Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo.

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.

Smart City Solutions from a Societal Perspective-A Case Study.

More and more cities are evolving towards the smart city concept, which brings about a tangible effect of raised life quality levels. This is confirmed by the satisfaction of inhabitants with the introduced smart solutions. It is their opinion on this issue that is crucial, as they are the main beneficiaries of these measures. This article presents a case study of a medium-sized city, which illustrates the smart city issue from an inhabitants' perspective. An analysis of objective indices classifies a city as smart, and it is included in European lists of smart cities. It is interesting, however, to see how the inhabitants themselves assess the city in the context of the smart solutions in place. Are they relevant to them? Has the quality of life increased? Are they satisfied with the individual aspects of city functioning? What solutions are they awaiting? What areas require changes? The involvement of inhabitants and the public participation level were also assessed. The questionnaire survey results indicated whether the city can be classified as a smart city of the future and identified the spheres of the city's activities that need improvement. The main conclusions are that inhabitants have a positive perception of a city's smart services provided that their implementation results in an enhanced quality of life. At the same time, even though inhabitants are aware of the smart services introduced, they do not fully use them, which may be due to their low attractiveness, a lack of promotion on the part of the city, or a lack of equipment readiness.

The COVID-19 epidemic in Poland and its influence on the quality of life of university students (young adults) in the context of restricted access to public spaces.

Aim: The aim of this study was to determine the impact of social distancing, self-isolation and limited access to public spaces during the COVID-19 pandemic on the quality of life and psychological well-being of young people. Subject and methods: The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus posed a new global challenge in 2020. The new coronavirus emerged locally, but it rapidly spread to all continents and also reached Poland. On 24 March, the Polish Ministry of Health enforced self-isolation measures to contain the transmission of the disease. The new regulations imposed restrictions on civic freedoms, including access to public spaces. These measures significantly affected the daily lives of Polish citizens. Public spaces play a fundamental role in catering to the citizens' basic needs. Access to public spaces is directly correlated with the quality of life, human relations and spontaneous interactions. Young people are highly socially active, and they are frequent users of public spaces. University students (young adults) who transitioned to online learning on 12 March were surveyed remotely with the Microsoft Forms online tool. The questionnaire contained several questions to elicit demographic information about the respondents, as well as 25 closed-ended questions relating to university students' quality of life and access to public spaces during the epidemic. The survey was carried out during the first Polish lockdown in April 2020. The study group was composed of 132 respondents who were university students aged 19-26. Results: The results revealed a strong correlation between the severity of lockdown measures during the epidemic and the students' activity levels in public spaces, a considerable deterioration in their physical and psychological well-being, and the overall quality of life. Conclusion: The respondents were significantly affected by the absence of direct social interactions which, in their opinion, can be only partially compensated for by remote contact.

The Theoretical Model of Decision-Making Behaviour Geospatial Analysis Using Data Obtained from the Games of Chess.

The game of chess offers a conducive setting to explore basic cognitive processes, including decision-making. The game exercises analytical cause-and-effect thinking skills regardless of the level of play. Moreover, chess portals provide information on the chess games played and serve as a vast database. The numbers of games played thus have the potential to be analyzed comprehensively, including for purposes other than analyzing chess matches only. The primary objective of this study is to develop a methodology for using information obtained from chess games for geospatial social analysis. The assumption is that the methodology will allow for general geographical variation in personality inference in the future, relying on big data from chess databases. Future large-scale studies of the geographical differentiation of personality traits using the developed methodology may be applicable in a number of ways. The results can be used wherever cross-sectional social analyses are needed in the context of personality traits (decision-making) to better understand their geographical background. In turn, the geographical distribution of these traits is accompanied by a range of important social, educational, health, political and economic implications.

3D optical illusion as visualisation tools in spatial planning and development.

Spatial planning and development use various visualisation methods. Technological advancements in visualisation techniques have allowed imaging to shift from 2 to 3D dimensions. 3D optical illusion, which converts information recorded in the digital form into a three-dimensional presentation, can be a new tool for presenting spatial development solutions. Since a optical illusion is a direct spatial presentation, it requires neither specialist preparation nor spatial imagination. For this reason, it can become an effective means of visual communication with the public in the area of spatial planning and development. This article shows an example of the imaging of a model element of spatial development (a building) using the 3D illusion technique. Collected opinions of the test group of viewers confirm the usefulness of this tool. The presented 3D visualisation effect evoked positive reactions among the viewers. The use of the hologram technique in spatial planning and development appears to be justified and is an interesting research trend.

