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CRISPR-Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR-Cas-based therapies. This review examines the current landscape of CRISPR-Cas implementation in clinical trials, with data from key registries, including the Australian New Zealand Clinical Trials Registry, the Chinese Clinical Trial Register, and ClinicalTrials.gov. Emphasis is placed on the mechanism of action of tested therapies, the delivery method, and the most recent findings of each clinical trial.

Currently, the CRISPR-Cas9 system serves as a prevalent tool for genome editing and gene expression regulation. Its therapeutic application is limited by off-target effects that can affect genomic integrity through nonspecific, undesirable changes in the genome. Various strategies have been explored to mitigate the off-target effects. Many approaches focus on modifying components of the system, namely, Cas9 and guide RNAs, to enhance specificity. However, a common challenge is that methods aiming to increase specificity often result in a significant reduction in the editing efficiency. Here, we introduce a novel approach to modifying crRNA to balance CRISPR-Cas9 specificity and efficiency. Our approach involves incorporating nucleoside modifications, such as replacing ribo- to deoxyribonucleosides and backbone modifications, using phosphoryl guanidine groups, specifically 1,3-dimethylimidazolidin-2-ylidene phosphoramidate. In this case, within the first 10 nucleotides from the 5' crRNA end, phosphodiester bonds are substituted with phosphoryl guanidine groups. We demonstrate that crRNAs containing a combination of deoxyribonucleosides and single or multiple phosphoryl guanidine groups facilitate the modulation of CRISPR-Cas9 system activity while improving its specificity in vitro .

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance., (© 2024. The Author(s).)

Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[amino(polyethylene glycol)-2000] (DSPE-PEG 2000 ) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.

Hepatitis is an inflammatory liver disease primarily caused by hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. The chronic forms of hepatitis resulting from HBV and HCV infections can progress to cirrhosis or hepatocellular carcinoma (HCC), while acute hepatitis can lead to acute liver failure, sometimes resulting in fatality. Viral hepatitis was responsible for over 1 million reported deaths annually. The treatment of hepatitis caused by viral infections currently involves the use of interferon-α (IFN-α), nucleoside inhibitors, and reverse transcriptase inhibitors (for HBV). However, these methods do not always lead to a complete cure for viral infections, and chronic forms of the disease pose significant treatment challenges. These facts underscore the urgent need to explore novel drug developments for the treatment of viral hepatitis. The discovery of the CRISPR/Cas9 system and the subsequent development of various modifications of this system have represented a groundbreaking advance in the quest for innovative strategies in the treatment of viral infections. This technology enables the targeted disruption of specific regions of the genome of infectious agents or the direct manipulation of cellular factors involved in viral replication by introducing a double-strand DNA break, which is targeted by guide RNA (spacer). This review provides a comprehensive summary of our current knowledge regarding the application of the CRISPR/Cas system in the regulation of viral infections caused by HAV, HBV, and HCV. It also highlights new strategies for drug development aimed at addressing both acute and chronic forms of viral hepatitis.

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.

Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.

At the present time, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has been widely adopted as an efficient genomic editing tool. However, there are some actual problems such as the off-target effects, cytotoxicity, and immunogenicity. The incorporation of modifications into guide RNAs permits enhancing both the efficiency and the specificity of the CRISPR-Cas9 system. In this study, we demonstrate that the inclusion of N 6 -methyladenosine, 5-methylcytidine, and pseudouridine in trans-activating RNA (tracrRNA) or in single guide RNA (sgRNA) enables efficient gene editing in vitro . We found that the complexes of modified guide RNAs with Cas9 protein promoted cleavage of the target short/long duplexes and plasmid substrates. In addition, the modified monomers in guide RNAs allow increasing the specificity of CRISPR-Cas9 system in vitro and promote diminishing both the immunostimulating and the cytotoxic effects of sgRNAs.

Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. The study of the pathogenesis of GBM, as well as the development of targeted oncolytic drugs, require the use of actual cell models, in particular, the use of 3D cultures or neurospheres (NS). During the formation of NS, the adaptive molecular landscape of the transcriptome, which includes various regulatory RNAs, changes. The aim of this study was to reveal changes in the expression of microRNAs (miRNAs) and their target mRNAs in GBM cells under conditions of NS formation. Neurospheres were obtained from both immortalized U87 MG and patient-derived BR3 GBM cell cultures. Next generation sequencing analysis of small and long RNAs of adherent and NS cultures of GBM cells was carried out. It was found that the formation of NS proceeds with an increase in the level of seven and a decrease in the level of 11 miRNAs common to U87 MG and BR3, as well as an increase in the level of 38 and a decrease in the level of 12 mRNA/lncRNA. Upregulation of miRNAs hsa-miR: -139-5p; -148a-3p; -192-5p; -218-5p; -34a-5p; and -381-3p are accompanied by decreased levels of their target mRNAs: RTN4, FLNA, SH3BP4, DNPEP, ETS2, MICALL1, and GREM1. Downregulation of hsa-miR: -130b-5p, -25-5p, -335-3p and -339-5p occurs with increased levels of mRNA-targets BDKRB2, SPRY4, ERRFI1 and TGM2. The involvement of SPRY4, ERRFI1, and MICALL1 mRNAs in the regulation of EGFR/FGFR signaling highlights the role of hsa-miR: -130b-5p, -25-5p, -335-3p, and -34a-5p not only in the formation of NS, but also in the regulation of malignant growth and invasion of GBM. Our data provide the basis for the development of new approaches to the diagnosis and treatment of GBM.

Glioma is the most common and heterogeneous primary brain tumor. The development of a new relevant preclinical models is necessary. As research moves from cultures of adherent gliomas to a more relevant model, neurospheres, it is necessary to understand the changes that cells undergo at the transcriptome level. In the present work, we used three patient-derived gliomas and two immortalized glioblastomas, while their cultivation was carried out under adherent culture and neurosphere (NS) conditions. When comparing the transcriptomes of monolayer (ML) and NS cell cultures, we used Enrichr genes sets enrichment analysis to describe transcription factors (TFs) and the pathways involved in the formation of glioma NS. It was observed that NS formation is accompanied by the activation of five common gliomas of TFs, SOX2, UBTF, NFE2L2, TCF3 and STAT3. The sets of transcripts controlled by TFs MYC and MAX were suppressed in NS. Upregulated genes are involved in the processes of the epithelial-mesenchymal transition, cancer stemness, invasion and migration of glioma cells. However, MYC/MAX-dependent downregulated genes are involved in translation, focal adhesion and apical junction. Furthermore, we found three EGFR and FGFR signaling feedback regulators common to all analyzed gliomas-SPRY4, ERRFI1, and RAB31-which can be used for creating new therapeutic strategies of suppressing the invasion and progression of gliomas.

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR-Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR-Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex's components of Cas9-RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9-RNA/DNA formation from the Cas9-RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR-Cas9 system.

The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR-Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.

Uracil-DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil-DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC 50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme's active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A-H1N1 and H3N2-and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0-M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.

CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2'-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/Cas9-mediated editing of box C/D snoRNA genes in Gas5 . We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2'-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m 6 A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene., (Copyright © 2019 Filippova, Matveeva, Zhuravlev, Balakhonova, Prokhorova, Malanin, Shah Mahmud, Grigoryeva, Anufrieva, Semenov, Vlassov and Stepanov.)

In response to the wide variety of external and internal signals, mammalian cells undergo apoptosis, programmed cell death. Dysregulation of apoptosis is involved in multiple human diseases, including cancer, autoimmunity, and ischemic injuries. Two types of apoptosis have been described: the caspase-dependent one, leading to digestion of cellular proteins, and caspase-independent apoptosis, resulting in DNA fragmentation. The latter type of apoptosis is executed by AIF protein and is believed to have appeared first during evolution. The key step in the caspase-independent apoptosis program is the dissociation of AIF from the outer mitochondrial membrane (OMM). However, the molecular mechanism of interaction between AIF and OMM remains poorly understood. In this study, we demonstrated that AIF can bind to OMM via mortalin protein. We confirmed interaction between AIF and mortalin both in vitro and in vivo and mapped the amino acid sequences that are important for the binding of these proteins. Next, we showed that apoptosis induction by chemotherapy leads to downregulation of AIF-mortalin interaction and dissociation of AIF from the OMM. Finally, a bioinformatic analysis demonstrated that a high level of mortalin expression correlates with a worse survival prognosis for glioma patients. Altogether, our data revealed that mortalin plays an important role in the regulation of the caspase-independent apoptotic pathway and allowed us to speculate that inhibition of AIF-mortalin interaction may induce a dissociation of AIF from the OMM and subsequent apoptosis of cancer cells.

