Evaluation of Gene Expression Knock-Down by Chemically and Structurally Modified Gapmer Antisense Oligonucleotides.

We analyzed the effect of modified nucleotides within gapmer antisense oligonucleotides on RNase H mediated gene silencing. Additionally, short hairpins were introduced into antisense oligonucleotides as structural motifs, and their influence on biological and physicochemical properties of pre-structured gapmers was investigated for the first time. The results indicate that two LNA residues in specified positions of the gap flanking regions are sufficient and favorable for efficient knock-down of the ß-actin gene. Furthermore, the introduction of other modified nucleotides, i. e. glycyl-amino-LNA-T, 2'-O-propagyluridine, polyamine functionalized uridine, and UNA, in specified positions, also increases the inhibition of ß-actin expression. Importantly, the presence of hairpins within the gapmers improves their silencing properties.

Allee effect in a manipulative parasite within poikilothermic host under temperature change.

Temperature and intraspecific competition are important factors influencing the growth of all organisms, including parasites. The temperature increase is suggested to stimulate the development of parasites within poikilothermic hosts. However, at high parasite densities, this effect could be diminished, due to stronger intraspecific competition. Our study, for the first time, addressed the joint effects of warming and parasite abundances on parasite growth in poikilothermic hosts. The growth of the common fish parasite larvae (trematode Diplostomum pseudospathaceum) within the rainbow trout at different infection intensities and temperatures (15°C and 18°C) was experimentally investigated. The results showed that temperature was positively correlated with both parasite infection success and growth rates. The growth rates increased much more compared to those in many free-living poikilothermic animals. Atypically for a majority of parasites, D. pseudospathaceum larvae grow faster when abundant (Allee effect). The possible causes for this phenomenon (manipulation cost sharing, etc.) are discussed in this study. Importantly, limited evidence of the interaction between temperature and population density was found. It is likely that temperature did not change the magnitude of the Allee effect but affected its timing. The impact of these effects is supposed to become more pronounced in freshwater ecosystems under current climate changes.

A Structural Potential of Rare Trinucleotide Repeat Tracts in RNA.

Among types of trinucleotide repeats, there is some disproportion in the frequency of their occurrence in the human exome. This research presents new data describing the folding and thermodynamic stability of short, tandem RNA repeats of 23 types, focusing on the rare, yet poorly analyzed ones. UV-melting experiments included the presence of PEG or potassium and magnesium ions to determine their effect on the stability of RNA repeats structures. Rare repeats predominantly stayed single-stranded but had the potential for base pairing with other partially complementary repeat tracts. A coexistence of suitably complementary repeat types in a single RNA creates opportunities for interaction in the context of the secondary structure of RNA. We searched the human transcriptome for model RNAs in which different, particularly rare trinucleotide repeats coexist and selected the GABRA4 and CHIC1 RNAs to study intramolecular interactions between the repeat tracts that they contain. In vitro secondary structure probing results showed that the UAA and UUG repeat tracts, present in GABRA4 3' UTR, form a double helix, which separates one of its structural domains. For the RNA CHIC1 ORF fragment containing four short AGG repeat tracts and the CGU tract, we proved the formation of quadruplexes that blocked reverse transcription.

Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene.

Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs.

Analysis of Deaths among HIV-Infected Patients Hospitalized in 2009-2018 in Main Centre of Infectious Disease in Region of Lower Silesia in Poland, Detailing Lesions in the Central Nervous System.

Background and Objectives: Patients living with HIV (PLWH), especially those diagnosed too late or not receiving treatment with antiretroviral drugs in the stage of advanced immunodeficiency AIDS for various reasons, develop additional opportunistic infections or AIDS-defining diseases that may contribute directly to the death of these patients. Material and Methods: In this work, we focused on disorders of the central nervous system (CNS) by retrospectively analyzing the symptoms, clinical and autopsy diagnoses of patients diagnosed with HIV infection who died in the provincial specialist hospital in the Lower Silesia region in Poland. Results: The autopsy was performed in 27.4% cases. The cause of death was determined to be HIV-related/AIDS-associated in 78% patients. The most common AIDS-defining CNS diseases in our cohort were toxoplasmosis and cryptococcosis. Conslusions: The presented results of the most common causes of changes in the central nervous system among deceased HIV-infected patients are comparable to the results of studies by other scientists cited in the publication.

Beyond G-Quadruplexes-The Effect of Junction with Additional Structural Motifs on Aptamers Properties.

