Structure-activity relationship study of mesyl and busyl phosphoramidate antisense oligonucleotides for unaided and PSMA-mediated uptake into prostate cancer cells.

Phosphorothioate (PS) group is a key component of a majority of FDA approved oligonucleotide drugs that increase stability to nucleases whilst maintaining interactions with many proteins, including RNase H in the case of antisense oligonucleotides (ASOs). At the same time, uniform PS modification increases nonspecific protein binding that can trigger toxicity and pro-inflammatory effects, so discovery and characterization of alternative phosphate mimics for RNA therapeutics is an actual task. Here we evaluated the effects of the introduction of several N-alkane sulfonyl phosphoramidate groups such as mesyl (methanesulfonyl) or busyl (1-butanesulfonyl) phosphoramidates into gapmer ASOs on the efficiency and pattern of RNase H cleavage, cellular uptake in vitro, and intracellular localization. Using Malat1 lncRNA as a target, we have identified patterns of mesyl or busyl modifications in the ASOs for optimal knockdown in vitro. Combination of the PSMA ligand-mediated delivery with optimized mesyl and busyl ASOs resulted in the efficient target depletion in the prostate cancer cells. Our study demonstrated that other N-alkanesulfonyl phosphoramidate groups apart from a known mesyl phosphoramidate can serve as an essential component of mixed backbone gapmer ASOs to reduce drawbacks of uniformly PS-modified gapmers, and deserve further investigation in RNA therapeutics.

Activity of nsp14 Exonuclease from SARS-CoV-2 towards RNAs with Modified 3'-Termini.

The COVID-19 pandemic has shown the urgent need for new treatments for coronavirus infections. Nucleoside analogs were successfully used to inhibit replication of some viruses through the incorporation into the growing DNA or RNA chain. However, the replicative machinery of coronaviruses contains nsp14, a non-structural protein with a 3'→5'-exonuclease activity that removes misincorporated and modified nucleotides from the 3' end of the growing RNA chain. Here, we studied the efficiency of hydrolysis of RNA containing various modifications in the 3'-terminal region by SARS-CoV-2 nsp14 exonuclease and its complex with the auxiliary protein nsp10. Single-stranded RNA was a preferable substrate compared to double-stranded RNA, which is consistent with the model of transfer of the substrate strand to the exonuclease active site, which was proposed on the basis of structural analysis. Modifications of the phosphodiester bond between the penultimate and last nucleotides had the greatest effect on nsp14 activity.

Polymer/magnetite carriers functionalized by HER2-DARPin: Avoiding lysosomes during internalization and controlled toxicity of doxorubicin by focused ultrasound induced release.

Nanomedicine has revolutionized the available treatment options during the last decade, but poor selectivity of targeted drug delivery and release is still poses a challenge. In this study, doxorubicin (DOX) and magnetite nanoparticles were encapsulated by freezing-induced loading, coated with polymeric shell bearing two bi-layers of polyarginine/dextran sulphate and finally modified with HER2-specific DARPin proteins. We demonstrated that the enhanced cellular uptake of these nanocarriers predominantly occurs by SKOV-3 (HER2+) cells, in comparison to CHO (HER2-) cells, together with the controlled DOX release using low intensity focused ultrasound (LIFU). In addition, a good ability of DARPin+ capsules to accumulate in the tumor and the possibility of combination therapy with LIFU were demonstrated. A relatively high sensitivity of the obtained nanocarriers to LIFU and their preferential interactions with mitochondria in cancer cells make these carriers promising candidates for cancer treatment, including novel approaches to overcome drug resistance.

[Experience of Teraligen usage in pediatric psychiatry]. / Opyt primeneniya Teralidzhena v psikhiatrii detskogo vozrasta.

