Directed Evolution of a Soluble Human IL-17A Receptor for the Inhibition of Psoriasis Plaque Formation in a Mouse Model

1. Download : Download high-res image (276KB)
2. Download : Download full-size image

Highlights? IL-17 is involved in many inflammatory diseases ? Directed evolution of IL-17A receptor results in improved IL-17A affinity ? The engineered receptors efficiently inhibit IL-17A induced cytokine secretion ? Engineered receptor promotes the recovery of psoriasis plaques in mouse model     

Stability and state of aggregation of aqueous fibrinogen and dipalmitoylphosphatidylcholine lipid vesicles

The stability and state of aggregation of aqueous fibrinogen (FB) and dipalmitoylphosphatidylcholine (DPPC) vesicles in water or buffer at 25 degrees C were studied with dynamic light scattering (DLS), UV-vis spectroturbidimetry (ST), and cryo-transmission electron microscopy (cryo-TEM). In water, when 1000 ppm (0.10 wt %) DPPC dispersions were prepared with a protocol including extensive sonication, they contained mostly vesicles and were quite clear, transparent, and stable for at least 30 days. FB mixtures with water (0.075 wt %) were quite unstable and biphasic. They formed large aggregates which eventually precipitated. The addition of DPPC vesicles into these unstable FB dispersions reversed FB aggregation and precipitation and produced stable translucent microdispersions. The inferred lipid/protein aggregates were limited in size, with average diameters ranging from 200 to 300 nm. In buffer, DPPC dispersions were also clear and quite stable, with average dispersed particles diameter of ca. 90 nm. FB dissolved in aqueous buffer and formed transparent and stable solutions. Adding salt to an aggregated FB dispersion in water reversed the aggregation. FB aggregated and redissolved in the presence of the citrate and after the citrate was removed. There was no effect of citrate (present in FB initially) in the FB aggregation or redissolution. FB molecules in buffer form dimers or higher aggregates. Their average aggregation number is 2, determined with Rayleigh scattering analysis of turbidity data. The average hydrodynamic diameter of FB solutions from DLS was 30 nm. Mixing a stable FB solution in buffer and a stable DPPC dispersion in buffer produced highly unstable mixtures, in which large aggregates precipitated. These results have implications in understanding the interactions of lipids and proteins in many biological applications and food processing applications.     

Effect of sonication and freezing-thawing on the aggregate size and dynamic surface tension of aqueous DPPC dispersions

The effect of sonication and freezing-thawing on the aggregate size and dynamic surface tension of aqueous dipalmitoylphosphatidylcholine (DPPC) dispersions was studied by cryogenic-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), UV-vis spectroturbidimetry, and surface tensiometry. When 1000 ppm (0.1 wt%) DPPC dispersions were prepared with a certain protocol, including extensive sonication, they contained mostly frozen vesicles and were quite clear, transparent, and stable for at least 30 days. The average dispersed vesicles diameter was 80 nm in water and 90 nm in standard phosphate saline buffer. After a freeze-thaw cycle, this dispersion became turbid, and precipitates of coagulated vesicles were observed with large particles of average size of 1.5x10(3) nm. The vesicle coagulation is due to the local salt concentration increase during the freezing of water. This dispersion has much higher equilibrium and dynamic surface tension than those before freezing. When this freeze-thawed dispersion was subjected to a resonication at 55 degrees C, smaller vesicles with sizes of ca. 70 nm were produced, and a lower surface tension behavior was restored as before freezing. Similar behavior was observed at 30 ppm DPPC. These results indicate that the freeze-thaw cycle causes substantial aggregation and precipitation of the vesicles. These results have implications for designing efficient protocols of lipid dispersion preparation and lung surfactant replacement formulations in treating respiratory disease and for effective administration.     

A unique paradigm for a Turn-ON near-infrared cyanine-based probe: noninvasive intravital optical imaging of hydrogen peroxide

The development of highly sensitive fluorescent probes in combination with innovative optical techniques is a promising strategy for intravital noninvasive quantitative imaging. Cyanine fluorochromes belong to a superfamily of dyes that have attracted substantial attention in probe design for molecular imaging. We have developed a novel paradigm to introduce a Turn-ON mechanism in cyanine molecules, based on a distinctive change in their π-electrons system. Our new cyanine fluorochrome is synthesized through a simple two-step procedure and has an unprecedented high fluorescence quantum yield of 16% and large extinction coefficient of 52,000 M(-1)cm(-1). The synthetic strategy allows one to prepare probes for various analytes by introducing a specific triggering group on the probe molecule. The probe was equipped with a corresponding trigger and demonstrated efficient imaging of endogenous hydrogen peroxide, produced in an acute lipopolysaccharide-induced inflammation model in mice. This approach provides, for the first time, an available methodology to prepare modular molecular Turn-ON probes that can release an active cyanine fluorophore upon reaction with specific analyte.     

Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing

The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process—and not only its endpoint—to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach.

Open image in new window

Graphical Abstract A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.

     

Perturbations of Multipliers of Systems of Periodic Ordinary Differential Equations

The paper deals with periodic systems of ordinary differential equations (ODEs). A new approach to the investigation of variations of multipliers under perturbations is suggested. It enables us to establish explicit conditions for the stability and instability of perturbed systems.