Background: The phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment., Methods: Here, we developed primary cultures of human breast tissue - normal cells (BN1), cancer cells (BC5), and cells from a chemotherapy-treated tumour (BrCCh1) to compare their response to conventional chemotherapeutics and to innate immunity stimulators with that of the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. Expression of the progesterone receptor (PGR), oestrogen receptor (ER) α and β, human epidermal growth factor receptor (HER) 2 and 3 and aromatase CYP19, as well as expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) mRNA in human breast cells were characterized., Results: We revealed that BC5 carcinoma cells were PGR low /ERb high /ERa - /Cyp19 + , the BrCCh1 cells that originated from the recurrent tumour were PGR - /ERb + /ERa - /Cyp19 + , and normal BN cells were PGR - /ERb + /ERa - /Cyp19 high . The treatment of primary culture cells with antitumour therapeutics revealed that BrCCh1 cells were doxorubicine-resistant and sensitive to cisplatin. BC5 cells exhibited low sensitivity to tamoxifen and cisplatin. The innate immunity activators interferon-α and an artificial small nucleolar RNA analogue increased expression of IFIT3 at different levels in primary cells and in the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. The relative level of activation of IFIT3 expression was inversely correlated with the baseline level of IFIT3 mRNA expression in breast cell lines., Conclusion: Our data demonstrated that primary cancer cells are a useful model for the development of novel cancer treatments. Our findings suggest that expression of IFIT3 mRNA can be used as a prognostic marker of breast cancer cell sensitivity to immunostimulating therapeutics.

This review considers approaches for detection of modified monomers in the RNA structure of living organisms. Recently, some data on dynamic alterations in the pool of modifications of the key RNA species that depend on external factors affecting the cells and physiological conditions of the whole organism have been accumulated. The recent studies have presented experimental data on relationship between the mechanisms of formation of modified/minor nucleotides of RNA in mammalian cells and the development of various pathologies. The development of novel methods for detection of chemical modifications of RNA nucleotides in the cells of living organisms and accumulation of knowledge on the contribution of modified monomers to metabolism and functioning of individual RNA species establish the basis for creation of novel diagnostic and therapeutic approaches. This review includes a short description of routine methods for determination of modified nucleotides in RNA and considers in detail modern approaches that enable not only detection but also quantitative assessment of the modification level of various nucleotides in individual RNA species.

It has been previously demonstrated that lactaptin, the proteolytic fragment of human milk protein κ-casein, induces the death of various cultured cancer cells. The recombinant analog of lactaptin, RL2, effectively induces the apoptosis of mouse hepatocarcinoma-1 (HA-1) tumor cells in vitro and suppress the growth of HA-1 tumors and metastases in vivo . The antitumor drug Lactaptin developed on the basis of RL2 has been successful in preclinical trials. Lactaptin shows its efficiency in relation to mouse and human cancer cells and tumors. However, Lactaptin, as with the majority of protein-based therapeutic drugs, is distributed evenly throughout the organism, which reduces its antitumor efficacy. To develop the targeted delivery of lactaptin, the present study selected tumor-specific peptides by screening a phage display peptide library in vivo on A/Sn strain mice with subcutaneously transplanted HA-1 cells. Two genetic constructs were made for the production of recombinant fusion proteins composed of RL2 and the selected tumor-targeting peptide. In vitro experiments involving HA-1, MDA-MB-231 and MCF-7 cells cultures demonstrated that the fusion proteins induce apoptotic death in mouse and human tumor cells, as with RL2. The in vivo experiments involving the mouse HA-1 tumor model demonstrated that the tumor fluorescence intensity of the Cy5-fusion protein conjugates is higher than that of RL2-Cy5. As conjugation of the tumor-specific peptides to RL2 provided retention of RL2 in the tumor tissues, fusion proteins composed of lactaptin and peptides specific for human tumors are deemed promising to improve the antitumor efficiency of lactaptin.

