Excessive oxidative stress drives lipid peroxidation and contributes to skeletal muscle atrophy in a range of musculoskeletal diseases. Polyunsaturated fatty acids (PUFAs) are essential components of muscle cell membrane phospholipids and are especially susceptible to peroxidation due to the presence of double bonds. Currently, therapeutic options targeting lipid peroxidation to prevent muscle wasting are limited. Substituting the hydrogen atom at the bis-allylic position with deuterium could conceivably limit lipid peroxidation while retaining enzymatic PUFA metabolism. Here we investigated the potential role of deuterated PUFAs (D-PUFAs) in protecting against muscle cell dysfunction under conditions of elevated oxidative stress. Both native (H-) and deuterated (D-) forms of long chain PUFAs including arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) stimulated in vitro muscle cell growth and development in the absence of oxidative stress. D-ARA, D-EPA, D-DPA, and D-DHA each protected cultured myotubes against the deleterious effects of direct exposure to reactive oxygen species (ROS) by limiting lipid peroxidation. In contrast, H-ARA, H-EPA, H-DPA, and H-DHA each increased sensitivity to ROS-induced lipid peroxidation and exacerbated oxidative stress-induced muscle cell dysfunction. Deuterated 18-carbon linoleic acid (D-LA), alpha linolenic acid (D-ALA), as well as D-ARA and D-EPA (but not D-DPA or D-DHA) also protected against the deleterious effects of ferroptosis inducer erastin on myogenic differentiation. Finally, D-PUFAs modulated local expression of endogenous antioxidant enzymes, muscle-specific protein ligases, and key enzymes involved in mitochondrial energy metabolism. Overall, our study suggests a promising role of D-PUFAs as novel therapeutics to protect against skeletal muscle dysfunction induced by oxidative stress.

Aims: Non-enzymatic autoxidation of polyunsaturated fatty acids (PUFAs), generating numerous toxic by-products implicated in neurodegeneration, aging, and other pathologies, is a key process in ferroptosis. Lipid peroxidation (LPO) can be inhibited by deuterated polyunsaturated fatty acids (D-PUFA), as the rate-limiting step of abstraction of bis-allylic hydrogen atoms is slowed down by replacing the bis-allylic hydrogens with deuteriums. Here, we aimed to assess the protective effect of monounsaturated fatty acids (MUFA), which do not undergo LPO, as compared to that of various D-PUFAs, in a liposomal model of LPO. Methods: To detect LPO induced by ferrous ions in liposomes, we used the LPO fluorescent probe C11-Bodipy (581/591), in addition to measuring conjugated diene and malondialdehyde accumulation. Results: By applying the C11-Bodipy (581/591) probe, we found that both 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-stearoyl-2-(11,11-d2-linoleoyl)-phosphatidylcholine (D2-Lin-PC) protect non-deuterated 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (H-Lin-PC) liposomes from LPO. Similarly, both POPC and 1-stearoyl-2-(11,11,14,14-D4-linolenyl)-phosphatidylcholine (D4-Lnn-PC) protect 1-stearoyl-2-linolenyl-phosphatidylcholine (H-Lnn-PC), and so does 1-stearoyl-2-(6,6,9,9,12,12,15,15,18,18-d10-docosahexaenoyl)-sn-glycero-3-phosphatidylcholine (D10-DHA-PC). The conjugated diene and malondialdehyde probes also showed similar protective effects of POPC and D-PUFA on LPO in H-Lnn-PC. Conclusion: Obviously, the presence of non-oxidizable lipids, such as POPC, similar to the deuterated lipids D2-Lin-PC, D4-Lnn-PC, and D10-DHA-PC, leads to a sharp decrease in the length of lateral propagation of chain reactions in lipid membranes, but they do not participate in LPO themselves.

A new approach to siRNA delivery using high-density lipoprotein-like nanoparticles (HDL NPs) was investigated, incorporating oligoamine and cholesterol-derived cationic lipids (CLs) to associate siRNA with the carrier. Newly designed or commercially available compounds, including GL67 and 3-beta-[N-(N ',N '-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), were tested for siRNA binding, cytotoxicity, and siRNA cellular uptake. GL67 emerged as the most promising CL for siRNA delivery via HDL NPs. While it contributed to substantial siRNA uptake and cytosolic delivery in HepG2 cells, gene silencing remained limited, indicating a need for further optimization. Despite this, the study highlights the potential of positively charged cholesterol derivatives for siRNA delivery using HDL NPs. An analysis of the relationship between CL head group structure and HDL NPs' siRNA binding efficiency and cytotoxicity showed that factors such as oligoamine molecule conjugation site, linker type, amine group ethylation, and alkyl chain length between amine groups are crucial for optimizing CL design. Furthermore, the phospholipid environment surrounding CLs significantly influences HDL NPs' performance, particularly in siRNA cellular uptake. The study also revealed that intracellular siRNA trafficking varies by cell type, emphasizing the importance of customizing HDL NP formulations for specific cells. These insights are important for designing more effective HDL NPs for siRNA therapeutic delivery.

