Study of the RNA splicing kinetics via in vivo 5-ethynyl uridine labelling.

Splicing, a process of intron removal from eukaryotic RNA transcripts, is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency giving rise to alternative splicing products. At the same time, splice sites might be utilised at a variable rate. We used 5-ethynyl uridine labelling to sequence a nascent transcriptome of HeLa cells and deduce the rate of splicing for each donor and acceptor splice site. The following correlation analysis allowed us to assess a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as splicing rate decrease with a decreased complementarity of donor splice site to U1 and acceptor sites to U2 snRNAs, or an acceleration of donor site usage if an upstream acceptor site is located at a shorter distance. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of acceptor splicing site utilization, or the differences in splicing rate between long, short and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the polyA site, which might be explained by a cooperativity of the splicing and polyadenylation. In addition, we performed the analysis of splicing kinetics of SF3B4 knockdown cells which suggested the impairment of U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field as it provides general splicing rate dependencies as well as helps justify the existence of slowly removed splice sites, e.g. to ensure alternative splicing.

An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Synthetic Activators of Autophagy.

Autophagy is a central process for degradation of intracellular components that do not operate correctly. Molecular mechanisms underlying this process are extremely difficult to study, since they involve a large number of participants. The main task of autophagy is redistribution of cellular resources in response to environmental changes, such as starvation. Recent studies show that autophagy regulation could be the key to achieve healthy longevity, as well as to create therapeutic agents for treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Thus, development of autophagy activators with established detailed mechanism of action is a really important area of research. Several commercial companies are at various stages of development of such molecules, and some of them have already begun to introduce autophagy activators to the market.

Natural Activators of Autophagy.

Autophagy is the process by which cell contents, such as aggregated proteins, dysfunctional organelles, and cell structures are sequestered by autophagosome and delivered to lysosomes for degradation. As a process that allows the cell to get rid of non-functional components that tend to accumulate with age, autophagy has been associated with many human diseases. In this regard, the search for autophagy activators and the study of their mechanism of action is an important task for treatment of many diseases, as well as for increasing healthy life expectancy. Plants are rich sources of autophagy activators, containing large amounts of polyphenolic compounds in their composition, which can be autophagy activators in their original form, or can be metabolized by the intestinal microbiota to active compounds. This review is devoted to the plant-based autophagy activators with emphasis on the sources of their production, mechanism of action, and application in various diseases. The review also describes companies commercializing natural autophagy activators.

Structure and Composition of Spermatozoa Fibrous Sheath in Diverse Groups of Metazoa.

The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.

A New Mouse Strain with a Mutation in the NFE2L2 (NRF2) Gene.

Transcription factor NRF2 is involved in inflammatory reactions, maintenance of redox balance, metabolism of xenobiotics, and is of particular interest for studying aging. In the present work, the CRISPR/Cas9 genome editing technology was used to generate the NRF2ΔNeh2 mice containing a substitution of eight amino acid residues at the N-terminus of the NRF2 protein, upstream of the functional Neh2 domain, which ensures binding of NRF2 to its inhibitor KEAP1. Heterozygote NRF2wt/ΔNeh2 mice gave birth to homozygous mice with lower than expected frequency, accompanied by their increased embryonic lethality and visual signs of anemia. Mouse embryonic fibroblasts (MEFs) from the NRF2ΔNeh2/ΔNeh2 homozygotes showed impaired resistance to oxidative stress compared to the wild-type MEFs. The tissues of homozygous NRF2ΔNeh2/ΔNeh2 animals had a decreased expression of the NRF2 target genes: NAD(P)H:Quinone oxidoreductase-1 (Nqo1); aldehyde oxidase-1 (Aox1); glutathione-S-transferase A4 (Gsta4); while relative mRNA levels of the monocyte chemoattractant protein 1 (Ccl2), vascular cell adhesion molecule 1 (Vcam1), and chemokine Cxcl8 was increased. Thus, the resulting mutation in the Nfe2l2 gene coding for NRF2, partially impaired function of this transcription factor, expanding our insights into the functional role of the unstructured N-terminus of NRF2. The obtained NRF2ΔNeh2 mouse line can be used as a model object for studying various pathologies associated with oxidative stress and inflammation.

