المؤلفون: Noreen Halimani, Mikhail Nesterchuk, Alexandra A. Tsitrina, Marat Sabirov, Irina N. Andreichenko, Nataliya O. Dashenkova, Elizaveta Petrova, Alexey M. Kulikov, Timofei S. Zatsepin, Roman A. Romanov, Arsen S. Mikaelyan, Yuri V. Kotelevtsev

المصدر: Scientific Reports, Vol 14, Iss 1, Pp 1-21 (2024)

مصطلحات موضوعية: Medicine, Science

الوصف: Abstract Hepatic fibrosis remains a significant clinical challenge due to ineffective treatments. 4-methylumbelliferone (4MU), a hyaluronic acid (HA) synthesis inhibitor, has proven safe in phase one clinical trials. In this study, we aimed to ameliorate liver fibrosis by inhibiting HA synthesis. We compared two groups of mice with CCl4-induced fibrosis, treated with 4-methylumbelliferone (4MU) and hyaluronan synthase 2 (HAS2) targeting siRNA (siHAS2). The administration of 4MU and siHAS2 significantly reduced collagen and HA deposition, as well as biochemical markers of hepatic damage induced by repeated CCl4 injections. The transcriptomic analysis revealed converging pathways associated with downstream HA signalling. 4MU- and siHAS2-treated fibrotic livers shared 405 upregulated and 628 downregulated genes. These genes were associated with xenobiotic and cholesterol metabolism, mitosis, endoplasmic reticulum stress, RNA processing, and myeloid cell migration. The functional annotation of differentially expressed genes (DEGs) in siHAS2-treated mice revealed attenuation of extracellular matrix-associated pathways. In comparison, in the 4MU-treated group, DEGs were related to lipid and bile metabolism pathways and cell cycle. These findings confirm that HAS2 is an important pharmacological target for suppressing hepatic fibrosis using siRNA.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2045-2322Test

الوصول الحر: https://doaj.org/article/bf1761999a1a4425962a2dc2b2b8b341Test

المؤلفون: Eugenia A. Panova, Denis A. Kleymenov, Dmitry V. Shcheblyakov, Evgeniia N. Bykonia, Elena P. Mazunina, Alina S. Dzharullaeva, Anastasia N. Zolotar, Artem A. Derkaev, Ilias B. Esmagambetov, Ivan I. Sorokin, Evgeny V. Usachev, Anatoly N. Noskov, Igor A. Ivanov, Timofei S. Zatsepin, Sergey E. Dmitriev, Vladimir A. Gushchin, Boris S. Naroditsky, Denis Y. Logunov, Alexander L. Gintsburg

المصدر: Frontiers in Immunology, Vol 14 (2023)

مصطلحات موضوعية: single-domain antibody, mRNA platform, BoNT/A inhibitor, VHH-Fc antibody, Clostridium botulinum, Immunologic diseases. Allergy, RC581-607

الوصف: Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/articles/10.3389/fimmu.2023.1098302/fullTest; https://doaj.org/toc/1664-3224Test

الوصول الحر: https://doaj.org/article/045a699a392047c2a0f489f33838e32aTest

المؤلفون: Vera S. Shashkovskaya, Polina I. Vetosheva, Arina G. Shokhina, Ilya O. Aparin, Tatiana A. Prikazchikova, Arsen S. Mikaelyan, Yuri V. Kotelevtsev, Vsevolod V. Belousov, Timofei S. Zatsepin, Tatiana O. Abakumova

المصدر: Biomedicines, Vol 11, Iss 7, p 1783 (2023)

مصطلحات موضوعية: reactive oxygen species, hepatocellular carcinoma, HyPer7, hydrocyanine, lipid nanoparticles, Biology (General), QH301-705.5