The field of delivering nucleic acids (NAs) via high-density lipoprotein-mimicking nanoparticles (HDL NPs) has shown promising advancements over the past two decades. HDL NPs are designed to efficiently bind NAs, safeguard them from degradation, and help navigate through various biological barriers to deliver them into the target cell's cytosol. Some HDL NPs allow direct cytosolic delivery of NAs by a selective mechanism with the involvement of HDL's natural receptor scavenger receptor class B type I (SR-B1). In contrast, others rely on endocytic uptake of the entire NA-loaded HDL NP. Owing to their highly biocompatible nature, ability to target clinically relevant receptors, and fine tunability, NA-loaded HDL NPs are applied to treat cancer, cardiovascular diseases, and brain malignancies. They are also emerging as potent vaccines against cancers and infectious diseases. This review focuses on various architectures of NA-loaded HDL NPs, their mechanisms for NA cellular uptake, and their therapeutic efficacy in vivo. It comprehensively covers the latest nanocarriers for NA delivery that contain HDL's apolipoproteins (ApoA-I, ApoE) or their mimetic peptides, which define the unique functional and targeting capabilities of HDL NPs.

The effects of pH and the solution composition on the photophysical properties of two carboxyfluorescein bifluorophores were studied along with a comparison of the fluorescence effect of these bifluorophores with that of monomer carboxyfluoresceins (5-FAM and 6-FAM) in conjugates with protein molecules. Both the bifluorophores were found to fluoresce well in an alkaline medium but the fluorescence intensity decreased at pH 6–7 and almost disappeared in acidic media. The fluorescence of meta-(6-FAM)2 is very sensitive to the solution composition, while the fluorescence of meta-(5-FAM)2 is more stable and depends only slightly on the solution properties. Upon formation of a conjugate of the bifluorophore with a protein, the fluorescence yield of the bifluorophore decreases to a greater extent in comparison with the FAM monomers but the total fluorescence yield is not less than the intensities of the two carboxyfluorescein monomer–protein conjugates due to the high extinction coefficient of the bifluorophore ( 140,000 M–1·cm–1).

In non-viscous aqueous solutions, the cyanine fluorescent dyes Cy3 and Cy5 have rather low fluorescence efficiency (the fluorescence quantum yields of Cy3 and Cy5 are 0.04 and 0.3, respectively [1, 2]) and short excited state lifetimes due to their structural features. In this work, we investigated the effect of solubility and rotational degrees of freedom on the fluorescence efficiency of Cy3 and Cy5 in several ways. We compared the fluorescence efficiencies of two cyanine dyes sCy3 and sCy5 with the introduction of a sulfonyl substituent in the aromatic ring as well as covalently bound to T10 oligonucleotides. The results show that because of the different lengths of the polymethine chains between the aromatic rings of the dyes, cis–trans-isomerization has a much greater effect on the Cy3 molecule than on the Cy5 molecule, while the effect of aggregation is also significant.

Arachidonic acid (ARA) is a major component of lipid bilayers as well as the key substrate for the eicosanoid cascades. ARA is readily oxidized, and its non-enzymatic and enzymatic oxidation products induce inflammatory responses in nearly all tissues, including lung tissues. Deuteration at bis-allylic positions substantially decreases the overall rate of ARA oxidation when hydrogen abstraction is an initiating event. To compare the effects of dosing of arachidonic acid (H-ARA) and its bis-allylic hexadeuterated form (D-ARA) on lungs in conventionally healthy mice and in an acute lung injury model, mice were dosed with H-ARA or D-ARA for six weeks through dietary supplementation and then challenged with intranasal lipopolysaccharide (LPS) for subsequent analysis of bronchoalveolar lavage fluid and lung tissue. Dosing on D-ARA resulted in successful incorporation of D-ARA into various tissues. D-ARA significantly reduced LPS-induced adverse effects on alveolar septal thickness and the bronchoalveolar area. Oral deuterated ARA is taken up efficiently and protects against adverse LPS-induced pathology. This suggests novel therapeutic avenues for reducing lung damage during severe infections and other pathological conditions with inflammation in the pulmonary system and other inflammatory diseases.

ABSTRACTCRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect onin vitrotarget DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

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 HPUFA 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.

A synthetic protocol was developed and optimal reagents have been selected for obtaining guide RNA oligonucleotides for the CRISPR/Cas system using ASM-2000 automatic synthesizer in 500 nmol scale. Methods for the isolation, purification and analytical control of synthetic RNA oligonucleotides have been developed. The improved technology has been used for preparation of guide RNAs for the CRISPR Cas12a system.

Lipid peroxidation (LPO) plays a key role in many age-related neurodegenerative disorders and other pathologies. introduction of deuterated polyunsaturated fatty acids (D-PUFA) into membrane lipids has been recently suggested as a promising approach for protection against LPO. We have previously demonstrated inhibition of the Fe2+ascorbate - initiated LPO, when about 20% of membrane lipids contained D-PUFA with deuterium atoms instead of protons at bis-allylic positions (Firsov et al. FEBS J. 2019). Here, we report on the protective action of D-PUFA upon the photodynamic induction (illumination in the presence of a photosensitizer) of LPO in liposomes via two experimental procedures: 1) sulforhodamine B leakage from liposomes, detected with an FCS setup by changes in the amplitude of autocorrelation function, and 2) formation of diene conjugates, measured by absorbance at 234 nm. Trisulfonated aluminum phthalocyanine (AlPcS3) was used as a photosensitizer in these experiments. in particular, in the case of liposomes formed from lipids without D-PUFA (dilinoleyl-sn-glycero-3-phosphatidylcholine), 1-minute illumination led to a dramatic decrease in the amplitude of the autocorrelation function (ACF) showing permeabilization of the liposomes. by contrast, no decrease in the ACF amplitude was observed in the case of 50% D-PUFA (1-palmitoyl-2-(11,11-D2-linoleyl)-sn-glycero-3-phosphatidylcholine) - containing liposomal lipids. Experiments with giant unilamellar liposomes (GUVs) confirmed the protective action of the D-PUFA on the membrane permeability compromised by photodymanic action. The light-induced changes of the GUV surface as well as phase-contrast intensity (related to sucrose leakage) were significantly decreased in the presence of D-PUFA. This work was supported by the Russian Science Foundation grant 19-74-00015.

Graphene oxide (GO) functionalization has great importance for its practical application in many fields, and is a key step for GO bioconjugation. Polydispersity, limited colloidal stability and changeable extinction of GO complicates functional group quantification and the modification study. To bypass the mentioned limitations, we have used GO immobilization on glass support to quantify its epoxy groups surface load, and to study sustainability of the functional groups introduced through epoxy-amine reaction during hydrazine reduction. It was found out that GO contains near 1 epoxy group per nm2, and the major part of the functional groups are remaining after reduction process allowing to use this modification to make functionalized reduced graphene oxide (rGO). Then we modified colloidal GO with azide groups by aminotetraethylene glycol azide (H2N-TEG-N3) in mild conditions to avoid aggregation, and characterized it by means of immobilization on glass surface. Functional group surface load of the functionalized GO was determined as 0,7 groups per nm2. Finally, we proved capability of azide-functionalized GO for conjugation by strain promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) with BCN derivative of organic fluorescent dye JOE.

An effective method for the preparation of water-soluble cyanine dye Cy5 using the soluble polymers supported liquid-phase organic synthesis (LPOS) was proposed. Poly (ethylene glycol) methyl ether with a molecular weight of 2000 was used as a polymer substrate, which allowed us to simplify the characterization of products at intermediate stages of synthesis by NMR spectroscopy. This approach makes it easy to obtain the necessary cyanine dyes, which are widely used as fluorescent labels and are popular modifying reagents in biochemistry and medicine.

We report herein improved version of the synthesis of hapten based on cholecalciferol and its active metabolite 25-hydroxycholecalcalferol. The methodology of obtaining high-molecular immunogenic conjugates of vitamin D 3 derivatives with bovine serum albumin and horseradish peroxidase conjugates for direct ELISA was optimized. Immunisation of rabbits was carried out and polyclonal antibodies to 25-hydroxycholecalceferol were obtained and tested in an enzyme-linked immunosorbent assay. To improve the accuracy of the method, the sample preparation procedure was optimized, including the release of vitamin D 3 and its active metabolites from complexes with vitamin D-binding protein.