The data of studies on the use of Teraligen in various fields of clinical medicine - neurology, dermatology, gastroenterology, anesthesiology and psychiatry are presented. According to studies conducted in psychiatry, Teraligen is a drug for the balanced treatment of anxiety-phobic, behavioral and sleep disorders in children, both within neurotic conditions and in the structure of autistic and schizophrenic spectrum disorders. Based on 100 case histories of children aged 3 to 16 years, the experience of using Teraligen as part of mono- and combination therapy at a dosage of 1.25 mg to 50 mg/day in the treatment of autism spectrum disorders (n=31), psychotic disorders (n=18), schizotypal disorders (n=41), behavioral and emotional disorders on residual organic soil (n=10). A good clinical effect of Teraligen has been shown in anxiety-phobic, affective, somatovegetative disorders and sleep disorders in all identified nosologies. Good tolerability of Teraligen, the possibility of its use as part of complex therapy and the almost complete absence of adverse events, indicating the softness and balance of the drug were noted.

Tachykinins amplify the action of capsaicin on central histaminergic neurons.

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

Determination of the Affinity of Eukaryotic DDX3 RNA Helicase to the Characteristic Elements of mRNA Secondary Structure.

DDX3 RNA helicase is involved in many processes of RNA metabolism in eukaryotic cells. Many studies of DDX3 have shown that it is also involved in the translation initiation process, both cap-dependent and IRES-dependent. However, the specificity of the secondary structure of mRNA 5'-UTRs, which require DDX3 RNA helicase for effective translation, has not yet been determined. We performed a bioinformatic analysis of the 5'-UTR secondary structures in the pool of DDX3-dependent mRNAs in silico and predicted that the length of 5'-UTRs for such mRNAs is less than the average for the genome and that there are also characteristic hairpin structures in the region of the first 50 nucleotides from the 5'-end of the mRNA.

Long Noncoding RNA LL35/Falcor Regulates Expression of Transcription Factor Foxa2 in Hepatocytes in Normal and Fibrotic Mouse Liver.

Long noncoding RNAs (lncRNA) play important roles in the regulation of transcription, splicing, translation, and other processes in the cell. Human and mouse lncRNA (DEANR1 and LL35/Falcor, respectively) located in the genomic environment in close proximity to the Foxa2 transcription factor were discovered earlier. In this work, tissue-specific expression of LL35/Falcor lncRNA has been shown in mouse liver and lungs. The use of antisense oligonucleotides allowed us to achieve LL35/Falcor lncRNA downregulation by 90%. As a result, the level of Foxa2 mRNA and protein dropped, which confirms the involvement of LL35/Falcor lncRNA in the regulation of transcription factor Foxa2. We have shown a decrease in the expression of LL35 lncRNA in liver fibrosis, which correlates with the previously published data for mRNA Foxa2. Thus, lncRNA LL35 regulates Foxa2 expression in the liver not only in normal conditions, but also during development of fibrosis, which allows one to consider lncRNA a biomarker of this pathological process.

[Cholangiocellular cancer: the state of the problem and ways to improve the results of surgical treatment]. / Kholangiotselliuliarnyi rak: sostoianie problemy i puti uluchsheniia khirurgicheskogo lecheniia.