Macro-TSH - tips and tricks for gynecologists.

Thyroid disorders are one of the most common endocrinopathies in women of reproductive age. Measurement of TSH (thyroid-stimulating hormone) concentration in women planning pregnancy/pregnant is a golden standard of thyroid function assessment. When the laboratory findings do not correspond with the clinical signs, it is reasonable to mark macro-TSH.

Indole-3-propionic acid, a tryptophan-derived bacterial metabolite, increases blood pressure via cardiac and vascular mechanisms in rats.

Recent evidence suggests that gut bacteria-derived metabolites interact with the cardiovascular system and alter blood pressure (BP) in mammals. Here, we evaluated the effect of indole-3-propionic acid (IPA), a gut bacteria-derived metabolite of tryptophan, on the circulatory system. Arterial BP, electrocardiographic, and echocardiographic (ECHO) parameters were recorded in male, anesthetized, 12-wk-old Wistar-Kyoto rats at baseline and after intravenous administration of either IPA or vehicle. In additional experiments, rats were pretreated with prazosin or pentolinium to evaluate the involvement of the autonomic nervous system in cardiovascular responses to IPA. IPA's concentrations were measured using ultra-high performance liquid chromatography tandem mass spectrometry. The reactivity of endothelium-intact and -denuded mesenteric resistance arteries was tested. Cells' viability and lactate dehydrogenase (LDH) cytotoxicity assays were performed on cultured cardiomyocytes. IPA increased BP with a concomitant bradycardic response but no significant change in QTc interval. The pretreatment with prazosin and pentolinium reduced the hypertensive response. ECHO showed increased contractility of the heart after the administration of IPA. Ex vivo, IPA constricted predilated and endothelium-denuded mesenteric resistance arteries and increased metabolic activity of cardiomyocytes. IPA increases BP via cardiac and vascular mechanisms in rats. Furthermore, IPA increases cardiac contractility and metabolic activity of cardiomyocytes. Our study suggests that IPA may act as a mediator between gut microbiota and the circulatory system.

The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer.

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.

RNA and DNA G-quadruplexes bind to human dicer and inhibit its activity.

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity.

The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3'-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3'-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated.

Turkey Tail Medicinal Mushroom, Trametes versicolor (Agaricomycetes), Crude Exopolysaccharides with Antioxidative Activity.

Crude Trametes versicolor exopolysaccharides (cEPS) were used for antioxidative activity testing. Obtained results revealed high ability of cEPS for DPPH free radical scavenging and high chelating ability at the highest tested concentration (20 mg/mL), while the reducing power was significantly lower. However, based on the EC50 values, antioxidative activities of the cEPS decreased in the following order: reducing power > DPPH scavenging ability > chelating ability. Due to the high carbohydrate and ß-glucan content it is assumed that they are the main carriers of cEPS antioxidative activities. D-glucose was the main monosaccharide (87.18 ± 0.27%) while the dominant amino acids were L-lysine (L-glutamic and L-aspartic acid), which are amino acids with taste similar to the monosodium glutamate. In addition, content of sweet tasting amino acids compared with the group of bitter tasting amino acid was 2.1 times higher, indicating favorable composition of cEPS protein fraction for food industry applying.

The RNA-RNA base pairing potential of human Dicer and Ago2 proteins.

The ribonuclease Dicer produces microRNAs (miRNAs) and small interfering RNAs that are handed over to Ago proteins to control gene expression by targeting complementary sequences within transcripts. Interestingly, a growing number of reports have demonstrated that the activity of Dicer may extend beyond the biogenesis of small regulatory RNAs. Among them, a report from our latest studies revealed that human Dicer facilitates base pairing of complementary sequences present in two nucleic acids, thus acting as a nucleic acid annealer. Accordingly, in this manuscript, we address how RNA structure influences the annealing activity of human Dicer. We show that Dicer supports hybridization between a small RNA and a complementary sequence of a longer RNA in vitro, even when both complementary sequences are trapped within secondary structures. Moreover, we show that under applied conditions, human Ago2, a core component of RNA-induced silencing complex, displays very limited annealing activity. Based on the available data from new-generation sequencing experiments regarding the RNA pool bound to Dicer in vivo, we show that multiple Dicer-binding sites within mRNAs also contain miRNA targets. Subsequently, we demonstrate in vitro that Dicer but not Ago2 can anneal miRNA to its target present within mRNA. We hypothesize that not all miRNA duplexes are handed over to Ago proteins. Instead, miRNA-Dicer complexes could target specific sequences within transcripts and either compete or cooperate for binding sites with miRNA-Ago complexes. Thus, not only Ago but also Dicer might be directly involved in the posttranscriptional control of gene expression.

