 ± 1.7% and 42.1 ± 0.3%, depending on the storage temperature. This suggested a measurable difference in the α‐amylase susceptibility between the A and B polymorphs of retrograded amylopectin.     

Environmental impact and human health risks of polychlorinated dibenzo-p-dioxins and dibenzofurans in the vicinity of a new hazardous waste incinerator: a case study

The purpose of this study was to assess the environmental impact of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in the vicinity of a new hazardous waste incinerator (HWI) 4 years after regular operation of the facility. A double approach was carried out. The PCDD/F congener profiles corresponding to environmental samples, soil and herbage, collected before the HWI (baseline) and 4 years after starting regular operations, as well as PCDD/F profiles of air emission samples, were compared. The potential health risks (carcinogenic and noncarcinogenic) due to PCDD/F exposure were assessed for adults and children living in the neighborhood of the facility. Human exposure to PCDD/Fs was mainly due to dietary food intake. Comparisons between the PCDD/F congener profiles corresponding to the baseline and current surveys, as well as data concerning the human health risk assessment, indicate that the HWI in question does not cause additional risks to the environment orto the population living in the vicinity of the facility.     

Efficient sequence-specific purification of Listeria innocua mRNA species by triplex affinity capture with parallel tail-clamps

Parallel clamps can interact in a sequence-specific manner with homopyrimidine DNA and RNA oligonucleotides to form triplexes. For longer nucleic acids, we have previously demonstrated the inhibitory effect of DNA-target secondary structures on triplex formation. We further designed a modification of these molecules-that is, tail-clamps formed by addition of a tail sequence to the parallel clamp-and proved efficient binding of the molecules with structured single-stranded DNA targets. Here we explore the possible application of the tail-clamp strategy for triplex formation with RNA targets, which are typically found as strongly folded single-stranded molecules. Efficient and specific binding of a tail-clamp designed to form a parallel triplex with Listeria innocua iap mRNA sequences has been verified by UV melting curves and triplex affinity capture techniques. Furthermore, we show for the first time the formation of stable complexes of mRNA with tail-clamps not only under acidic but also under neutral and slightly basic pH conditions. These results signify a further step towards the possible applications of triplexes with mRNA molecules; research, analytical, and therapeutic uses can be envisaged. As an example, our tail-clamp-based triplex affinity capture assay allowed the specific capture and recovery of iap mRNA molecules from an L. innocua total RNA solution with 45 % yield.     

Engineering Potato Virus X Particles for a Covalent Protein Based Attachment of Enzymes

Plant virus nanoparticles are often used to display functional amino acids or small peptides, thus serving as building blocks in application areas as diverse as nanoelectronics, bioimaging, vaccination, drug delivery, and bone differentiation. This is most easily achieved by expressing coat protein fusions, but the assembly of the corresponding virus particles can be hampered by factors such as the fusion protein size, amino acid composition, and post‐translational modifications. Size constraints can be overcome by using the Foot and mouth disease virus 2A sequence, but the compositional limitations cannot be avoided without the introduction of time‐consuming chemical modifications. SpyTag/SpyCatcher technology is used in the present study to covalently attach the Trichoderma reesei endoglucanase Cel12A to Potato virus X (PVX) nanoparticles. The formation of PVX particles is confirmed by western blot, and the ability of the particles to display Cel12A is demonstrated by enzyme‐linked immunosorbent assays and transmission electron microscopy. Enzymatic assays show optimal reaction conditions of 50 °C and pH 6.5, and an increased substrate conversion rate compared to free enzymes. It is concluded that PVX displaying the SpyTag can serve as new scaffold for protein display, most notably for proteins with post‐translational modifications.     

3D optical coherence tomography as new tool for microscopic investigations of nucleate boiling on heated surfaces

Boiling phenomena are an important aspect in security and efficiency for technical applications with high heat flux like nuclear reactors. This study presents optical coherence tomography (OCT) as a novel modality for three-dimensional and two-dimensional time resolved imaging of nucleate boiling on heated surfaces on a microscopic scale with high spatial (<10 μm) and temporal (>25 frames per second) resolution. Within this study, a borosilicate glass plate coated with an optically transparent and electrically conductive indium tin oxide (ITO) layer with a thickness of approximately 100 nm was used as heating surface. The combination of these two properties allows optical inspection of the nucleate boiling from the backside by OCT focused on the formation, growth and detachment of single bubbles. We demonstrated for the first time that OCT is an excellent tool to acquire two-dimensional and three-dimensional images of the base of vapor bubbles from the backside of the heated surface. The acquired images allow for instance the temporally resolved measurement of the bubble diameter, diameter of the bubble base and the contact angle. Exploiting the phase information of the acquired OCT signal stacks allows imaging the movement of the bubble surrounding fluid. We think that OCT will provide many new insights into the boiling phenomena at the bubble base. The recent enhancement of the acquisition rates of OCT systems will facilitate four-dimensional imaging of single bubble evaporation procedures in the nearer future.     

Asymmetric hybrid silica nanomotors for capture and cargo transport: towards a novel motion-based DNA sensor

An innovative self-propelled nanodevice able to perform motion, cargo transport, and target recognition is presented. The system is based on a mesoporous motor particle, which is asymmetrically functionalized by the attachment of single-stranded DNA onto one of its faces, while catalase is immobilized on the other face. This enzyme allows catalytic decomposition of hydrogen peroxide to oxygen and water, giving rise to the driving force for the motion of the whole system. Moreover the motor particles are able to capture and transport cargo particles functionalized with a noncomplementary single-stranded DNA molecule, only if a specific oligonucleotide sequence is present in the media. Functionalization with characteristic oligonucleotide sequences in the system implies a potential for further developments for lab-on-chip devices with applications in biomedical applications.     

Wild Lactobacillus strains: Technological characterisation and design of Coalho cheese lactic culture

To design a specific lactic culture for the controlled manufacture of coalho cheese, 13 Lactobacillus rhamnosus, two Lactobacillus fermentum and one Lactobacillus plantarum strains isolated from artisanal coalho cheeses were identified and characterised. Two Lb. rhamnosus, one Lb. plantarum and one Lb. fermentum were selected and grouped in pairs designing four different culture formulations that demonstrated a good performance in cheesemaking experiments at pilot scale. Further studies to adjust the balance of strains used are necessary to attain adequate sensorial and technological attributes as expected for artisanal cheeses.