Fragments of small nucleolar RNAs (snoRNAs) were found among various non-coding RNAs (ncRNAs) circulating in human blood. Currently, the function of such cell-free sno-derived-RNAs is not clearly defined. This work is aimed at identifying regulatory pathways controlled by extracellular snoRNAs. In order to determine the molecular targets and pathways affected by artificial snoRNAs, we performed Illumina array analysis of MCF-7 human adenocarcinoma cells transfected with box C/D RNAs. The genes related to the innate immune response and apoptotic cascades were found to be activated in transfected cells compared with control cells. Intriguingly, the transfection of MCF-7 cells with artificial box C/D snoRNAs also increased the transcription of several microRNAs, such as mir-574, mir-599 and mir-21. Our data demonstrated that extracellular snoRNAs introduced into human cells may function as gene expression modulators, with activation of microRNA genes being one of the regulatory mechanisms.

Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.

Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2'-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs.

In this study we obtained and characterized the recombinant analogue of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation and mitosis. The influence ofnucleophosmin 1 on extrcellular RNAs accumulation in human adenocarcinoma cells MCF-7 was analyzed. It was found that incubation of AluY RNA (n > 300 nt), U24 snoRNA analogues (n ~ 80 nt) with Npm1-His6 resulted in RNA-protein non-covalent complexes formation, but not in case of the short oligoribonucleotide (n = 22 nt). It was shown that interaction of AluY RNA analogue with Npm1-His6 significantly increases transfection efficacy of the RNA into MCF-7 human cells. Altogether, these data allow us to conclude, that nucleophosmin 1 not only binds RNA with complex secondary structure, but also promotes uptake and internalization of such RNA by human cells.

Box C/D small nucleolar RNAs (snoRNAs) are known to guide the 2'-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct 2'-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA (rRNA) biogenesis. Box C/D snoRNAs guide the site-specific 2'-O-ribose methylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number of box C/D snoRNAs and their fragments have recently been reported to regulate post-transcriptional modifications and the alternative splicing of pre-mRNA. Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18S rRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein (hsc70), were designed and synthesized in this study. It was found that after the transfection of MCF-7 human cells with artificial box C/D RNAs in complex with lipofectamine, snoRNA analogues penetrated into cells and accumulated in the cytoplasm and nucleus. It was demonstrated that the transfection of cultured human cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partial splicing impairments. It was found that transfection with artificial snoRNAs directed to 18S and 28S rRNA nucleotides, significant for ribosome functioning, induce a decrease in MCF-7 cell viability.

This work is been based upon the experience of performing 100 reconstructive microsurgical operations with transplantation of combined vascular-neural autografts, in 24 cases--with introduction cellular material into zone of transplantation. The complex approach aimed to creating optimal conditions for the functional regeneration of the spinal cord (SC), including neurosurgical methods restoring anatomical integrity of the organ and also multicomponent and staging rehabilitation of patients has been developed. On the base of complex approach during a postoperative period there has been put synchronous multifactorial action, consisting of functional multichannel electrical pacing of muscles simultaneously participating in tumble. The patient concentrates all his attention on the trial to tumble with paralized extremities by himself. At that the series and the site of application of these electrical impulses forms the algorithm of this tumble. Synchronism of produced afferent and efferent impulses creates new possibilities in activating of regenerating processes in the damaged area of the spinal cord. As a result of treatment within the bounds of the developed program the authors get clinically and electrophysiologically proved certain improvement that they can not only predict but guarantee patients of this group. Presented here results of the complex treatment of patients with trauma of the spinal cord within the framework of research international program confidently testify advantages of the new approach to solving of this problem. For the first time in history of medicine there is created close cooperation and regular continuity between molecular biology, neurosurgery and rehabilitation. This scientific alliance permits in proper time to keep a close watch, quickly correct and improve every stage of treatment. The effect of implement of innovations is been summarized at the each next stage of proposed complex treatment and eventually significantly elevates an ultimate result of medical rehabilitation of invalids with trauma of the spinal cord.