G-quadruplexes constitute an important type of nucleic acid structure, which can be found in living cells and applied by cell machinery as pivotal regulatory elements. Importantly, robust development of SELEX technology and modern, nucleic acid-based therapeutic strategies targeted towards various molecules have also revealed a large group of potent aptamers whose structures are grounded in G-quadruplexes. In this review, we analyze further extension of tetraplexes by additional structural elements and investigate whether G-quadruplex junctions with duplex, hairpin, triplex, or second G-quadruplex motifs are favorable for aptamers stability and biological activity. Furthermore, we indicate the specific and pivotal role of the G-quadruplex domain and the additional structural elements in interactions with target molecules. Finally, we consider the potency of G-quadruplex junctions in future applications and indicate the emerging research area that is still waiting for development to obtain highly specific and effective nucleic acid-based molecular tools.

G4 Matters-The Influence of G-Quadruplex Structural Elements on the Antiproliferative Properties of G-Rich Oligonucleotides.

G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich sequences of DNA or RNA that have attracted increased attention as anticancer agents. This systematic study aimed to investigate the anticancer potential of five G4-forming, sequence-related DNA molecules in terms of their thermodynamic and structural properties, biostability and cellular uptake. The antiproliferative studies revealed that less thermodynamically stable G4s with three G-tetrads in the core and longer loops are more predisposed to effectively inhibit cancer cell growth. By contrast, highly structured G4s with an extended core containing four G-tetrads and longer loops are characterized by more efficient cellular uptake and improved biostability. Various analyses have indicated that the G4 structural elements are intrinsic to the biological activity of these molecules. Importantly, the structural requirements are different for efficient cancer cell line inhibition and favorable G4 cellular uptake. Thus, the ultimate antiproliferative potential of G4s is a net result of the specific balance among the structural features that are favorable for efficient uptake and those that increase the inhibitory activity of the studied molecules. Understanding the G4 structural features and their role in the biological activity of G-rich molecules might facilitate the development of novel, more potent G4-based therapeutics with unprecedented anticancer properties.

Gapmer Antisense Oligonucleotides Containing 2',3'-Dideoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxofuranosyl Nucleotides Display Site-Specific RNase†H Cleavage and Induce Gene Silencing.

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.

Trematode cercariae as prey for zooplankton: effect on fitness traits of predators.

Removal of parasite free-living stages by predators has previously been suggested an important factor controlling parasite transmission in aquatic habitats. Experimental studies of zooplankton predation on macroparasite larvae are, however, scarce. We tested whether trematode cercariae, which are often numerous in shallow waters, are suitable prey for syntopic zooplankters. Feeding rates and survival of freshwater cyclopoids (Megacyclops viridis, Macrocyclops distinctus), calanoids (Arctodiaptomus paulseni), cladocerans (Sida crystallina) and rotifers Asplanchna spp., fed with cercariae of Diplostomum pseudospathaceum, a common fish trematode, were studied. In additional long-term experiments, we studied reproduction of cyclopoids fed with cercariae. All tested zooplankton species consumed cercariae. The highest feeding rates were observed for cyclopoids (33 ± 12 cercariae ind-1 h-1), which actively reproduced (up to one egg clutch day-1) when fed ad libitum with cercariae. Their reproductive characteristics did not change significantly with time, indicating that cercariae supported cyclopoids' dietary needs. Mortality of rotifers and cladocerans was high (25-28% individuals) when exposed to cercariae in contrast to cyclopoids and calanoids (<2%). Cercariae clogged the filtration apparatus of cladocerans and caused internal injuries in predatory rotifers, which ingested cercariae. Observed trophic links between common freshwater zooplankters and cercariae may significantly influence food webs and parasite transmission in lentic ecosystems.

G-Quadruplex-Forming Aptamers-Characteristics, Applications, and Perspectives.

G-quadruplexes constitute a unique class of nucleic acid structures formed by G-rich oligonucleotides of DNA- or RNA-type. Depending on their chemical nature, loops length, and localization in the sequence or structure molecularity, G-quadruplexes are highly polymorphic structures showing various folding topologies. They may be formed in the human genome where they are believed to play a pivotal role in the regulation of multiple biological processes such as replication, transcription, and translation. Thus, natural G-quadruplex structures became prospective targets for disease treatment. The fast development of systematic evolution of ligands by exponential enrichment (SELEX) technologies provided a number of G-rich aptamers revealing the potential of G-quadruplex structures as a promising molecular tool targeted toward various biologically important ligands. Because of their high stability, increased cellular uptake, ease of chemical modification, minor production costs, and convenient storage, G-rich aptamers became interesting therapeutic and diagnostic alternatives to antibodies. In this review, we describe the recent advances in the development of G-quadruplex based aptamers by focusing on the therapeutic and diagnostic potential of this exceptional class of nucleic acid structures.