الوصف: Reactive oxygen species (ROS) are highly reactive products of the cell metabolism derived from oxygen molecules, and their abundant level is observed in many diseases, particularly tumors, such as hepatocellular carcinoma (HCC). In vivo imaging of ROS is a necessary tool in preclinical research to evaluate the efficacy of drugs with antioxidant activity and for diagnosis and monitoring of diseases. However, most known sensors cannot be used for in vivo experiments due to low stability in the blood and rapid elimination from the body. In this work, we focused on the development of an effective delivery system of fluorescent probes for intravital ROS visualization using the HCC model. We have synthesized various lipid nanoparticles (LNPs) loaded with ROS-inducible hydrocyanine pro-fluorescent dye or plasmid DNA (pDNA) with genetically encoded protein sensors of hydrogen peroxide (HyPer7). LNP with an average diameter of 110 ± 12 nm, characterized by increased stability and pDNA loading efficiency (64 ± 7%), demonstrated preferable accumulation in the liver compared to 170 nm LNPs. We evaluated cytotoxicity and demonstrated the efficacy of hydrocyanine-5 and HyPer7 formulated in LNP for ROS visualization in mouse hepatocytes (AML12 cells) and in the mouse xenograft model of HCC. Our results demonstrate that obtained LNP could be a valuable tool in preclinical research for visualization ROS in liver diseases.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2227-9059/11/7/1783Test; https://doaj.org/toc/2227-9059Test

الوصول الحر: https://doaj.org/article/351f4c6f437349af9b0647befbdc787fTest

المؤلفون: Ekaterina S. Komarova, Zoya S. Chervontseva, Ilya A. Osterman, Sergey A. Evfratov, Maria P. Rubtsova, Timofei S. Zatsepin, Tatiana A. Semashko, Elena S. Kostryukova, Alexey A. Bogdanov, Mikhail S. Gelfand, Olga A. Dontsova, Petr V. Sergiev

المصدر: Microbial Biotechnology, Vol 13, Iss 4, Pp 1254-1261 (2020)

مصطلحات موضوعية: Biotechnology, TP248.13-248.65

الوصف: Summary Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next‐generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100‐fold range.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1751-7915Test

الوصول الحر: https://doaj.org/article/74952a128b1e4632b64c756fc07dca1aTest

المؤلفون: Noreen Halimani, Mikhail Nesterchuk, Irina N. Andreichenko, Alexandra A. Tsitrina, Andrey Elchaninov, Anastasia Lokhonina, Timur Fatkhudinov, Nataliya O. Dashenkova, Vera Brezgina, Timofei S. Zatsepin, Arsen S. Mikaelyan, Yuri V. Kotelevtsev

المصدر: Cells, Vol 11, Iss 16, p 2498 (2022)

مصطلحات موضوعية: macrophages, polarization, siRNA, IRF5, IRF3, EGR2, Cytology, QH573-671

الوصف: Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone-marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2073-4409/11/16/2498Test; https://doaj.org/toc/2073-4409Test

الوصول الحر: https://doaj.org/article/15fdbb37cfd446388d971bcc2e8cf3f5Test

المؤلفون: Vladimir B. Tsvetkov, Timofei S. Zatsepin, Anton V. Turaev, Valentina M. Farzan, Galina E. Pozmogova, Andrey V. Aralov, Anna M. Varizhuk

المصدر: Computational and Structural Biotechnology Journal, Vol 17, Iss , Pp 527-536 (2019)