Effect of the consumption of brazzein and monellin, two recombinant sweet-tasting proteins, on rat gut microbiota.

Sweet-tasting proteins (SPs) are proteins of plant origin initially isolated from tropical fruits. They are thousands of times sweeter than sucrose and most artificial sweeteners. SPs are a class of proteins capable of causing a sweet taste sensation in humans when interacting with the T1R2/T1R3 receptor. SP thaumatin has already been introduced in the food industry in some countries. Other SPs, such as monellin and brazzein, are promising products. An important stage in researching SPs, in addition to confirming the absence of toxicity, mutagenicity, oncogenicity, and allergenic effects, is studying their influence on gut microbiota. In this paper we describe changes in the composition of rat gut microbiota after six months of consuming one of two recombinant SPs-brazzein or monellin. A full length 16S gene sequencing method was used for DNA library barcoding. The MaAsLin2 analysis results showed noticeable fluctuations in the relative abundances of Anaerocella delicata in brazzein-fed rat microbiota, and of Anaerutruncus rubiinfantis in monellin-fed rat microbiota, which, however, did not exceed the standard deviation. The sucrose-fed group was associated with an increase in the relative abundance of Faecalibaculum rodentium, which may contribute to obesity. Overall, prolonged consumption of the sweet proteins brazzein and monellin did not significantly change rat microbiota and did not result in the appearance of opportunistic microbiota. This provides additional evidence for the safety of these potential sweeteners.

Hybrid Molecules of Azithromycin with Chloramphenicol and Metronidazole: Synthesis and Study of Antibacterial Properties.

The sustained rise of antimicrobial resistance (AMR) causes a strong need to develop new antibacterial agents. One of the methods for addressing the problem of antibiotic resistance is through the design of hybrid antibiotics. In this work, we proposed a synthetic route for the conjugation of an azithromycin derivative with chloramphenicol and metronidazole hemisuccinates and synthesized two series of new hybrid molecules 4a-g and 5a-g. While a conjugation did not result in tangible synergy for wild-type bacterial strains, new compounds were able to overcome AMR associated with the inducible expression of the ermC gene on a model E. coli strain resistant to macrolide antibiotics. The newly developed hybrids demonstrated a tendency to induce premature ribosome stalling, which might be crucial since they will not induce a macrolide-resistant phenotype in a number of pathogenic bacterial strains. In summary, the designed structures are considered as a promising direction for the further development of hybrid molecules that can effectively circumvent AMR mechanisms to macrolide antibiotics.

Triphenylphosphonium Analogs of Short Peptide Related to Bactenecin 7 and Oncocin 112 as Antimicrobial Agents.

Antimicrobial peptides (AMPs) have recently attracted attention as promising antibacterial agents capable of acting against resistant bacterial strains. In this work, an approach was applied, consisting of the conjugation of a peptide related to the sequences of bactenecin 7 (Bac7) and oncocin (Onc112) with the alkyl(triphenyl)phosphonium (alkyl-TPP) fragment in order to improve the properties of the AMP and introduce new ones, expand the spectrum of antimicrobial activity, and reduce the inhibitory effect on the eukaryotic translation process. Triphenylphosphonium (TPP) derivatives of a decapeptide RRIRPRPPYL were synthesized. It was comprehensively studied how the modification of the AMP affected the properties of the new compounds. It was shown that while the reduction in the Bac7 length to 10 a.a. residues dramatically decreased the affinity to bacterial ribosomes, the modification of the peptide with alkyl-TPP moieties led to an increase in the affinity. New analogs with structures that combined a decapeptide related to Bac7 and Onc112-Bac(1-10, R/Y)-and TPP attached to the C-terminal amino acid residue via alkylamide linkers, inhibited translation in vitro and were found to be more selective inhibitors of bacterial translation compared with eukaryotic translation than Onc112 and Bac7. The TPP analogs of the decapeptide related to Bac7 and Onc112 suppressed the growth of both Gram-negative bacteria, similar to Onc112 and Bac7, and Gram-positive ones, similar to alkyl-TPP derivatives, and also acted against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, was transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either a decylamide or ethylamide linker caused B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog was proven to be non-toxic for mammalian cells using the MTT test.