Bivalent EGFR-Targeting DARPin-MMAE Conjugates

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).     

NUMERICAL ANALYSIS OF THE EFFECTS OF LOCAL HYDRODYNAMICS ON MASS TRANSFER IN HETEROGENEOUS POROUS MEDIA

Many engineering problems require the estimation of mass transfer coefficients in porous materials. In heterogeneous materials or in cases where mass transfer sites are not spatially uniform, empirical equations for mass transfer coefficients vary widely, and the origin of these differences is not well understood. In this article, we use a stochastic algorithm to model mass transfer from single particles in a two-dimensional heterogeneous packed bed. The computed mass transfer coefficients are used to generate a distribution of local Peclet numbers in the bed. Detailed hydrodynamics are then used to interpret variations in the local Peclet number. The results show clear relationships between pore structure, streamline patterns, and mass transfer rates.     

A Synthetic Approach to Star‐Like Polymers of Styrene and Butyl Acrylate by Linking Reactions using Functionalized Methacrylates

Summary: Coupling reactions between terminal functionalized polymer chains were chosen for the synthesis of star‐like polymers consisting of polystyrene and polystyrene‐block‐poly[styrene‐co‐(butyl acrylate)] arms. For the preparation of terminal functionalized polymer chains a side reaction of the 2,2,6,6‐tetramethylpiperidine‐N‐oxyl (TEMPO) mediated free radical polymerization of methacrylates could be used successfully to convert TEMPO terminated polymers into end functionalized polymers. The number of functionalized monomer units attached to the polymer chain is directly related to the TEMPO concentration during this reaction. Different polystyrenes and polystyrene‐block‐poly[styrene‐co‐(butyl acrylate)] block copolymers were functionalized with a variable number of epoxide and alcohol groups at the chain end. For the determination of the optimal reaction parameters for the coupling reactions between these polymer chains, epoxy functionalized polystyrenes were converted with hydroxy functionalized polystyrenes under basic and acidic conditions. By activation with sodium hydride or boron trifluoride star‐like polymers were synthesized under mild conditions. The transfer of the reaction conditions to coupling reactions between end functionalized polystyrene‐block‐poly[styrene‐co‐(butyl acrylate)] copolymers showed that star‐like polymers with more than 12 arms were formed using boron trifluoride as activating agent.

     

Measurement of Micro Kinetics of Hydrogenation in Liquid Phase Using Raman Spectroscopy

Hydrogenation of liquid organic hydrogen carriers is usually carried out in liquid phase. To measure the kinetics of this hydrogenation, an experimental setup using in situ Raman spectroscopy for analysis of the reaction mixture is proposed. With this setup it is possible to perform hydrogenation reactions at temperatures of up to 573 K and pressures up to 25 MPa. For validation of the experimental setup the hydrogenation of 1‐octene was measured in liquid phase. The reaction progress can be monitored in detail by Raman spectroscopy. To determine kinetic parameters from the experimental data, two modeling approaches were applied: a classic kinetic model and a thermodynamic kinetic model. The results were compared to literature data.     

Quasi-spherical focal spot in two-photon scanning microscopy by three-ring apodization

We present a beam-shaping technique for two-photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three-ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi-spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot-fluorescence efficiency.     

Host Recognition by the Specialist Hoverfly <Emphasis Type="Italic">Microdon mutabilis</Emphasis>, a Social Parasite of the Ant <Emphasis Type="Italic">Formica lemani</Emphasis>

The larva of the hoverfly Microdon mutabilis is a specialist social parasite of the ant Formica lemani that is adapted to local groups of F. lemani colonies but mal-adapted to colonies of the same species situated only a few hundred meters away. At a study site in Ireland, F. lemani shares its habitat with four other ant species. All nest under stones, making the oviposition choice by M. mutabilis females crucial to offspring survival. In this study, we tested the hypothesis that, as an extreme specialist, M. mutabilis should respond to cues derived from its host rather than from its microenvironment, a phenomenon that has hitherto only been addressed in the context of herbivorous insects and their parasitoids. In behavioral assays, M. mutabilis females reacted to volatiles from F. lemani colonies by extending their ovipositors, presumably probing for an oviposition substrate. This behavior was not observed toward negative controls or volatiles from colonies of Myrmica scabrinodis, the host ant of the closely related Microdon myrmicae. Coupled gas chromatography-electroantennography (GC-EAG) that used antennal preparations of M. mutabilis located a single physiologically active compound within an extract of heads of F. lemani workers. Coupled GC-mass spectrometry (GC-MS) tentatively identified the compound as a methylated methylsalicylate. GC co-injection of the extract with authentic samples showed that of the four possible isomers (methyl 3-, 4-, 5-, and 6-methylsalicylate), only methyl 6-methylsalicylate co-eluted with the EAG-active peak. Furthermore, the response to methyl 6-methylsalicylate was four times higher than to those of the other isomers. Coupled GC-EAG and GC-MS also revealed physiological responses to two constituents, 3-octanone and 3-octanol, of the M. scabrinodis alarm pheromone. However, the behavioral trials did not reveal any behavior that could be attributed to these compounds. Results are discussed in the context of four phases of host location behavior, and of the characteristics, which volatile cues should provide to be useful for an extreme specialist such as M. mutabilis.     