Synthesis and hybridization properties of oligonucleotide analogues with novel acyclic triazole internucleotide linkages.

Herein, we describe synthesis of novel acyclic dinucleotide analogues connected via triazole linkage in CuAAC reaction. Synthesis pathway starting from previously obtained building blocks containing alkyne or azide functional group is described. Further functionalization and application of dinucleotide analogues in DNA phosphoramidite solid-phase synthesis is also explained. Additionally, we have examined the influence of novel modifications on DNA duplex thermodynamic stability.

Thermodynamic, structural and fluorescent characteristics of DNA hairpins containing functionalized pyrrolo-2'-deoxycytidines.

Herein, we present comprehensive physicochemical and structural analysis of various DNA hairpins modified with pyrrolo-2'-deoxycytidine (Py-dC) derivatives. The introduction of modified Py-dC in most cases causes minor decrease of hairpin thermodynamic stability. The energetically unfavorable effect is more pronounced when modified residue is present within hairpin loop. Our studies indicate that thermodynamic effects induced by all Py-dC derivatives are net results of increased stacking interactions caused by larger surface of pyrrolo-2'-deoxycytidine aromatic ring and unfavorable effect implied by the presence of additional side chains. The CD spectra of all modified hairpins are similar to unmodified hairpin indicating that the presence of Py-dC derivatives does not disrupt the secondary structure of DNA. Interestingly, the presence of various side chains can increase fluorescent discrimination of paired and unpaired regions of DNA. The fluorescence observed for hairpins modified within loop is significantly quenched when Py-dC derivative is present in the stem region.

Chemogenetic silencing of neurons in retrosplenial cortex disrupts sensory preconditioning.

An essential aspect of episodic memory is the formation of associations between neutral sensory cues in the environment. In light of recent evidence that this critical aspect of learning does not require the hippocampus, we tested the involvement of the retrosplenial cortex (RSC) in this process using a chemogenetic approach that allowed us to temporarily silence neurons along the entire rostrocaudal extent of the RSC. A viral vector containing the gene for a synthetic inhibitory G-protein-coupled receptor (hM4Di) was infused into RSC. When the receptor was later activated by systemic injection of clozapine-N-oxide, neural activity in RSC was transiently silenced (confirmed using a patch-clamp procedure). Rats expressing hM4Di and control rats were trained in a sensory preconditioning procedure in which a tone and light were paired on some trials and a white noise stimulus was presented alone on the other trials during the Preconditioning phase. Thus, rats were given the opportunity to form an association between a tone and a light in the absence of reinforcement. Later, the light was paired with food. During the test phase when the auditory cues were presented alone, controls exhibited more conditioned responding during presentation of the tone compared with the white noise reflecting the prior formation of a tone-light association. Silencing RSC neurons during the Preconditioning phase prevented the formation of an association between the tone and light and eliminated the sensory preconditioning effect. These findings indicate that RSC may contribute to episodic memory formation by linking essential sensory stimuli during learning.

Watson-Crick hydrogen bonding of unlocked nucleic acids.

We herein describe the synthesis of two new unlocked nucleic acid building blocks containing hypoxanthine and 2,6-diaminopurine as nucleobase moieties and their incorporation into oligonucleotides. The modified oligonucleotides were used to examine the thermodynamic properties of UNA against unmodified oligonucleotides and the resulting thermodynamic data support that the hydrogen bonding face of UNA is Watson-Crick like.

Unlocked nucleic acids: implications of increased conformational flexibility for RNA/DNA triplex formation.

Unlocked nucleic acids (UNAs) have been introduced at specific positions in short model DNA hairpins and RNA/DNA triplexes for the first time. UNA residues destabilize the hairpins and decrease triplex thermodynamic stability or suppress triplex formation for most of the evaluated structures. Nevertheless, the incorporation of UNA residues at certain positions of dsDNA was found to be energetically favourable or at least did not affect triplex stability. Notably, the most thermodynamically stable UNA-modified triplexes exhibited improved stability at both acidic and physiological pH. The specificity of the interactions between the triplex-forming oligonucleotide and dsDNA was characterized using EMSA for the most thermodynamically stable structures, and triplex dissociation constants were determined. One of the modified triplexes exhibited an improved Kd in comparison with the unmodified triplex. CD and thermal difference spectra indicated that UNA residues do not alter the overall structure of the most thermodynamically stable triplexes. In addition, incubation of the modified oligonucleotides with human serum indicated that the UNAs demonstrate the potential to improve the biological stability of nucleic acids.

