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2’-OMe-RNA analogues and LNA point modifications of DNA oligonucleotides were applied for the modulation of the G-quadruplex topology and enhancement of peroxidase activity of the resulting DNAzymes. The effect of the 2’-OMe-RNA analogue was studied for full length modified oligonucleotides with various sequences. In the case of LNA-point modification, we have chosen a telomeric DNA sequence and investigated various numbers of modifications. Our main goal was to prove that the application of these modifications can influence the activity of DNAzyme, especially those, which normally form poor DNAzymes. As an example, we have chosen the telomeric HT22 sequence which is known to form DNAzyme characterized by low activity. In all cases, the DNAzymes formed by a telomeric sequence with the application of the 2’-OMe-RNA analogue as well as LNA-point modification, showed significantly higher peroxidase activity. We were also able to shift the formation of hybrid or antiparallel topology to parallel topology. These results are important for the development of probes for biological applications as well as for the design of probes based on DNA sequences that normally form DNAzymes with low activity. This paper also provides information on how the application of nucleotide analogues can transform the topology of G-quadruplexes.

Non‐syndromic cleft lip with or without cleft palate (nsCL/P) is a common craniofacial anomaly with a complex and heterogeneous aetiology. Knowledge regarding specific genetic factors underlying this birth defect is still not well understood. Therefore, we conducted an independent replication analysis for the top‐associated variants located within the DLG1 locus at chromosome 3q29, which was identified as a novel cleft‐susceptibility locus in our genome‐wide association study (GWAS). Mega‐analysis of the pooled individual data from the GWAS and replication study confirmed that common DLG1 variants are associated with the risk of nsCL/P. Two single nucleotide polymorphisms (SNPs), rs338217 and rs7649443, were statistically significant even at the genome‐wide level (Ptrend = 9.70E−10 and Ptrend = 8.96E−09, respectively). Three other SNPs, rs9826379, rs6805920 and rs6583202, reached a suggestive genome‐wide significance threshold (Ptrend < 1.00E−05). The location of the strongest individual SNP in the intronic sequence of the gene encoding DLG1 antisense RNA suggests that the true causal variant implicated in the risk of nsCL/P may affect the DLG1 gene expression level rather than structure of the encoded protein. In conclusion, we identified a novel cleft‐susceptibility locus at chromosome 3q29 with a DLG1 as a novel candidate gene for this common craniofacial anomaly. [ABSTRACT FROM AUTHOR]

Bioluminescent high-throughput screening (HTS) assays, based largely on the activity of firefly (FLuc), Renilla (RLuc), and/or NanoLuc (NLuc) luciferases, are widely utilised in research and drug discovery. In this study, we quantify the luciferase-based real-life HTS assay interference from biologically and environmentally relevant metal ions ubiquitously present in buffers, environmental and biological matrices, and as contaminants in plastics and compound libraries. We also provide insights into the cross-effects of metal ions and other key experimental and biological reagents (e.g., buffer types, EDTA, and glutathione) to inform HTS assay design, validation, and data interpretation. A total of 21 ions were screened in three robust HTS assays ("SC" assays) based on the luminescence of FLuc, RLuc, and NLuc luciferases. Three newly optimised HEPES buffer variants ("H" assays) were developed for direct luciferase comparison. Interference in bioluminescent signal generation was quantified by calculating the IC 50 values from concentration-dependent experiments for selected highly active and relevant metal ions. Metal ion inhibition mechanisms were probed by variations in specific reagents, EDTA, GSH, and the sequence of addition and buffer composition. In this study, we revealed a significant impact of metal ions' salts on luciferase-mediated bioluminescence, even at biologically and environmentally relevant concentrations. The extent of signal interference largely aligned with the Irving-Williams series of metal ion-ligand affinities (Cu > Zn > Fe > Mn > Ca > Mg), supporting previous reports on metal ion-dependent FLuc inhibition. However, the absolute magnitude and relative extent of signal reduction by metal ions' salts differed between SC and H assays and between luciferases, suggesting a complex network of metal ions' interactions with enzymes, substrates, reactants, and buffer elements. The diversity of the tested conditions and variability of responses provided insights into potential interference mechanisms and synergies that may exacerbate or alleviate interference. The beneficial influence of EDTA and the impact of glutathione, present natively in cells, on bioluminescence readout were pinpointed. Given the ubiquity of metal ions in analysed samples, the causative role in false-positive generation in drug discovery, and the wide breadth of luciferase-based assays used in screening, awareness and quantification of metal influence are crucial for developing assay validation protocols and ensuring reliable screening data, ultimately increasing the critical robustness of bioluminescence-based HTS assays., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Canyelles i Font, Żukowski, Khan, Kwiatek and Kolanowski.)