SiC ceramic micropatterns from polycarbosilanes

Micropatterned SiC ceramics were fabricated from polycarbosilanes applying a softlithographic replication technique. A polydimethylsiloxane mould replicated from a photolithographic microstructured silicon wafer was used as master structure. The polydimethylsiloxane mould was coated with a solution containing a mixture of two different polycarbosilanes in n-octane. After treatment at 200–400 °C the cross-linked polycarbosilane films were debonded and pyrolysed at 900 °C in nitrogen and subsequently crystallised at temperatures up to 1500 °C in argon. The cross-linking and thermal degradation behaviour of the polycarbosilanes was investigated by Fourier-transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis. X-ray diffractrometry showed the expected development of a nanocrystalline β-SiC (3 nm) as the main phase with increasing temperature. However, traces of α-SiO₂ derived from the polycarbosilane precursors were also detected by X-ray analysis. Removal of the α-SiO₂ dioxide with hydrofluoric acid in the pyrolysed samples and subsequent increased the crystallite size to 7 nm. The Young's modulus determined by nanoindentation was increased from 3 GPa after cross-linking to 110 GPa after crystallisation. Scanning electron microscopy revealed, that the initial micropatterns were fully retained in the pyrolysed and crystallised SiC ceramics. The micropatterned cross-linked and crystallised β-SiC based substrates exhibited light scattering characteristics, which qualify them as promising candidates for diffractive optical elements in microoptical applications.     

Submicron droplet formation in the human lung

The exhaled breath of humans contains droplets originating from the lung lining fluid. An analysis of these droplets for non-volatile proteinaceous biomarkers holds potential as a non-invasive diagnosis of lung diseases. To ease the interpretation of the diagnostic results, the source strength of the particles should be known und therefore an understanding of the particle generation process is required. It is assumed that during reopening of a collapsed terminal airway a liquid bridge of the lung lining fluid ruptures and droplets are generated. The objective of our experimental and theoretical study was to clarify the mechanisms of droplet generation for quiet breathing patterns by investigating in detail the number flux and the particle size distribution in the exhaled breath. The process of liquid film rupture is modelled by computational fluid dynamics analysis from which the droplet size distribution is calculated. In addition the number emission flux and the droplet size distribution are systematically measured in the exhaled breath of healthy volunteers. The strong increase of the particle emission flux with tidal volume and the good agreement between measured and calculated droplet number distribution both showing droplets primarily in the submicron range confirm the present hypothesis that reopening of collapsed airway structures associated with the rupture of a surfactant film is the physical mechanism of droplet generation. This was hypothesized previously in the literature.     

Thermobaric stratification in very deep Norwegian freshwater lakes

Thermobaric stratification was investigated in very deep lakes of elongated shape. We considered the spring time configuration when large freshwater lakes may have their deepest recirculation. In five very deep freshwater lakes in Norway, profiles were measured in spring 2006 with multiparameter probes. Temperature profiles confirmed thermobaric stratification in all of these lakes, i.e. the temperature profiles crossed the 4 °C line and thus were affected by pressure effects on the temperature of maximum density. Electrical conductance was low in all of the investigated lakes, and vertical gradients were too small to play a role in the density stratification. In all lakes, deep water was well supplied with oxygen. Three out of five lakes showed a temperature gradient close to Eklund's stability theorem. However, the remaining two lakes clearly opposed the theorem. Bottom temperatures were more closely related to lake length than their maximum depths. Oxygen saturation profiles documented recent intensive gas exchange with the atmosphere. They agree well with the possible formation of deep water from density driven flows at horizontal temperature gradients close to 4 °C.