Indole-3-Propionic Acid, a Tryptophan-Derived Bacterial Metabolite, Reduces Weight Gain in Rats.

Recent evidence suggests that tryptophan, an essential amino acid, may exert biological effects by means of tryptophan-derived gut bacteria products. We evaluated the potential contribution of tryptophan-derived bacterial metabolites to body weight gain. The study comprised three experimental series performed on separate groups of male, Sprague-Dawley rats: (i) rats on standard laboratory diet treated with water solution of neomycin, an antibiotic, or tap water (controls-1); (ii) rats on standard diet (controls-2) or tryptophan-high (TH) or tryptophan-free (TF) diet; and (iii) rats treated with indole-3-propionic acid (I3P), a bacterial metabolite of tryptophan, or a vehicle (controls-3). (i) Rats treated with neomycin showed a significantly higher weight gain but lower stool and blood concentration of I3P than controls-1. (ii) The TH group showed significantly smaller increases in body weight but higher stool and plasma concentration of I3P than controls-2. In contrast, the TF group showed a decrease in body weight, decreased total serum protein and a significant increase in urine output. (iii) Rats treated with I3P showed significantly smaller weight gain than controls-3. Our study suggests that I3P, a gut bacteria metabolite of tryptophan, contributes to changes in body weight gain produced by antibiotics and tryptophan-rich diet.

Colonic indole, gut bacteria metabolite of tryptophan, increases portal blood pressure in rats.

Portal hypertension (PH) is a potentially life-threatening condition. We investigated the effects of indole and dietary tryptophan, a substrate for gut bacterial production of indole, on portal blood pressure (PBP), portal blood flow (PBF), and arterial blood pressure (ABP) in Sprague-Dawley rats (SD) and SD with PH induced by liver cirrhosis (SD-PH). Hemodynamics were recorded in anesthetized male 28-wk-old SD and SD-PH at baseline and after the administration of either a vehicle or indole into the colon. Blood levels of tryptophan and its bacterial metabolites were evaluated using chromatography coupled with mass spectrometry. Indole at lower doses increased PBP and PBF. Indole at higher doses produced a transient increase in PBP, which was accompanied by a decrease in ABP. Portal blood levels of indole, indole-3-propionic, indole-3-lactic, and indole-3-acetic acids were higher in SD-PH, suggesting an increased gut-blood barrier permeability. Rats on a tryptophan-rich diet showed a significantly higher PBP and portal blood level of indoles than rats on a tryptophan-free diet. In conclusion, a tryptophan-rich diet and intracolonic indole increase PBP and portal blood level of indole. Rats with PH show an increased penetration of indoles from the colon to the circulation. Intracolonic indole production may be of therapeutic importance in PH.

Selective Cytotoxic Activity of Se-Methyl-Seleno-L-Cysteine- and Se-Polysaccharide-Containing Extracts from Shiitake Medicinal Mushroom, Lentinus edodes (Agaricomycetes).

Numerous formulations derived from the shiitake medicinal mushroom, Lentinus edodes, demonstrate anticancer activities. We hypothesized that isolates from selenium (Se)-enriched mycelia of L. edodes would possess stronger cancer-preventive properties than current preparations. The aim of this study was to investigate whether the presence of Se-methyl-seleno-L-cysteine in mycelial extracts of L. edodes affects their cytotoxic activity (makes them stronger) or whether they are as effective as Se-containing polysaccharides. Extracts were prepared from Se-containing mycelia under various conditions and assayed for cytotoxic activity in cancer (PC3 and HeLa) and normal (HMEC-1) cell lines. The chemical composition of the extracts was examined; specifically, the amounts of potentially cytotoxic Se compounds (methylselenocysteine, selenomethionine, and Se-containing polysaccharides) were measured. The relationship between extract composition and biological activity was characterized. Mycelial cultures were cultivated in a 10-L bioreactor in medium enriched with sodium selenite. Mycelial extracts were prepared either at 100°C or at 4°C in acidic solution. Total Se content was determined using the atomic absorption spectrometry method, and methylselenocysteine and selenomethionine contents were measured using reverse-phase high-performance liquid chromatography. Protein, carbohydrate, and polyphenolic contents were determined with spectrophotometric methods, and Se-containing polysaccharides were measured with the use of precipitation. Anticancer activity of mycelial extracts was examined using the MTT cell viability assay. Extracts containing Se-methyl-seleno-L-cysteine or Se-polysaccharides prepared at 4°C and 100°C, respectively, display moderate, time-dependent, specific cytotoxic activity in HeLa and PC3 cell lines. The effect in HeLa cells is more pronounced in the extract prepared at 4°C than at 100°C. The effect is almost equal for the PC3 cell line. However, both extracts have no effect or only slightly stimulate normal (HMEC-1) cell viability. The selective cytotoxic activity of L. edodes extracts in cancer (PC3 and HeLa) cells is due to the presence of both Se-methyl-seleno-L-cysteine and selenated polysaccharides, perhaps in combination with other active ingredients.