Bright fluorescent probes with enhanced intensities in the fluorescein channel are of great value for plenty of biological applications. To design effective probes one should introduce as many as possible fluorophores to the biomolecule while leaving its native structure as intact as possible. To reach this compromise, we designed and synthesized fluorescein bifluorophores on the 3,5-diaminobenzoic acid scaffold, which allows for insertion of two fluorophores at one modification site of a biomolecule. Rigid structure of the branching linker group allows to minimize self-quenching the fluorophores. However, despite the structure similarities of fluorescein isomers (5-FAM and 6-FAM), different photophysical behavior was observed for the corresponding bifluorophores. Here we made efforts to get insight into these effects with the focus on the media viscosity impact.

We report herein improved version of the synthesis of hapten based on cholecalciferol and its active metabolite 25-hydroxycholecalcalferol. The methodology of obtaining high-molecular immunogenic conjugates of vitamin D3 derivatives with bovine serum albumin and horseradish peroxidase conjugates for direct ELISA was optimized. Immunisation of rabbits was carried out and polyclonal antibodies to 25-hydroxycholecalceferol were obtained and tested in an enzyme-linked immunosorbent assay. To improve the accuracy of the method, the sample preparation procedure was optimized, including the release of vitamin D3 and its active metabolites from complexes with vitamin D-binding protein.

Lipid peroxidation (LPO) is causative for pathogenesis of many diseases, including neurodegenerative and inflammatory processes. To suppress LPO, it has recently been proposed to use polyunsaturated fatty acids (PUFAs) containing deuterium atoms instead of protons at bis-allylic positions (D-PUFAs). The presence of less than 20% D-PUFA in the total amount of PUFA was shown to completely stop LPO in yeast cells under oxidative stress. Here, we report results of studying the impact of phospholipids containing D-PUFAs on LPO in model bilayer lipid membranes by using two approaches: 1) monitoring diene conjugates in liposomal membranes and 2) measuring permeabilization of liposomes. The latter was estimated with the fluorescence correlation spectroscopy method from changes in the amplitude of the autocorrelation function of liposomes loaded with the fluorescent dye sulforhodamine B. Both approaches revealed a strong protective action of small amounts of deuterated PUFA against LPO in bilayer membranes. The extent of protection depended on the nature of D-PUFAs, being determined predominantly by the total level of deuterated bis-allylic (CD2) groups. For each D-PUFA, there was found a threshold percentage (about 20-25% for 1-acyl-2-(11,11-D2-linoleyl)-sn-glycero-3-phosphatidylcholine and much less for 1-acyl-2-(6,6,9,9,12,12,15,15,18,18-D10-docosahexaenoyl)-sn-glycero-3-phosphatidylcholine) in the H-PUFA matrix that inhibited LPO, which could be attributed to an ability of D-PUFA-derived radicals to interrupt LPO chain reactions in a lipid bilayer. The protecting effect of D-PUFAs was compared to the action of various free-radical scavengers, e.g. trolox. This work was supported by the Russian Science Foundation grant 19-74-00015.

— A comparative analysis of the interaction of the chimeric protein BCR-ABL, of the normal type and with the T315I mutation, with known inhibitors as well as compounds potentially capable of inhibiting the mutant protein has been carried out by computer modeling. It has been shown that the compounds proposed are incorported into the structure of the protein with the retention of the basic hydrogen bonds and intermolecular interactions. Two structures containing the pyrrole cycle have been synthesized, which, according to the results of computer modeling, appear to be most promising.

Autoxidation of polyunsaturated fatty acids (PUFAs) damages lipid membranes and generates numerous toxic by-products implicated in neurodegeneration, aging, and other pathologies. Abstraction of bis-allylic hydrogen atoms is the rate-limiting step of PUFA autoxidation, which is inhibited by replacing bis-allylic hydrogens with deuterium atoms (D-PUFAs). In cells, the presence of a relatively small fraction of D-PUFAs among natural PUFAs is sufficient to effectively inhibit lipid peroxidation (LPO). Here, we investigate the effect of various D-PUFAs on the stability of liposomes under oxidative stress conditions. The permeability of vesicle membranes to fluorescent dyes was measured as a proxy for bilayer integrity, and the formation of conjugated dienes was monitored as a proxy for LPO. Remarkably, both approaches reveal a similar threshold for the protective effect of D-PUFAs in liposomes. We show that protection rendered by D-PUFAs depends on the structure of the deuterated fatty acid. Our findings suggest that protection of PUFAs against autoxidation depends on the total level of deuterated bi-sallylic (CD2) groups present in the lipid bilayer. However, the phospholipid containing 6,6,9,9,12,12,15,15,18,18-d(10)-docosahexaenoic acid exerts a stronger protective effect than should be expected from its deuteration level. These findings further support the application of D-PUFAs as preventive/therapeutic agents in numerous pathologies that involve LPO.