Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Cholic Acid (MT921) after a Subcutaneous Injection in the Submental Area to Humans.

This study aimed to explore pharmacokinetics, pharmacodynamics, and safety/tolerability of MT921, an injectable cholic acid, after a single subcutaneous administration to healthy volunteers. A randomized, double-blinded, placebo-controlled, single dose-ascending phase 1 study enrolled 24 subjects who were assigned to three groups (60 mg, 120 mg, and 150 mg) of MT921. Blood samples were obtained for a 24-h period before and after injecting MT921 to the submental fat area. Plasma concentrations of cholic acid and deoxycholic acid were determined for pharmacokinetic analysis. Levels of free fatty acid, triglyceride, and total cholesterol were measured for pharmacodynamic analysis. Safety and tolerability were assessed until 21 days post-dose. While systemic exposure to cholic acid tended to increase as the MT921 dose increased, pharmacokinetic profiles of deoxycholic acid were similar among dose groups without showing significant changes. Pharmacodynamic profiles were comparable when measured at baseline and post-dose. The most frequent adverse events were injection site pain and edema. All adverse drug reactions resolved without treatment. MT921 appeared to be well-tolerated after an injection to the submental area at a dose up to 150 mg. Systemic exposure to cholic acid increased as the dose increased. Blood lipid profiles and deoxycholic acid levels were not affected by MT921 treatment.

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating.

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

Electric Triggering for Enhanced Control of Droplet Generation.

Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its "off-time" is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.

Separation Phenomena in Tailored Micro- and Nanofluidic Environments.

Separations of bioanalytes require robust, effective, and selective migration phenomena. However, due to the complexity of biological matrices such as body fluids or tissue, these requirements are difficult to achieve. The separations field is thus constantly evolving to develop suitable methods to separate biomarkers and fractionate biospecimens for further interrogation of biomolecular content. Advances in the field of microfabrication allow the tailored generation of micro- and nanofluidic environments. These can be exploited to induce interactions and dynamics of biological species with the corresponding geometrical features, which in turn can be capitalized for novel separation approaches. This review provides an overview of several unique separation applications demonstrated in recent years in tailored micro- and nanofluidic environments. These include electrokinetic methods such as dielectrophoresis and electrophoresis, but also rather nonintuitive ratchet separation mechanisms, continuous flow separations, and fractionations such as deterministic lateral displacement, as well as methods employing entropic forces for separation.

Deterministic Ratchet for Sub-micrometer (Bio)particle Separation.

Resolving the heterogeneity of particle populations by size is important when the particle size is a signature of abnormal biological properties leading to disease. Accessing size heterogeneity in the sub-micrometer regime is particularly important to resolve populations of subcellular species or diagnostically relevant bioparticles. Here, we demonstrate a ratchet migration mechanism capable of separating sub-micrometer sized species by size and apply it to biological particles. The phenomenon is based on a deterministic ratchet effect, is realized in a microfluidic device, and exhibits fast migration allowing separation in tens of seconds. We characterize this phenomenon extensively with the aid of a numerical model allowing one to predict the speed and resolution of this method. We further demonstrate the deterministic ratchet migration with two sub-micrometer sized beads as model system experimentally as well as size-heterogeneous mouse liver mitochondria and liposomes as model system for other organelles. We demonstrate excellent agreement between experimentally observed migration and the numerical model.

Degenerative Calcification of Pericardial Bioprostheses: Comparison of Five Implantation Methods in a Rabbit Model.

BACKGROUND AND AIM OF THE STUDY: The degenerative calcification of bioprosthetic heart valves remains a clinical challenge, especially among young adults and children. Animal models that are based on subcutaneous and intramuscular implantation and are typically used to assess interventions to prevent bioprosthetic heart valve calcification do not reflect actual hemodynamic stress and lack direct blood contact. Thus, the study aim was to investigate bioprosthesis calcification at different implantation sites. METHODS: The calcification degrees of five valve implantation methods, namely subcutaneous, intramuscular and intravenous implantation, and arterial and venous patch angioplasty, were simultaneously investigated in 10 New Zealand White rabbits. RESULTS: Ultrasonography and computed tomography images showed vascular patency to be well maintained in all implanted vessels. Histologically, cellular infiltrates around the implant and within the collagen fibers were only found in the intravenous implantation group, which also had the highest calcium level among the methods. CONCLUSION: The present study was the first to compare the degree of calcification after applying five implantation methods simultaneously in one animal species. The rabbit intravenous implantation model, which involved direct contact with blood factors, is expected to serve as a useful animal model for research into the prevention of bioprosthetic heart valve degeneration.

Enhanced switching behavior of iron oxide nanoparticle-doped liquid-crystal display.

It is well known that doping nanoparticles (NPs) in liquid crystals (LCs) can easily change the physical and electro-optical properties of LC mixture. In this paper, we demonstrate homogeneous, aligned nematic LC (N-LC) system dispersed in iron oxide (Fe2O3) NPs. The prepared Fe2O3 NPs have an average particle size of 50 nm. By changing the doping concentration of Fe2O3 NPs, we observed the characteristics of LC systems. Electrooptical (EO) characteristics included faster rising and falling times (2.14 ms and 10.24 ms, respectively) and lower driving voltage (1.45 V) compared with a pure N-LC cell. We demonstrated these results via the relationship between dielectric con- stant and LC device properties. The results were verified by software simulation based on general physical properties. Moreover, we observed that LC system with Fe2O3 NPs could be accomplished without capacitance hysteresis by capturing charged impurities. Superior performance of LC cell with Fe2O3 NPs indicates that the proposed LC system have strong potential for use in the production of advanced LC displays.

Superior properties of homogeneously aligned twisted nematic liquid crystals on nanoscale molybdenum trioxide surfaces.

Homogeneously aligned twisted nematic liquid crystals (TN-LCs) were produced on molybdenum trioxide (MoO3) thin films via ion beam (IB) irradiation. Control of the pretilt angle was achieved by varying the IB incident angle. X-ray photoelectron spectroscopy (XPS) analysis revealed that the intensity of the Mo-O and O-Mo bonds at various IB incident angles exhibited the tendency that was opposite to that of the pretilt angle and the lowest intensity was produced at 45°. Superior electro-optical (EO) characteristics were also observed. Furthermore, a TN-LC cell fabricated with the MoO3 thin films exhibited high thermal stability. Such stability could be maintained at temperatures greater than 150 °C.

Superior optical properties of homogeneous liquid crystal alignment on a tin (IV) oxide surface sequentially modulated via ion beam irradiation.

We first investigated the alignment characteristics of tin (IV) oxide (SnO(2)) thin films deposited by radio-frequency (RF) magnetron sputtering. This study demonstrates that liquid crystal (LC) molecules could be aligned homogeneously by controlling the Ion Beam (IB) irradiation energy densities. We also show that the pretilt angle of the LC molecules has a close relation with the surface energy. X-ray photoelectron spectroscopy (XPS) indicates that a non-stoichiometric SnO(2-x) surface converted by ion beam irradiation can horizontally align the LC molecules. The measured electro-optical (EO) characteristics showed high performance, comparable with those of rubbed and ion-beam irradiated polyimide (PI) layers.