AIM: To improve the outcomes in patients with resectable biliary cancer. MATERIAL AND METHODS: There were 263 procedures for cholangiocellular carcinoma (CCC) for the period 1998­2017. Adjuvant chemotherapy was performed in 102 (38.8%) patients. Extensiveliver resections (78.9%) prevailed for intrahepatic cholangiocellular carcinoma (n=128), 6 (4.7%) patients required vascular resection. Seventy-seven pancreatoduodenectomies were performed for common bile duct cancer, portal vein resection was done in 8 (10.4%) patients. In case of Klatskin tumor (n=58) liver resection combined with bile duct resection (n=52) prevailed. Portal vein resection was done in 16 (27.6%) patients. RESULTS: Postoperative morbidity in patients with intrahepatic CCC was revealed in 68 (53.1%) cases, mortality ­ in 5 (3.9%) cases. Among patients with Klatskin tumor morbidity was revealed in 51 (87.9%) cases, mortality ­ in 6 (10.3%) cases. In patients with common bile duct cancer morbidity was revealed in 53 (68.8%) cases, mortality ­ in 4 (5.2%) cases. In whole cohort median overall survival was 30 months. R0-resection was associated with better long-term results (median 37 months) compared with R1­R2 resection (20 months; p=0.01). Lymph node involvement is associated with significantly worse prognosis (p=0.016), however 5-year survival is observed (25.6%). Adjuvant chemotherapy in R0-resection significantly improved long-term results: median was 46 months (vs. 30 in group without chemotherapy; p=0.02). In intrahepatic CCC patients multiple lesions or mechanical jaundice did not aggravate long-term results. CONCLUSION: R0-resection including lymphadenectomy, resection of adjacent organs and vessels is advisable for CCC. Isolated bile duct resection should be used as an exception. Adjuvant therapy improved long-term results. Multiple lymph node lesion or bile duct infiltration are not contraindications to surgery in intrahepatic CCC patients.

Possible Role of Escherichia coli Protein YbgI.

Proteins containing the NIF3 domain are highly conserved and are found in bacteria, eukaryotes, and archaea. YbgI is an Escherichia coli protein whose gene is conserved among bacteria. The structure of YbgI is known; however, the function of this protein in cells remains obscure. Our studies of E. coli cells with deleted ybgI gene suggest that YbgI is involved in formation of the bacterial cell wall.

[Early diagnosis of stress-induced hypertension in young employees of state law enforcement agencies].

Psychoemotional stress is one of the leading cardiovascular risk factors. The aim of this study was to explore manifestations of arterial hypertension (AH) in young men employed in the stress associatedfield works. Material and methods. A total of 68 young men, exposed to job stress (JS) of different severity during 1 to 5 years were surveyed. The control group was formed of persons whose daily work was unrelated to operational activities. The study included ambulatory monitoring (AM) blood pressure (BP) on different days of the week, comparing the results of the office and DMBP determination and assessment of resistance to a variety of diagnostic loads. Results. The study revealed an increase of AMBP indicators during the working day and their normalization at weekend. The comparison of the results of office and ambulatory determination of BP demonstrated that different forms of stressful AH (stable, isolated office and latent) occurred significantly more often than in the control group. These changes almost completely disappeared at weekends. The tolerance ofpsychoemotional testing was much worse than that of traditional exercises. It is proposed to more extensively use AMBP during regular medical examinations of young men exposed to JS in order to establish a timely differential diagnosis of various forms of stressful hypertension.

mRNA-Based Therapeutics - Advances and Perspectives.

In this review we discuss features of mRNA synthesis and modifications used to minimize immune response and prolong efficiency of the translation process in vivo. Considerable attention is given to the use of liposomes and nanoparticles containing lipids and polymers for the mRNA delivery. Finally we briefly discuss mRNAs which are currently in the clinical trials for cancer immunotherapy, vaccination against infectious diseases, and replacement therapy.

[Pathogenetic Relationship between Endothelial Dysfunction and Disorders of Blood Coagulation Potential in Pregnancy Complicated by Pre-Eclampsia].

