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.

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.

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.

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.

[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.

Chronotropic and inotropic components of cardiac reflexes in cats.

The relationship between chronotropic and inotropic components of cardiac reflexes were studied in cats using intravenous blood injections, occlusion of the abdominal aorta, pressing on eyeballs (Aschner maneuver), and occlusion of the carotid arteries. Inotropic reactions were characterized by the contractility index DPxHR/MSAP. Different reflexes were shown to be characterized by different ratio of chronotropic and inotropic components, in addition, heart rate and contractility were often altered in opposite directions.

Effect of atropine, propranolol, and atenolol on wave structure of heart rate oscillations in rats.

Were studied the effects of atropine and beta-adrenoblockers on the power of very low (<0.25 Hz), low (0.25-0.70 Hz), and high frequency (0.7-1.8 Hz) waves of the heart rhythm in wakeful rats. Atropine reduced the power of all waves in 100% cases. Propranolol on average decreased the power of very low frequency waves and increased the power of low and high frequency waves, although opposite effects were observed in many cases. Atenolol produced similar effects. Some animals demonstrated spontaneous moderation of respiratory rate to a level corresponding to low-frequency oscillations of the heart rhythm accompanied by elevation of the relative power of low frequency waves. Inconsistency of the effects of beta-adrenoblockers in rats can be related to variability of sympathetic tone and spontaneous deceleration of respiration with the corresponding changes of low frequency waves. Augmentation of the high frequency waves during application of beta-adrenoblockers is not related to their action on CNS.

Chronotropic and dromotropic components of cardiac reflexes in the cat.

The relationship between dromotropic and chronotropic components of various cardiac reflexes was studied in cats. Intravenous infusion of blood was mainly accompanied by unidirectional negative chronotropic and dromotropic effects, but the dynamics of these effects was different. Clumping of the carotid arteries in most animals induced unidirectional negative chronotropic and dromotropic effects. Their dynamics was also different and differed from that observed during intravenous blood infusion. Pulsatile increase in blood pressure in the carotid artery was accompanied by a unidirectional negative effect in the majority of animals. The opposite chronotropic and dromotropic effects with similar temporal dynamics were revealed in 1/3 animals. The ratio of positive and negative effects was similar during clumping of the abdominal aorta (1/3 unidirectional, 1/3 opposite, and 1/3 isolated chronotropic and dromotropic effects). Aschner test was characterized by the prevalence of isolated chronotropic effect (negative effect in the majority of animals; positive effect in 1/3 animals). Hence, different cardiac reflexes are characterized by different ratio between chronotropic and dromotropic components.

Effect of pentobarbital on spectral characteristics and phase ratios of wave oscillations of cardiac contraction period and time of atrioventricular conduction in cats.

Effects of pentobarbital on spectral characteristics and phase ratios of wave oscillations of the cardiac contraction period (RR interval) and time of atrioventricular conduction (AV interval) were studied in experiments of cats. Pentobarbital moderately reduced the mean values of both intervals and significantly reduced their standard deviations and spectral powers in all frequency bands (high-frequency, low-frequency, and very low-frequency). Pentobarbital treatment led to deceleration of breathing, the frequency range of respiratory oscillations of RR and AV intervals shifted in some cases from high to low frequencies; evaluation of spectral power in the intermittent band corresponding to respiration frequency (instead of standard fixed high-frequency band) showed that pentobarbital suppressed the respiratory oscillations in these bands. Pentobarbital induced inversion of phase ratio between respiratory oscillations of RR and AV intervals: oscillations of both intervals before pentobarbital coincided by phase, while after pentobarbital injection they were in antiphase. The mechanisms of the latter phenomenon deserve further investigation.

Chronodromotropic coordination in cats.

The coordinated nervous influences on HR and atrioventricular conduction velocity (chronodromotropic coordination) were examined in wakeful cats. The wave structure and reflex reactions of RR and atrioventricular (AV) intervals to stress noise stimulation were studied under normal conditions and during the action of blockers of peripheral receptors in ANS. Variations of both intervals had similar wave structure (power spectrum) and similar reactions to noise stimulus. Atropine pronouncedly decreased all components of the spectra in the high, low, and very low frequency ranges. It eliminated the reactions of both intervals to noise stimulation. In RR intervals, the high-frequency spectrum component decreased more strongly than the low-frequency ones. By contrast, in AV intervals atropine most greatly decreased the very-low-spectrum component, while the high frequency was decreased less of all. Propranolol produced no effect on the response to noise. It did not decrease components of the wave structure in both intervals, except for the very-low-frequency peak of AV interval. The nervous chronotropic and dromotropic influences were largely coordinated, although they were not obligatorily parallel.

Role of sympathetic and parasympathetic nervous systems in heart rate regulation in cats.

The effects of atropine and beta-adrenoceptor blockers on mean HR, wave structure of the cardiac rhythm, and chronotropic reaction to noise stress were examined in cats. Atropine (0.5 mg/kg) increased the mean HR and significantly decreased the spectrum power of HF, LF, and VLF oscillations. The decrease in HF power was most pronounced, which enhanced LF/HF ratio. Propranolol (0.5 mg/kg) decreased the mean HR and slightly increased the power of HF, LF, and VLF oscillations. Atenolol (2 mg/kg) exerted similar but more pronounced effects. beta-Adrenoceptor blockers increased HF power to a greater extent than LF and VLF power, which led to a decrease in LF/HF ratio. Atropine markedly decreased the chronotropic reaction to stress. beta-Adrenoceptor blockers produced no effect on the amplitude of this reaction, but accelerated restoration of initial HR. It is established that the changes in power spectrum of HR and the phase reflex reactions in cats are mediated by parasympathetic influences; the sympathetic system is involved only in the control of mean HR, probably in response to the level of animal activity. The changes in LF/HF ratio during blockade of sympathetic and parasympathetic systems are caused by opposite influences of these systems on HF oscillations, but not by hypothetic "sympathetic" and "parasympathetic" nature of LF and HF oscillations of the heart rhythm.

Chronotropic and dromotropic components of cardiac reflexes in rabbits.

The relationships between dromotropic and chronotropic components of five reflexes were studied in rabbits: intravenous and intraarterial blood injections, occlusion of the carotid arteries, Aschner maneuver, and stimulation of depressors. All these stimuli reduced heart rate (except carotid artery occlusion, which induced approximately equal number of tachi- and bradycardic responses). The former three stimuli also reduced atrioventricular (AV) conduction velocity, the changes in these two parameters were proportional. Changes in heart rate induced by Aschner maneuver were more pronounced than changes in AV conduction. Stimulation of depressor induced co-directed shifts in these parameters during the first seconds, but then AV conduction increased, while heart rate remained decreased; bradycardia and AV acceleration persisted for long time after termination of stimulation. Our findings attest to independent regulation of heart rate and AV conduction velocity and to the absence of a strict relationship between these two parameters.