FerryCalin: an Engineered Lipocalin Protein Directed against the Transferrin Receptor with Potential for Brain Drug Delivery

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin variants were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin ⋅ Fe³⁺), among those an Anticalin – dubbed FerryCalin – exhibiting a dissociation constant (K_D) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to the fast k_on rate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.     

Inactivation of avian influenza virus by heat and high hydrostatic pressure

Avian influenza viruses threaten the life of domestic terrestrial poultry and contaminate poultry meat and eggs. Recently, these viruses rarely infected humans but had a high mortality rate in Southeast Asia, the Middle East, and Egypt. Thereby, these viruses caused high economic costs for production of poultry and health protection. We inactivated a highly pathogenic avian influenza A virus of subtype H7N7 in cell culture medium and chicken meat by heat and high hydrostatic pressure. Because heat and pressure inactivation curves of the H7N7 virus showed deviations from first-order kinetics, a reaction order of 1.1 had to be selected. A mathematical inactivation model has been developed that is valid between 10 and 60 degrees C and up to 500 MPa, allowing the prediction of the reduction in virus titer in response to pressure, temperature, and treatment time. Incubation at 63 degrees C for 2 min and 500 MPa at 15 degrees C for 15 s inactivated more than 10(5) PFU/ml, respectively. Thus, we suggest high-pressure treatment of poultry and its products to avoid the possible health threat by highly pathogenic avian influenza viruses.     

Tip-enhanced Raman scattering (TERS) from hemozoin crystals within a sectioned erythrocyte

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.     

Uniformly gold nanoparticles derived from P2VP-b-PCHMA block copolymer templates with different reduction methods

The micellization of poly(2-vinylpyridine)-block-poly(cyclohexyl methacrylate) (P2VP-b-PCHMA) in THF can be induced by the complexation between the P2VP blocks and HAuCl4, forming composite polymeric micelles with PCHMA being the shell and P2VP/HAuCl4 complex being the core. In order to obtain regular arrays of gold nanoparticles (Au NPs), monolayer of HAuCl4-loaded surface micelles have been produced by spin-coating the micellar solution, and Au NPs in different size have been obtained by oxygen plasma with different reduction processes. In addition, pyrole (PY) has been used as an efficient reducing agent to fabricate dispersed Au NPs within micellar structure in a short reducing time, resulting in a raspberry-like morphology of the Au-polymer composites. With the addition of annealing processes or longer reducing time (one month), different shapes of Au NPs have been observed in the cast films. Furthermore, core-shell nanostructures of gold-polypyrole (Au-PPY) have also been observed by employing vapor phase polymerization of PY onto HAuCl4-loaded polymeric solution-cast films.     

On the ignition and flame development in a spray-guided direct-injection spark-ignition engine

High-speed fuel, flow, and flame imaging are combined with spark discharge measurements to investigate the causes of rare misfires and partial burns in a spray-guided spark-ignited direct-injection (SG-SIDI) engine over a range of nitrogen dilution levels (0–26% by volume). Planar laser induced fluorescence (PLIF) of biacetyl is combined with planar particle image velocimetry (PIV) to provide quantitative measurements of equivalence ratio and flow velocity within the tumble plane of an optical engine. Mie scattering images used for PIV are also used to identify the enflamed region to resolve the flame development. Engine parameters were selected to mimic low-load idle operating conditions with stratified fuel injection, which provided stable engine performance with the occurrence of rare misfire and partial burn cycles. Nitrogen dilution was introduced into the intake air, thereby displacing the oxygen, which destabilized combustion and increased the occurrence of poor burning cycles. Spark measurements revealed that all cycles exhibited sufficient spark energy and duration for successful ignition. High-speed PLIF, PIV, and Mie scattering images were utilized to analyze the spatial and temporal evolution of the fuel distribution and flow velocity on flame kernel development to better understand the nature of poor burning cycles at each dilution level. The images revealed that all cycles exhibited a flammable mixture near the spark plug at spark timing and a flame kernel was present for all cycles, but the flame failed to develop for misfire and partial burn cycles. Improper flame development was caused by slow flame propagation which prevented the flame from consuming the bulk of the fuel mixture within the piston bowl, which was a crucial step to achieve further combustion. The mechanisms identified in this work that caused slower flame development are: (1) lean mixtures, (2) external dilution, and (3) convection velocities that impede transport of the flame into the fuel mixture.     