Preliminary study of themes of meaning and psychosocial service use among informal cancer caregivers.

OBJECTIVE: The burden experienced by informal caregivers (ICs) of patients with advanced cancer is well documented. ICs are at risk for anxiety and depression, as well as existential concerns that arise when a loved one is facing a terminal illness. Few psychosocial interventions focus on existential concerns of ICs. However, a growing body of literature indicates that finding meaning in the experience of being an IC for a person with cancer has the potential to buffer against burden. The purpose of this study was to collect preliminary descriptive data regarding caregiver burden, meaning, and psychosocial service use to inform the adaptation of a meaning-centered intervention for ICs. METHOD: Twenty-five caregivers and 32 patients completed brief, anonymous questionnaires that asked about their role as a caregiver or their perception of their loved one as a caregiver, caregiver burden, and psychosocial service use. RESULTS: Caregivers and patients identified anxiety and depression as top correlates of burden experienced by caregivers, whereas guilt, issues with role/sense of identity, and self-care were additional areas of concern. The majority of caregivers were not receiving psychosocial services, although they almost unanimously reported desiring services. A greater proportion of patients than caregivers believed that an intervention designed to enhance meaning would ameliorate burden, but, nevertheless, close to three quarters of caregivers reported interest in participating in such an intervention. SIGNIFICANCE OF RESULTS: These study findings provide further support for, at a minimum, engaging ICs of persons with advanced cancer in interventions that address existential issues, mental health, self-care, and service use. Such interventions are likely to improve the quality of life of both patients with cancer and their ICs.

Revisiting the role of behavior-mediated structuring in the survival of populations in hostile environments.

Increasing the population density of target species is a major goal of ecosystem and agricultural management. This task is especially challenging in hazardous environments with a high abundance of natural enemies such as parasites and predators. Safe locations with lower mortality have been long considered a beneficial factor in enhancing population survival, being a promising tool in commercial fish farming and restoration of threatened species. Here we challenge this opinion and revisit the role of behavior structuring in a hostile environment in shaping the population density. We build a mathematical model, where individuals are structured according to their defensive tactics against natural enemies. The model predicts that although each safe zone enhances the survival of an individual, for an insufficient number of such zones, the entire population experiences a greater overall mortality. This is a result of the interplay of emergent dynamical behavioral structuring and strong intraspecific competition for safe zones. Non-plastic structuring in individuals' boldness reduces the mentioned negative effects. We demonstrate emergence of non-plastic behavioral structuring: the evolutionary branching of a monomorphic population into a dimorphic one with bold/shy strains. We apply our modelling approach to explore fish farming of salmonids in an environment infected by trematode parasites.

Influence of polarized light, disruptive visual patterns, and chemical cues on oviposition in the aquatic midge, Chironomus riparius (Diptera: Chironomidae).

Chironomid nonbiting midges are common in many waterbodies, occurring at high densities in sediment and also when flying adults emerge. Although important for food webs and ecosystem processes, the mass emergences of chironomid adults make some species nuisance pests. As part of an effort to develop "push-pull" strategies for managing midge populations, we investigated the importance of visual and chemical cues used by gravid females when selecting sites for oviposition. Field and laboratory oviposition choice tests with Chironomus riparius (Meigen) were used to assess the attractiveness of dark container colors and polarized light for females seeking water for egg laying. Females were not sensitive to increased intensity of polarized light, but they laid fewer eggs in containers with white color above the water's edge but black below the surface. A disruptive pattern of vertical black and white stripes at the water's edge reduced egg laying to a similar degree as white. To investigate the importance of olfactory or gustatory cues in oviposition decisions, we tested 3 potential chemical repellents (damaged larvae simulating predator presence, azadiractin, and picaridin) and 5 potential attractants (a tannin-molasses mixture, leaf detritus, live Hydrilla plants, periphyton, and fermented alfalfa infusions used to bait mosquito oviposition traps). Chemical cues appeared to play a weak role, if any, in oviposition choices. Onlyazadiractin (0.02 and 0.11 ppm) reduced the number of egg ropes deposited, but the mechanism may have been from mechanical trapping and drowning, not deterrence.