مصطلحات موضوعية: Biotechnology, TP248.13-248.65

الوصف: I-motif structures, adopted by cytosine-rich DNA strands, have attracted considerable interest as possible regulatory elements in genomes. Applied science exploits the advantages of i-motif stabilization under acidic conditions: i-motif-based pH sensors and other biocompatible nanodevices are being developed. Two key characteristics of i-motifs as core elements of nanodevices, i.e., their stability under physiological conditions and folding/unfolding rates, still need to be improved. We have previously reported a phenoxazine derivative (i-clamp) that enhances the thermal stability of the i-motif and shifts the pH transition point closer to physiological values. Here, we performed i-clamp guanidinylation to further explore the prospects of clamp-like modifications in i-motif fine-tuning. Based on molecular modeling data, we concluded that clamp guanidinylation facilitated interstrand interactions in an i-motif core and ultimately stabilized the i-motif structure. We tested the effects of guanidino-i-clamp insertions on the thermal stabilities of genomic and model i-motifs. We also investigated the folding/unfolding kinetics of native and modified i-motifs under moderate, physiologically relevant pH alterations. We demonstrated fast folding/unfolding of native genomic and model i-motifs in response to pH stimuli. This finding supports the concept of i-motifs as possible genomic regulatory elements and encourages the future design of rapid-response pH probes based on such structures. Incorporation of guanidino-i-clamp residues at/near the 5′-terminus of i-motifs dramatically decreased the apparent unfolding rates and increased the thermal stabilities of the structures. This counterplay between the effects of modifications on i-motif stability and their effects on kinetics should be taken into account in the design of pH sensors. Keywords: DNA secondary structure, I-motif, Phenoxazine derivative, Thermal stability, Kinetics, pH sensor

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2001037019300492Test; https://doaj.org/toc/2001-0370Test

الوصول الحر: https://doaj.org/article/42b74ec692d54b3a9b30b26a15aed2b7Test

المؤلفون: Mayya V. Monakhova, Elena A. Kubareva, Kirill K. Kolesnikov, Viktor A. Anashkin, Egor M. Kosaretskiy, Maria I. Zvereva, Elena A. Romanova, Peter Friedhoff, Tatiana S. Oretskaya, Timofei S. Zatsepin

المصدر: Molecules, Vol 27, Iss 8, p 2438 (2022)

مصطلحات موضوعية: regioselectivity, DNA modification, DNA–protein complex, modified oligonucleotide, crosslinking, DNA mismatch repair, Organic chemistry, QD241-441

الوصف: Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1420-3049/27/8/2438Test; https://doaj.org/toc/1420-3049Test

الوصول الحر: https://doaj.org/article/a814b76993eb444783e3801142ca0d1bTest

المؤلفون: Marina V. Novoselova, Tatiana O. Abakumova, Boris N. Khlebtsov, Timofei S. Zatsepin, Ekaterina N. Lazareva, Valery V. Tuchin, Vladimir P. Zharov, Dmitry A. Gorin, Ekaterina I. Galanzha

المصدر: Photoacoustics, Vol 20, Iss , Pp 100186- (2020)

مصطلحات موضوعية: Photoacoustic (optoacoustic) imaging, Angiography, Lymphography, Optical clearing, lymphatic drug delivery, Physics, QC1-999, Acoustics. Sound, QC221-246, Optics. Light, QC350-467

الوصف: Photoacoustic (PA) imaging (PAI) is an emerging powerful tool for noninvasive real-time mapping of blood and lymphatic vessels and lymph nodes in vivo to diagnose cancer, lymphedema and other diseases. Among different PAI instruments, commercially available raster-scanning optoacoustic mesoscopy (RSOM) (iThera Medical GmbH., Germany) is useful for high-resolution imaging of different tissues with high potential of clinical translation. However, skin light scattering prevents mapping vessels and nodes deeper than 1–2 mm, that limits diagnostic values of PAI including RSOM. Here we demonstrate that glycerol-based tissue optical clearing (TOC) overcomes this challenge by reducing light scattering that improves RSOM depth penetration. In preclinical model of mouse limb in vivo, the replacement of conventional acoustic coupling agents such as water on the mixture of 70 % glycerol and 30 % ultrasound (US) gel resulted in the increase of tissue imaging depth in 1.5–2 times with 3D visualization of vessels with diameter down to 20 μm. To distinguish blood and lymphatic networks, we integrated label-free PA angiography (i.e., imaging of blood vessels), which uses hemoglobin as endogenous contrast agent, with PA lymphography based on labeling of lymphatic vessels with exogenous PA contrast agents. Similar to well-established clinical lymphography, contrast agents were injected in tissue and taken up by lymphatic vessels within a few minutes that provided quick RSOM lymphography. Furthermore, co-injection of PA contrast dye and multilayer nanocomposites as potential low-toxic drug-cargo showed selective prolonged accumulation of nanocomposites in sentinel lymph nodes. Overall, our findings open perspectives for deep and high resolution 3D PA angio- and lymphography, and for PA-guided lymphatic drug delivery using new RSOM & TOC approach.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2213597920300264Test; https://doaj.org/toc/2213-5979Test