UNLABELLED: Until now, there is no systematic information on the role of endothelial dysfunction in the mechanisms of disorders of blood coagulation potential and microcirculation in different organs and tissues in preeclampsia. OBJECTIVE: Our aim was to extend the existing principles of diagnosis of pre-eclampsia by establishing the role of endothelial dysfunction in the mechanisms of blood coagulation potential violations. METHODS: A prospective comparative study was performed. Condition of coagulation processes studied by conventional techniques, parameters of a functional endothelium (nitric oxide metabolites, endothelin 1, thrombospondin, thrombomodulin and intercellular adhesion molecules in blood plasma)--by ELISA. RESULTS: The study group included 55 patients with moderate preeclampsia and 49 pregnant women with severe pre-eclampsia, in the control group--40 women with physiological pregnancy. In patients with pre-eclampsia moderate observed increase in plasma endothelin-1 (p < 0.001), thrombospondin (p < 0.001), intercellular adhesion molecules (p < 0.001) while reducing the level of nitrogen oxide (p < 0.001), increase in time of fibrinolysis (p < 0.050) and decreased international normalized ratio (p < 0.050) compared with the control group. With increasing severity of preeclampsia the researchers detected in blood plasma of patients a progressive increase in endothelin 1 (p1 < 0.020), thrombospondin (p1 < 0.001), intercellular adhesion molecules (p1 < 0.001) and decrease of nitric oxide metabolites (p1 < 0.001) and thrombomodulin (p1 < 0.001); the last combined with the activation of procoagulant hemostasis. CONCLUSION: There is a pathogenetic relationship between the development of endothelial dysfunction, impaired blood coagulation potential and the severity of clinical signs ofpreeclampsia. To widen the number of existing techniques to diagnose the severity of pre-eclampsia we recommende to mesure endothelin 1, thrombomodulin, thrombospondin, intercellular adhesion molecules and nitric oxide metabolites in the blood plasma, and use traditional indicators to assess the hemostatic system.

What do we know about ribosomal RNA methylation in Escherichia coli?

A ribosome is a ribonucleoprotein that performs the synthesis of proteins. Ribosomal RNA of all organisms includes a number of modified nucleotides, such as base or ribose methylated and pseudouridines. Methylated nucleotides are highly conserved in bacteria and some even universally. In this review we discuss available data on a set of modification sites in the most studied bacteria, Escherichia coli. While most rRNA modification enzymes are known for this organism, the function of the modified nucleotides is rarely identified.

Effect of adrenoblockers on slow (LF) waves in rabbit heart rate.

Propranolol and atenolol were used to examine the role of the adrenergic system in the genesis of slow HR variations (waves) in rabbits, the animals characterized by pronounced sympathetic influences. The control experiments were performed with physiological solution and long-term records in intact rabbits. The harmonic (spectrum) analysis was performed with fast Fourier transform. The adrenoblockers produced no decrease in the power of slow waves. The study did not found the sympathetic genesis of slow variations in rabbit HR.

[Ribosome: lessons of a molecular factory construction].

Ribosome is a macromolecular complex, which is responsible for protein biosynthesis. Two bacterial ribosomal subunits contain more than 4000 RNA nucleotides and 50 proteins. Ribosome assembly is a complicated multi-step process, vitally important for cell. In this review we summarised present-day conceptions about the mechanism of the bacterial ribosome assembly in the cell and in vitro model systems. Some details of the assembly of this machinery are still-unknown.

Benzodiazepine-site pharmacology on GABAA receptors in histaminergic neurons.

BACKGROUND AND PURPOSE: The histaminergic tuberomamillary nucleus (TMN) of the posterior hypothalamus controls the cognitive aspects of vigilance which is reduced by common sedatives and anxiolytics. The receptors targeted by these drugs in histaminergic neurons are unknown. TMN neurons express nine different subunits of the GABAA receptor (GABAA R) with three α- (α1, α2 and α5) and two γ- (γ1, γ 2) subunits, which confer different pharmacologies of the benzodiazepine-binding site. EXPERIMENTAL APPROACH: We investigated the actions of zolpidem, midazolam, diazepam, chlordiazepoxide, flumazenil (Ro15-1788) and methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM) in TMN neurons using mouse genetics, electrophysiological and molecular biological methods. KEY RESULTS: We find the sensitivity of GABAA R to zolpidem, midazolam and DMCM significantly reduced in TMN neurons from γ2F77I mice, but modulatory activities of diazepam, chlordiazepoxide and flumazenil not affected. Potencies and efficacies of these compounds are in line with the dominance of α2- and α1-subunit containing receptors associated with γ2- or γ1-subunits. Functional expression of the γ1-subunit is supported by siRNA-based knock-down experiments in γ2F77I mice. CONCLUSIONS AND IMPLICATIONS: GABAA R of TMN neurons respond to a variety of common sedatives with a high affinity binding site (γ2F77I) involved. The γ1-subunit likely contributes to the action of common sedatives in TMN neurons. This study is relevant for understanding the role of neuronal histamine and benzodiazepines in disorders of sleep and metabolism.

