Incautious design of shRNAs for stable overexpression of miRNAs could result in generation of undesired isomiRs.

shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.

Pyrimidine thioethers: A novel class of antidepressant agents, endowed with anxiolytic, performance enhancing and nootropic activity.

A series of new pyrimidine thioethers, recognized as the key intermediates in the synthesis of S-DABO antivirals, were prepared and evaluated both in vivo and in silico. The purpose of this evaluation was to find novel structural analogues of the known antihypoxic drug Isothiobarbamine endowed with improved pharmacological profile. The in vivo studies led to the identification of compounds 5c, 5e, and 5f endowed with antidepressant/anxiolytic, performance enhancing, and nootropic properties. Compounds 5c and 5f were further tested in mice affected by social depression and were able to increase motor and tentative search activity compared to control groups, along with higher interaction frequency and better results in a sucrose preference test. Overall, these data suggested a better psychoemotional state of the animals, treated with compounds 5c, and 5f. Moreover, 5c and 5f exhibited minimal acute toxicity, lower than Fluoxetine hydrochloride. Molecular modelling studies finally indicated the plausible biomolecular mechanism of action of compounds 5c, 5e, and 5f, which seem to bind GABA-A, melatonin, and sigma-1 receptors. Moreover, three-dimensional structure-activity relationships enabled to define a SAR model that will be of great utility for the design of further structurally optimized compounds of the above mentioned chemotype.

Application of the Balance Model in the Analysis of Factors Responsible for Depressive Disorders among Women in the COVID-19 Pandemic.

The COVID-19 pandemic has significantly affected the lives and mental health of people around the world, and it has become clinically essential to define risk factors in order to provide adequate prevention and support. The aim of the study was to describe coping strategies in Polish women related to the COVID-19 pandemic using the balance model, one of the most important concepts of positive psychotherapy (PPT after Peseschkian since 1977). The analysis included 735 women at the mean age of 39.61 years. The survey was conducted using the questionnaire form on the website. Based on Beck's depression test, depressive disorders were disclosed in 32.65%, and both the presence and severity of depressive syndromes were inversely correlated with age. Using a cluster analysis, three adaptation strategies could be identified, related to the different prevalence of depressive disorders. Relationships proved the most crucial area of the balance model, responsible for the effectiveness of the coping strategy. Based on the obtained results, it has to be concluded that preventive measures should primarily concern women aged < 25 years old and focus on strengthening the relationships area.

Effect of α-Methoxy Substitution on the Anti-HIV Activity of Dihydropyrimidin-4(3 H)-ones.

Conformational restriction applied to dihydrobenzylpyrimidin-4-(3 H)-ones (DABOs) by the intoduction of a methyl group at the α-benzylic position is known to massively improve the anti-HIV-1 activity of these compounds. Here, we report the effects of methoxy substitution at the α-benzylic position in S-, NH-, and N, N-DABOs carrying 2,6-difluoro, 2-chloro-6-fluoro, or 2,6-dichloro substituted benzyl moieties. The various α-methoxy DABO series (12-14) present different SAR at the dihalo benzyl substitution, with the most potent compounds (12d,e and 13c) showing similar (picomolar/nanomolar) anti-HIV-1 potency as the corresponding α-methyl analogues against wt HIV-1, and 10-100-fold increased potency (up to low nanomolar) against clinically relevant K103N, Y181C, Y188L, IRLL98, and K103N+Y181C HIV-1 mutant strains, highlighting the importance of the α-methoxy substitution to provide highly efficient DABOs as "second generation" NNRTIs. HPLC enantioseparation of three of the most potent derivatives (12d, 13c, and 14c) provided single enantiomers with significant enantioselectivity in HIV-1 inhibition. Computational studies allowed to correlate the best antiviral activity with the ( R) absolute configuration at the α-methoxy stereogenic center.