الوصول الحر: https://doaj.org/article/0f2f94dc7eb84298af3f1144a413f36aTest

المؤلفون: Alena Y. Streletskaia, Viacheslav V. Senichkin, Tatiana A. Prikazchikova, Timofei S. Zatsepin, Boris Zhivotovsky, Gelina S. Kopeina

المصدر: Frontiers in Cell and Developmental Biology, Vol 8 (2020)

مصطلحات موضوعية: apoptosis, cell cycle, splicing switch, Mcl-1, BH3-mimetics, Biology (General), QH301-705.5

الوصف: As an important regulator of apoptosis, Mcl-1 protein, a member of the Bcl-2 family, represents an attractive target for cancer treatment. The recent development of novel small molecule compounds has allowed Mcl-1-inhibitory therapy to proceed to clinical trials in cancer treatment. However, the possible adverse effects of either direct inhibition of Mcl-1 or upregulation of Mcl-1S, proapoptotic isoform resulting from alternative splicing of Mcl-1, remain unclear. Here, we investigated changes in Mcl-1S levels during cell cycle and the cell cycle-related functions of Mcl-1 isoforms to address the above-mentioned concerns. It was shown that an anti-mitotic agent monastrol caused accumulation of Mcl-1S mRNA, although without increasing the protein level. In contrast, both mRNA and protein levels of Mcl-1S accrued during the premitotic stages of the normal cell cycle progression. Importantly, Mcl-1S was observed in the nuclear compartment and an overexpression of Mcl-1S, as well as knockdown of Mcl-1, accelerated the progression of cells into mitosis and resulted in DNA damage accumulation. Surprisingly, a small molecule inhibitor of Mcl-1, BH3-mimetic S63845, did not affect the cell cycle progression or the amount of DNA damage. In general, upregulated Mcl-1S protein or genetically inhibited Mcl-1L were associated with the cell cycle perturbations and DNA damage accumulation in normal and cancer cells. At the same time, BH3-mimetic to Mcl-1 did not affect the cell cycle progression, suggesting that direct inhibition of Mcl-1 is devoid of cell-cycle related undesired effects.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/article/10.3389/fcell.2020.543066/fullTest; https://doaj.org/toc/2296-634XTest

الوصول الحر: https://doaj.org/article/fe281dc5d1934d109ba17179daeb6267Test

المؤلفون: Viacheslav V. Senichkin, Nikolay V. Pervushin, Alexey V. Zamaraev, Elena V. Sazonova, Anton P. Zuev, Alena Y. Streletskaia, Tatiana A. Prikazchikova, Timofei S. Zatsepin, Olga V. Kovaleva, Elena M. Tchevkina, Boris Zhivotovsky, Gelina S. Kopeina

المصدر: Cancers, Vol 14, Iss 1, p 181 (2021)

مصطلحات موضوعية: Bcl-2 family proteins, BH3-mimetics, Mcl-1, cancer therapy, sensitivity, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282

الوصف: BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2072-6694/14/1/181Test; https://doaj.org/toc/2072-6694Test

الوصول الحر: https://doaj.org/article/4d20d04e09eb46a3a7f13559f0a60005Test