[The first episode of psychosis in juvenile patients: personality features and cognitive functioning].

A systemic study of personality features and cognitive processes based on the conception of pathopsychological syndrome has been conducted in patients with the first episode of juvenile endogenous psychosis. The association between characteristics of a pathopsychological syndrome and clinically determined premorbid features has been identified. The highest expression of schizotypal features (reduced selectivity, social perception) and cognitive impairment (motivation, mental activity regulation, adequate self-esteem, emotional reaction to success/failure) were characteristic of a group named "deficit variant of pseudopsychopathy" which contained a large number of passive schizoids. It has been shown that premorbid personality features are most distinct in initial stages of disease ("pathos" factors). "Nosos" factors are implicit and can be only presumably regarded as predictors of disease course.

The bile steroid chenodeoxycholate is a potent antagonist at NMDA and GABA(A) receptors.

The bile steroids (BS) cholic acid and chenodeoxycholic acid are produced in hepatocytes and in the brain. Nothing is known about neuronal actions of BS. Deficiency in a 27-hydroxylase enzyme coincides with reduced production of chenodeoxycholic acid (CDCA) and a relative increase in cholic acid in an inherited lipid storage disease, cerebrotendinous xanthomatosis, characterized by neurological dysfunctions, which can be treated by dietary CDCA. We have examined the modulation of hypothalamic network activity by nine common BS. Cholate and CDCA significantly reduced the firing of hypothalamic neurons and synchronized network activity with CDCA being nearly 10 times more potent. The synthetic BS dehydrocholate synchronized the activity without affecting the firing rate. Gabazine, a GABA(A) receptor antagonist, occluded synchronization by BS. Whole-cell patch clamp recordings revealed a block of NMDA- and GABA(A)-receptors by BS. Potencies of nine common BS differed between NMDA and GABA(A) receptors, however in both cases they correlated with BS affinities for albumin but not with their lipophilicity, supporting a direct action at ligand gated ion channels. GABAergic synaptic currents displayed a faster decay under BS. Our data provide new insight into extrahepatic functions of BS revealing their neuroactive potential.

Molecular properties of hybrid macromolecular antioxidants: dextran hydrophobically modified by sterically hindered phenols.

The conformation properties of clinically relevant hybrid macromolecular antioxidants (dextran hydrophobically modified by sterically hindered phenols) in aqueous solution were characterized by a combination of dynamic light scattering (DLS), size exclusion chromatography (SEC), and small-angle neutron scattering (SANS). We were able to split and analyze separately two different types of polydispersity -polydispersity over molecular weights and the one over substitution degree. The properties of the hybrid macromolecules are determined by the number of hydrophobic antioxidants in a single molecule. An insertion of hydrophobic groups into a hydrophilic chain changes the conformation of a single conjugate macromolecule. We have established that with the increasing of a number of hydrophobic antioxidant groups, a conformational transition occurs where a single conjugate undergoes a transition from a Gaussian coil conformation to a more compact structure.

[Systems biology approach to the functional role of enzymatic modification of bacterial ribosome].

In this work we describe methodology for studying the role of bacterial ribosome modification in the regulation of gene expression. Ribosomal components modification influences translation efficiencies of certain mRNAs. Proteome analysis allows us to identify cellular protein composition change caused by ribosome modification gene knockout. Particular stage of gene expression responsible for certain protein concentration change could be found using reporter constructs. After identification of mRNA species, whose translation is influenced by ribosome modification we can determine exact mRNA region responsible for the observed changes. The developed methodology can be applied for studying other translational control mechanisms.