Deuterated polyunsaturated fatty acids inhibit photoirradiation-induced lipid peroxidation in lipid bilayers.

Lipid peroxidation (LPO) plays a key role in many age-related neurodegenerative conditions and other disorders. Light irradiation can initiate LPO through various mechanisms and is of importance in retinal and dermatological pathologies. The introduction of deuterated polyunsaturated fatty acids (D-PUFA) into membrane lipids is a promising approach for protection against LPO. Here, we report the protective effects of D-PUFA against the photodynamically induced LPO, using illumination in the presence of the photosensitizer trisulfonated aluminum phthalocyanine (AlPcS3) in liposomes and giant unilamellar vesicles (GUV), as assessed in four experimental models: 1) sulforhodamine B leakage from liposomes, detected with fluorescence correlation spectroscopy (FCS); 2) formation of diene conjugates in liposomal membranes, measured by absorbance at 234 nm; 3) membrane leakage in GUV assessed by optical phase-contrast intensity observations; 4) UPLC-MS/MS method to detect oxidized linoleic acid (Lin)-derived metabolites. Specifically, in liposomes or GUV containing H-PUFA (dilinoleyl-sn-glycero-3-phosphatidylcholine), light irradiation led to an extensive oxidative damage to bilayers. By contrast, no damage was observed in lipid bilayers containing 20% or more D-PUFA (D2-Lin or D10-docosahexanenoic acid). Remarkably, addition of tocopherol increased the dye leakage from liposomes in H-PUFA bilayers compared to photoirradiation alone, signifying tocopherol's pro-oxidant properties. However, in the presence of D-PUFA the opposite effect was observed, whereby adding tocopherol increased the resistance to LPO. These findings suggest a method to augment the protective effects of D-PUFA, which are currently undergoing clinical trials in several neurological and retinal diseases that involve LPO.

Molecular Beacon DNA Probes with Fluorescein Bifluorophore.

An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of "molecular beacon"-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or "click reaction" (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔCt = 1-2. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1134/S1068162021030055.

5-(Perylen-3-ylethynyl)uracil Derivatives Inhibit Reproduction of Respiratory Viruses.

In this work, we describe the synthesis of 5-(perylen-3-ylethynyl)uridine and its ability to effectively inhibit the replication of respiratory disease pathogens in cell culture, namely: influenza A virus (IVA); type 3 parainfluenza virus (PIV-3); and human respiratory syncytial virus (RSV). Related known compounds were also analyzed: 5-(perylen-3-ylethynyl)-2'-deoxy-uridine; 5-(perylen-3-ylethynyl)-arabino-uridine; and 1-carboxymethyl-3-pivaloyloxymethyl-5-(perylen-3-ylethynyl)uracil.

Highly Sensitive Immunochromatographic Identification of Tetracycline Antibiotics in Milk.

A rapid immunochromatographic assay was developed for the control of tetracycline (TC). The assay is based on the competition between immobilized TC-protein conjugate and TC in a tested sample for binding with polyclonal anti-TC antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. Conjugation of colloidal gold and the total immunoglobulin (IgG) fraction of polyclonal antibodies was used to increase the assay sensitivity to ensure low content of specific antibodies in the conjugate. This allowed effective inhibition of free TC and conjugate binding in the strip test zone. Photometric marker registration allows control of the reduction of binding, thereby enhancing detection sensitivity. The proposed assay allows TC to be detected at concentrations up to 20 ng/mL, exceeding the limit of detection of the known analogues, in a wide working range (more than two orders) of 60 pg/mL to 10 ng/mL, ensured through the use of polyclonal antibodies. The assay time is 10 min. The efficiency of the designed assay is shown to identify TC in milk; the degree of recovery of TC ranges from 90 to 112%. The precision of the concentrations measurements was no more than 10%.

Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(meth)acrylic acid.

BACKGROUND: Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising. RESULTS: New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(meth)acrylic acid. The beads contain up to 0.31 micromol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents. CONCLUSIONS: An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides.

Blocking of non-specific sorption in ELISA on formylated polystyrene beads.

The effect of pH of the blocking solution on the non-specific sorption of peroxidase conjugates to the 0.63-cm formylated polystyrene beads was studied. Anti-G-HRP and BSA-HRP sorption was shown to dramatically depend on pH of blocking protein solutions and was minimum at pH=pI. Sorption of G-HRP weakly depended on pH of the blocking buffer. The blocking efficiency of proteins on unmodified beads, as well as of Tween-20 on formylated beads appeared to be pH independent. The optimized blocking step resulted in a multiple decrease in the non-specific sorption of conjugates on formylated beads.