HTD Biosystems (United States)

Peroxisome proliferator-activated receptors (PPARs) modulate bile metabolism and are important therapeutic options in cholestatic diseases. This study was aimed at understanding the effects of single and multiple doses of seladelpar, a PPARδ (peroxisome proliferator-activated receptor delta) agonist, on plasma C4 (a freely diffusible metabolite accepted as a proxy for de novo bile acid biosynthesis), Fibroblast Growth Factor 21 (Fgf21), and gene expression changes in the liver of male and female mice. C57BL/6 mice were treated with seladelpar 10 mg/kg/day or vehicle through oral gavage before lights out on Day 1 (single dose) or from Day 1 to Day 7 (multiple doses). Liver samples were obtained at 0, 1, 2, 4, 8, 12, 16, and 24 h postdosing, and plasma C4 and Fgf21 levels were measured. In vehicle-treated mice, C4 levels were higher in the dark cycle compared to the light cycle, with higher levels in females than in males. Plasma Fgf21 did not vary substantially over the dark-light cycle or show a sex-specific expression pattern. Seladelpar treatment significantly reduced plasma C4 and increased Fgf21 levels in both sexes, which coincided with a decrease in cholesterol 7α-hydroxylase mRNA and an increase in Fgf21 mRNA in the livers. Untargeted RNA sequencing revealed a strong correlation between the genes differentially expressed after single- and multiple-dose seladelpar treatment. PPAR-responsive genes, including pyruvate dehydrogenase kinase 4, acyl-CoA thioesterase 2, and angiopoietin-like 4, were upregulated. No changes in nuclear receptors, clock genes, and sex-specific genes were observed. Overall, these results are consistent with a model where seladelpar treatment reduces bile acid synthesis by upregulating Fgf21 and modulating other PPAR-responsive genes.

We use a microfluidic method to estimate the water permeability coefficient (p) of membranes. As model lipid membranes we employ giant unilamellar vesicles (GUVs) composed of palmitoyloleoyl phosphatidylcholine and cholesterol (10 mol%). We have developed a microfluidic device with multiple chambers to trap GUVs and allow controlled osmotic exchange. Each chamber has a ring-shaped pressure-controlled valve which upon closure allows isolation of the GUVs in a defined volume. Opening the valves leads to a rapid fluid exchange between the trapping region and the microchannel network outside, thus allowing precise control over solution concentration around the GUVs contrary to other experimental approaches for permeability measurements reported in the literature. The area and volume changes of individual vesicles are monitored with confocal microscopy. The solute concentration in the immediate vicinity of the GUVs, and thus the concentration gradient across the membrane, is independently assessed. The data are well fitted by a simple model for water permeability which assumes that the rate of change in volume of a GUV per unit area is linearly proportional to concentration difference with permeability as the proportionality constant. Experiments of GUV osmotic deflation with hypertonic solutions yield the permeability of POPC/cholesterol 9/1 membranes to be p = 15.7 ± 5.5 μm s-1. For comparison, we also show results using two other approaches, which either do not take into account local concentration changes and/or do not resolve the precise vesicle shape. We point out the errors associated with these limitations. Finally, we also demonstrate the applicability of the microfluidic device for studying the dynamics of vesicles under flow.

We describe the engineering and product development of the chemiluminescent ZstatFlu-II Test kit for influenza diagnostics. The reaction vessel is a chemical implementation device with a polystyrene bottom chamber and a polypropylene top chamber that screw together. The patient's specimen is dispersed in a proprietary diluent and mixed inside the bottom chamber with the influenza viral neuraminidase-specific substrate, 1,2-dioxetane-4,7-dimethoxy-Neu5Ac. Neuraminidase catalysis releases the dioxetane. The top chamber contains 40% NaOH and is sealed at the top with an ABS plastic plug-crush pin assembly. The top chamber floor is 85% thinner at the centre, forming a frangible flap. An automated imaging device serves as an incubator for the chemical implementation devices and also facilitates the piercing of the flap by the crush pin. This action results in NaOH flushing into the bottom chamber, initiating chemiluminescence. The imaging device also exposes the Polaroid high-speed detector film to chemiluminescence. At the end of exposure, the film is automatically processed and ejected. Chemiluminescence from an influenza virus-positive specimen produces a "+"-shaped white image, archiving the diagnostic outcome. The modular ZstatFlu-II test kit components are easily adaptable for the chemiluminescent detection of a wide range of analytes.

This chapter contains sections titled: Introduction Analytical and Preparative Enzymatic Cleavage of DNA Analysis of DNA Methylation Nucleic Acid Enzymology Telomerase Assays Polynucleotide Kinase Assays Uracil DNA Glycosylase Assays Protein Nucleic Acid Interaction Mapping: Footprinting Nucleic Acid Tagging DNA Chromatography with Intercalating Dyes

Some biological properties of bovine and recombinant human Cu,Zn superoxide dismutase (bSOD and rhSOD)-poly-ethylene glycol (PEG) adducts prepared by coupling 1-9 strands of high molecular weight PEG (35,000-120,000 daltons) are compared to SOD adducts coupled with 7 or 15 strands of low molecular weight PEG (5,000 daltons). Plasma clearance after i.v. injection was measured in mice and dogs. Conjugates of bSOD with 2 strands of PEG 40,000, 3 strands of PEG 72,000 or 1 strand of PEG 100,000 demonstrated half-lives of about 36 hours in mice, whereas the half-life of a conjugate with 7 strands of PEG 5,000 was about 24 hours. A PEG-bSOD with an average of 3.3 strands of PEG 41,000 was cleared from plasma with a terminal half-life of 36 to 48 hours after intraperitoneal injection in mice. PEG-SODs prepared from bSOD and rhSOD with 3 strands of PEG 50,000 each had plasma half-lives of approximately five days in dogs. An enzyme immunoassay (ELISA) was employed to measure cross-reactivity with a rabbit antibody directed against bSOD. A series of bSOD adducts with 2 to 9 strands of PEG 35,000-120,000 were compared to PEG-bSODs with 7 or 15 strands of PEG 5,000. Attaching larger PEG strands was at least 3 times more effective in reducing antigenicity, compared to PEG 5,000. Ability to induce sensitizing antibodies was measured using subcutaneous sensitization followed by i.v. or s.c. challenge in mice. Some bSOD conjugates with either 7 or 15 strands of PEG 5,000 induced sensitization reactions before the sixth challenge. Fewer than 1% of the animals tested with bSOD or rhSOD adducts with 3 or 4 strands of PEG 65,000, or 3 strands of PEG in the 30,000-50,000 molecular weight range, showed signs of anaphylaxis during six or seven challenges. In a passive cutaneous anaphylaxis test (optimized to measure mouse IgE), neither bSOD coupled to 2 strands of PEG 120,000 nor bSOD coupled to 9 strands of PEG 35,000 induced detectable antibodies against either of these PEG-bSOD preparations or against bSOD; however, the adduct with 2 strands of PEG 120,000 reacted weakly with pre-formed antibodies to bSOD. The high molecular weight PEG-bSODs tested were not immunogenic, but were weakly antigenic, compared to bSOD.

Abstract Analytical tube and slab gel electrophoresis has frequently been used to monitor protein purification procedures. the ABI Model 230A HPECTM micropreparative electrophoresis system was used to separate carbonic anhydrase and fractions were analyzed for purity using the ABI Model 270A CE system and compared to silver stained slab gels. Capillary electrophoresis provides a simple, rapid method for the analysis of protein samples.