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Abstract Reactive oxygen/nitrogen species (ROS/RNS) are formed during normal cellular metabolism and contribute to its regulation, while many pathological processes are associated with ROS/RNS imbalances. Modern methods for measuring ROS/RNS are mainly based on the use of inducible fluorescent dyes and protein-based sensors, which have several disadvantages for in vivo use. Intravital electrochemical nanosensors can be used to quantify ROS/RNS with high sensitivity without exogenous tracers and allow dynamic ROS/RNS measurements in vivo. Here, we developed a method for quantifying total ROS/RNS levels in the liver and evaluated our setup in live mice using three common models of liver disease associated with ROS activation: acute liver injury with CCl4, partial hepatectomy (HE), and induced hepatocellular carcinoma (HCC). We have demonstrated using intravital electrochemical detection that any exposure to the peritoneum in vivo leads to an increase in total ROS/RNS levels, from a slight increase to an explosion, depending on the procedure. Analysis of the total ROS/RNS level in a partial hepatectomy model revealed oxidative stress, both in mice 24 h after HE and in sham-operated mice. We quantified dose-dependent ROS/RNS production in CCl4-induced injury with underlying neutrophil infiltration and cell death. We expect that in vivo electrochemical measurements of reactive oxygen/nitrogen species in the liver may become a routine approach that provides valuable data in research and preclinical studies.

A template-assisted assembly approach to a C24 fullerene-like double-stranded DNA polyhedral shell is proposed. The assembly employed a supramolecular oligonucleotide dendrimer as a 3D template that was obtained via the hybridization of siRNA strands and a single-stranded DNA oligonucleotide joined to three- or four-way branched junctions. A four-way branched oligonucleotide building block (a starlet) was designed for the assembly of the shell composed of three identical self-complementary DNA single strands and a single RNA strand for hybridization to the DNA oligonucleotides of the template. To prevent premature auto-hybridization of the self-complementary oligonucleotides in the starlet, a photolabile protecting group was introduced via the N3-substituted thymidine phosphoramidite. Cleavable linkers such as a disulfide linkage, RNase A sensitive triribonucleotides, and di- and trideoxynucleotides were incorporated into the starlet and template at specific points to guide the post-assembly disconnection of the shell from the template, and enzymatic disassembly of the template and the shell in biological media. At the same time, siRNA strands were modified with 2′-OMe ribonucleotides and phosphorothioate groups in certain positions to stabilize toward enzymatic digestion. We report herein a solid-phase synthesis of branched oligodeoxy and oligoribonucleotide building blocks for the DNA/RNA dendritic template and the branched DNA starlet for a template-assisted construction of a C24 fullerene-like DNA shell after initial molecular modeling, followed by the assembly of the shell around the DNA-coated RNA dendritic template, and visualization of the resulting nanostructure by transmission electron microscopy.

Abstract Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.

Abstract Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, protein tyrosine kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC.

Abstract Background HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. Results We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. Conclusions Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

Carbon-encapsulated iron nanoparticles (Fe@C) with a mean diameter of 15 nm have been synthesized using evaporation–condensation flow–levitation method by the direct iron-carbon gas-phase reaction at high temperatures. Further, Fe@C were stabilized with bovine serum albumin (BSA) coating, and their electromagnetic properties were evaluated to test their performance in magnetic hyperthermia therapy (MHT) through a specific absorption rate (SAR). Heat generation was observed at different Fe@C concentrations (1, 2.5, and 5 mg/mL) when applied 331 kHz and 60 kA/m of an alternating magnetic field, resulting in SAR values of 437.64, 129.36, and 50.4 W/g for each concentration, respectively. Having such high SAR values at low concentrations, obtained material is ideal for use in MHT.

Glucose and lipid metabolism are crucial functional systems in eukaryotes. A large number of experimental studies both in animal models and humans have shown that long non-coding RNAs (lncRNAs) play an important role in glucose and lipid metabolism. Previously, human lncRNA DEANR1/linc00261 was described as a tumor suppressor that regulates a variety of biological processes such as cell proliferation, apoptosis, glucose metabolism and tumorigenesis. Here we report that murine lncRNA Falcor/LL35, a proposed functional analog of human DEANR1/linc00261, is predominantly expressed in murine normal hepatocytes and downregulated in HCC and after partial hepatectomy. The application of high-throughput approaches such as RNA-seq, LC-MS proteomics, lipidomics and metabolomics analysis allowed changes to be found in the transcriptome, proteome, lipidome and metabolome of hepatocytes after LL35 depletion. We revealed that LL35 is involved in the regulation of glycolysis and lipid biosynthesis in vitro and in vivo. Moreover, LL35 affects Notch and NF-κB signaling pathways in normal hepatocytes. All observed changes result in the decrease in the proliferation and migration of hepatocytes. We demonstrated similar phenotype changes between murine LL35 and human linc00261 depletion in vitro and in vivo that opens the opportunity to translate results for LL35 from a liver murine model to possible functions of human lncRNA linc00261.