Functional characterization of a supramolecular affinity switch at the single molecule level

Surface-immobilized and switchable resorcin[4]arene receptor molecules were quantitatively investigated with atomic force microscopy (AFM) and AFM-single molecule force spectroscopy (AFM-SMFS). The upper rim of the supramolecular receptor cavities was modified with two photodimerizable anthracene moieties. The molecular constitution can be externally switched and controlled by exposure to ultraviolet (UV) light and heat. The topography as well as the complexation affinity against small ammonium guest ions of the two isomers were investigated at the single molecule level. Our results demonstrate the feasibility to externally control the supramolecular receptor's affinity and simultaneously quantify and associate these binding properties with the structural change of the resorcin[4]arenes structure on the basis of the measured molecule corrugation height.     

Effect of the carbon ion energy on the microstructure of ta-C/Cr multilayers

The effect of the carbon ion energy applied in a pulsed arc deposition process on the morphology of the interface between Cr layers and subsequently deposited tetrahedral bonded carbon (ta-C) coatings was investigated. The carbon ion energy was varied through the bias voltage between ~ 25 and 500 eV. Small-angle and wide-angle X-ray scattering (SAXS and WAXS), high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS) were used as the central experimental methods for microstructure analysis. In order to improve the scattering power of the ta-C/Cr interfaces for SAXS, the samples were prepared in the form of ta-C/Cr multilayers. SAXS revealed density and thickness of individual layers in the multilayer stack, and the roughness and morphology of the ta-C/Cr interfaces. The density of ta-C layers ranged between 2.72 and 3.15 g/cm³, which was related to the amount of sp³ bonds between 37% and 71%. The amount of the sp³ bonds calculated from the density of the ta-C layers agreed well with the amount of the sp³ bonds determined using EELS. HRTEM confirmed both the increase of the interface roughness with increasing carbon ion energy and the changes in the correlation of the interface corrugations observed by SAXS. Furthermore, HRTEM approved the crystallinity of Cr layers revealed by WAXS.     

Liquid–liquid slug flow: Hydrodynamics and pressure drop

In this paper, the hydrodynamics and the pressure drop of liquid–liquid slug flow in round microcapillaries are presented. Two liquid–liquid flow systems are considered, viz. water-toluene and ethylene glycol/water-toluene. The slug lengths of the alternating continuous and dispersed phases were measured as a function of the slug velocity (0.03–0.5 m/s), the organic-to-aqueous flow ratio (0.1–4.0), and the microcapillary internal diameter (248 and 498 μm). The pressure drop is modeled as the sum of two contributions: the frictional and the interface pressure drop. Two models are presented, viz. the stagnant film model and the moving film model. Both models account for the presence of a thin liquid film between the dispersed phase slug and the capillary wall. It is found that the film velocity is of negligible influence on the pressure drop. Therefore, the stagnant film model is adequate to accurately predict the liquid–liquid slug flow pressure drop. The influence of inertia and the consequent change of the slug cap curvature are accounted for by modifying Bretherton’s curvature parameter in the interface pressure drop equation. The stagnant film model is in good agreement with experimental data with a mean relative error of less than 7%.     

Analysis and demonstration of a control concept for a heat integrated simulated moving bed reactor

Successful operation of a simulated moving bed reactor requires a strategy to control switching of the inlet and outlet positions between different fixed-bed segments. This is due to the narrow operation window in which an ignited state can be maintained and complete conversion is assured in exothermal irreversible reactions. This work compares three control concepts, which are either based on monitoring the reaction front or the leading thermal front. Another feature investigated is the length of the observation time considering a single switching period and a complete cycle. For the concept favored some efforts regarding parameterization are made. The wider range of temperature set-points applicable can be related to stability limits of an associated hypothetical true moving bed reactor. Responses to step inputs in feed concentration and total flow rate are investigated experimentally in order to evaluate the performance of the controller suggested. An ignited state of the reactor could be maintained successfully for 3.5 days.