The fate of human fetal stem/progenitor cells transplanted into rat brain depends on conditions of preculturing (long or short) and state and site of transplantation. Human nestin-positive stem cells cultured according to the short protocol did not migrate into hypoxic and normal brain after transplantation, but actively migrated in damaged spinal cord. After transplantation of long-cultured cells into the brain mainly committed neuroblasts and solitary nestin-positive cells migrated from the site of transplantation into the brain.

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.

In vitro grown neural stem cells from human fetal brain were transplanted to adult rats with spinal trauma. The spinal cord was examined morphologically using histological and immunohistochemical methods on days 5, 15, 30, and 110. Human neural stem/progenitor cells were viable, migrated, and differentiated into neurons and glia in the traumatized spinal cord in adult rats.

Methods of detection of tuberculous infection in 127 children and adolescents treated in special hospitals and sanatoria have seen analyzed. The diagnosis was made using tuberculin in 62.2%, epidemiological evidence in 12.6%, fluorographic findings in 7.8%, survey of risk groups in 7.1%. 10.2% of the patients sought medical advice. A favourable course of the disease was seen in cases detected at tuberculin diagnosis and risk group examination. Complications occurred primarily in those who sought medical advice.

Main practical directions in rehabilitation of feet after microsurgical transplantations of different transplants from feet to hands are described. Four main groups of operations with different specific features of rehabilitation of feet both during operation and in the postoperative period were distinguished on the basis of an experience with the treatment of 46 patients.

The abdominal aorta in 56 white rats has been sutured by means of a microsurgical technique in order to study changes that the smooth muscle cells (SMC) of the vascular wall undergo during the restorative process of its integrity and intima formation. The material for histological investigation is fixed in 10% formalin or in Karnoy fluid and embedded in paraffin. For the electron microscopic investigation the specimens of the aorta are treated in 2% and 5% glutaraldehyde solution and in 1% tetraoxic osmium solution and embedded in epon and araldit mixture. The muscular wall SMC actively participate in the reparative processes when the vascular suture is performed by means of the microsurgical technique. The vascular wall SMC ensure the rat aorta restoration in such a way that already 3 weeks after the operation the cut line can be defined only by the presence of the suture thread and by a slight sheet of the connective tissue. Migration and accumulation of the SMC under the endothelium restored its integrity results in the intima formation. Simultaneously, an intercellular substance is forming, that is directly connected with the SMC development.

These authors describe indications, techniques, short- and long-term results of toe to hand transfers using the vein grafts. 145 toe to hand transfers were performed in 115 patients. Fifty cases necessitated 44 vein grafts in 37 patients; there were 41 vein to artery and 3 vein to vein grafts carried out. The grafts were used to bridge defects of a vessel over 55 mm (usually in patients with heavy posttraumatic scar formation and/or distrophic changes resulting from burns and frost-bite in the vessel stumps, or in the main vessel nourishing the transplant), both in primary surgery and in re-operation following the resection of the thrombosed microanastomosis. Success was achieved in 28 patients who underwent primary interposition of vein grafts, with 34 survivals (90%) of 38 transferred toes. That exceeded the average survival rate of 84%. In 9 patients vein grafting of arteries followed the resection of the thrombosed microanastomoses and resulted in 5 survivals of 12 transferred toes. In 6 cases vein grafts to arteries were performed both intraoperatively and in re-operation. In this group of patients 7 of 8 transferred toes survived for re-operation had been prompt. Venous grafting in toe to hand transfers carried out without delay and in full conformity with the indications, produced results statistically similar to those obtained in procedures involving no venous grafting.

A specialised department for vascular microsurgery was established in the Research Institute for Clinical and Experimental Surgery in 1973. 58 replantations of fingers in 42 patients were made. Total percentage of healing was 52 (30 fingers). Doppler's flowmetry is very useful for the control of microanastomoses of the arteries in fingers. Reconstruction of vessels in fingers was made with an operative microscope (magnification 16--24 times), special microtechnique and 10/0 sutures. Successful replantation is possible even 10--12 hours after severance, it the finger is kept in hypothermia at + 4 degrees C. Rapid admission of the patients to the hospital, i.e. improved information and emergency transport may shorten ischaemic time. The first stage of replantation is the marking of arteries and veins in the finger and in the stump. It is essential to suture not less than 2--3 veins for one artery and nerve. Thorough rehabilitation is of utmost importance to get the best functional results.