Nucleic acid aptamers specific to S-protein and its receptor binding domain (RBD) of SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virions are of high interest as potential inhibitors of viral infection and recognizing elements in biosensors. Development of specific therapy and biosensors is complicated by an emergence of new viral strains bearing amino acid substitutions and probable differences in glycosylation sites. Here, we studied affinity of a set of aptamers to two Wuhan-type RBD of S-protein expressed in Chinese hamster ovary cell line and Pichia pastoris that differ in glycosylation patterns. The expression system for the RBD protein has significant effects, both on values of dissociation constants and relative efficacy of the aptamer binding. We propose glycosylation of the RBD as the main force for observed differences. Moreover, affinity of a several aptamers was affected by a site of biotinylation. Thus, the robustness of modified aptamers toward new virus variants should be carefully tested.

DDX3 RNA helicase is intensively studied as a therapeutic target due to participation in the replication of some viruses and involvement in cancer progression. Here we used transcriptome analysis to estimate the primary response of hepatocytes to different levels of RNAi-mediated knockdown of DDX3 RNA helicase both in vitro and in vivo. We found that a strong reduction of DDX3 protein (>85%) led to similar changes in vitro and in vivo—deregulation of the cell cycle and Wnt and cadherin pathways. Also, we observed the appearance of dead hepatocytes in the healthy liver and a decrease of cell viability in vitro after prolonged treatment. However, more modest downregulation of the DDX3 protein (60–65%) showed discordant results in vitro and in vivo—similar changes in vitro as in the case of strong knockdown and a different phenotype in vivo. These results demonstrate that the level of DDX3 protein can dramatically influence the cell phenotype in vivo and the decrease of DDX3, for more than 85% leads to cell death in normal tissues, which should be taken into account during the drug development of DDX3 inhibitors.

The progress of the cell cycle is directly regulated by modulation of cyclins and cyclin-dependent kinases. However, many proteins that control DNA replication, RNA transcription and the synthesis and degradation of proteins can manage the activity or levels of master cell cycle regulators. Among them, RNA helicases are key participants in RNA metabolism involved in the global or specific tuning of cell cycle regulators at the level of transcription and translation. Several RNA helicases have been recently evaluated as promising therapeutic targets, including eIF4A, DDX3 and DDX5. However, targeting RNA helicases can result in side effects due to the influence on the cell cycle. In this review, we discuss direct and indirect participation of RNA helicases in the regulation of the cell cycle in order to draw attention to downstream events that may occur after suppression or inhibition of RNA helicases.

New lipid conjugates of DNA and RNA incorporating one to four [(4-dodecylphenyl)sulfonyl]phosphoramidate or (hexadecylsulfonyl)phosphoramidate groups at internucleotidic positions near the 3′ or 5′-end were synthesized and characterized. Low cytotoxicity of the conjugates and their ability to be taken up into cells without transfection agents were demonstrated. Lipid-conjugated siRNAs targeting repulsive guidance molecules a (RGMa) have shown a comparable gene silencing activity in PK-59 cells to unmodified control siRNA when delivered into the cells via Lipofectamine mediated transfection.

Ribosome biogenesis is among the founding processes in the cell. During the first stages of ribosome biogenesis, polycistronic precursor of ribosomal RNA passes complex multistage maturation after transcription. Quality control of preribosomal RNA (pre-rRNA) processing is precisely regulated by non-ribosomal proteins and structural features of pre-rRNA molecules, including modified nucleotides. However, many participants of rRNA maturation are still unknown or poorly characterized. We report that RNA m6A methyltransferase Mettl3 interacts with the 5′ external transcribed spacer (5′ETS) of the 47S rRNA precursor and modifies adenosine 196. We demonstrated that Mettl3 knockdown results in the increase of pre-rRNA processing rates, while intracellular amounts of rRNA processing machinery components (U3, U8, U13, U14, and U17 small nucleolar RNA (snoRNA)and fibrillarin, nucleolin, Xrn2, and rrp9 proteins), rRNA degradation rates, and total amount of mature rRNA in the cell stay unchanged. Increased efficacy of pre-rRNA cleavage at A’ and A0 positions led to the decrease of 47S and 45S pre-rRNAs in the cell and increase of mature rRNA amount in the cytoplasm. The newly identified conserved motif DRACH sequence modified by Mettl3 in the 5′-ETS region is found and conserved only in primates, which may suggest participation of m6A196 in quality control of pre-rRNA processing at initial stages demanded by increased complexity of ribosome biogenesis.