Bacteriophage-encoded functions engaged in initiation of homologous recombination events.

Recombination plays a significant role in bacteriophage biology. Functions promoting recombination are involved in key stages of phage multiplication and drive phage evolution. Their biological role is reflected by the great variety of phages existing in the environment. This work presents the role of recombination in the phage life cycle and highlights the discrete character of phage-encoded recombination functions (anti-RecBCD activities, 5' --> 3' DNA exonucleases, single-stranded DNA binding proteins, single-stranded DNA annealing proteins, and recombinases). The focus of this review is on phage proteins that initiate genetic exchange. Importance of recombination is reviewed based on the accepted coli-phages T4 and lambda models, the recombination system of phage P22, and the recently characterized recombination functions of Bacillus subtilis phage SPP1 and mycobacteriophage Che9c. Key steps of the molecular mechanisms involving phage recombination functions and their application in molecular engineering are discussed.

Synthesis and conformational analysis of novel trimeric maleimide cross-linking reagents.

Nine homotrifunctional cross-linking reagents are presented. Their synthesis and chemical properties as well as their characterization by classical mechanical conformational searching techniques is reported. Mixed Low Mode and Monte Carlo searching techniques were used to exhaustively sample the OPLS2005/GBSA(water) potential energy surface of trisubstituted cyclohexane and benzene derivatives of C3 symmetry. Geometric structure, molecular length, and hydrogen-bonding patterns were analyzed. Nonaromatic compounds exhibited exclusively chair conformations at low energies, with a preference for axial or equatorial arms depending upon the presence of additional ring substituent Me groups. Increasing chain length often resulted in overall shorter molecular length due to additional chain flexibility. These results were consistent with one- and two-dimensional temperature-dependent NMR studies.

Synthesis and anti-HIV activity of trivalent CD4-mimetic miniproteins.

A series of trivalent CD4-mimetic miniproteins was synthesized, in which three CD4M9 miniprotein moieties were tethered on a threefold-symmetric scaffold. The trivalent miniproteins were designed to target the CD4-binding sites displayed in the trimeric gp120 complex of HIV-1. The synthesis takes advantage of the highly efficient ligation between a cysteine-tagged CD4M9 miniprotein and a suitable trivalent maleimide that varied in the nature and length of spacer. Antiviral assay revealed that most of the synthetic trivalent miniproteins demonstrated significantly enhanced anti-HIV activities over the monomeric CD4M9 against both R5- and X4-tropic viruses, indicating the beneficial multivalent effects. One compound that possesses a hydrophobic linker was shown to be 140-fold more active than CD4M9 against HIV-1(Bal) infection, implicating a positive contribution of the lipid portion to the antiviral activity. It was also found that most of the trivalent miniproteins showed comparable anti-HIV activities in comparison with a typical bivalent miniprotein, regardless of the length of the linker. The results implicate a novel mechanism of the interactions between the multivalent inhibitors and the trimeric gp120 complex.

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment.

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.

A distinct single-stranded DNA-binding protein encoded by the Lactococcus lactis bacteriophage bIL67.

Single-stranded binding proteins (SSBs) are found to participate in various processes of DNA metabolism in all known organisms. We describe here a SSB protein encoded by the Lactococcus lactis phage bIL67 orf14 gene. It is the first noted attempt at characterizing a SSB protein from a lactococcal phage. The purified Orf14(bIL67) binds unspecifically to ssDNA with the same high affinity as the canonical Bacillus subtilis SSB. Electrophoretic mobility-shift assays performed with mutagenized Orf14(bIL67) protein derivatives suggest that ssDNA-binding occurs via a putative OB-fold structure predicted by three-dimensional modeling. The native Orf14(bIL67) forms homotetramers as determined by gel filtration studies. These results allow distinguishing the first lactococcal phage protein with single-strand binding affinity, which defines a novel cluster of phage SSBs proteins. The possible role of Orf14(bIL67) in phage multiplication cycle is also discussed.