With the development of medicine and the aging population, the demand for more effective therapies is escalating. A tool that facilitates the discovery and introduction of new therapeutic drugs is High-Throughput Screening (HTS). These tests, consisting of a wide set of various assays, allow testing hundreds of thousands of compounds in a short period of time. The aim is to accurately identify active compounds that could become potential therapeutic candidates in the pharmaceutical industry. HTS is the first step in the quest for potential drugs, therefore it is one of the crucial tests that determine whether a particular drug candidate will be discovered. In this review, different stages of high-throughput screening studies will be described, as well as methods utilized in these studies. The necessary steps in the optimization of these tests, selection of equipment, automation and key quality control parameters for reliably performed screening will also be presented.

Background: The single-step model is becoming increasingly popular for national genetic evaluations of dairy cattle due to the benefits that it offers such as joint breeding value estimation for genotyped and ungenotyped animals. However, the complexity of the model due to a large number of correlated effects can lead to significant computational challenges, especially in terms of accuracy and efficiency of the preconditioned conjugate gradient method used for the estimation. The aim of this study was to investigate the effect of pedigree depth on the model's overall convergence rate as well as on the convergence of different components of the model, in the context of the single-step single nucleotide polymorphism best linear unbiased prediction (SNP-BLUP) model., Results: The results demonstrate that the dataset with a truncated pedigree converged twice as fast as the full dataset. Still, both datasets showed very high Pearson correlations between predicted breeding values. In addition, by comparing the top 50 bulls between the two datasets we found a high correlation between their rankings. We also analysed the specific convergence patterns underlying different animal groups and model effects, which revealed heterogeneity in convergence behaviour. Effects of SNPs converged the fastest while those of genetic groups converged the slowest, which reflects the difference in information content available in the dataset for those effects. Pre-selection criteria for the SNP set based on minor allele frequency had no impact on either the rate or pattern of their convergence. Among different groups of individuals, genotyped animals with phenotype data converged the fastest, while non-genotyped animals without own records required the largest number of iterations., Conclusions: We conclude that pedigree structure markedly impacts the convergence rate of the optimisation which is more efficient for the truncated than for the full dataset., (© 2023. The Author(s).)

BSCL2 encodes seipin, a transmembrane endoplasmic reticulum protein associated with lipodystrophy and severe metabolic complications, including diabetes and hepatic steatosis. In pigs, BSCL2 expression increases during adipocyte differentiation. In the present study, we identified significant gene variants associated with fat deposition (FD)-related processes based on subcutaneous fat tissue RNA-seq data. In the association study, to prove our hypothesis, three Polish pig breeds were included: Złotnicka White (ZW, n = 72), Polish Landrace (PL, n = 201), and Polish Large White (PLW, n = 169). Based on variant calling analysis and χ 2 tests, BSCL2 mutations showing significantly different genotype/allele distribution between high- and low-fat pigs were selected for a comprehensive association study. Four interesting BSCL2 variants (rs346079334, rs341493267, rs330154033, and rs81333153) belonging to downstream and missense mutations were investigated. Our study showed a significant decrease in minor allele frequency for two BSCL2 variants (rs346079334 and rs341493267) in PL pigs in 2020-2021. In ZW, BSCL2 mutations significantly affected loin and ham fats, meat redness, and growth performance traits, such as feed conversion and daily feed intake. Similar observations were noted for PLW and PL, where BSCL2 mutations influenced fat depositions and meat traits, such as loin eye area, loin mass and fat, carcass yield, and growth performance traits. Based on the observation in pigs, our study supports the theory that BSCL2 expressed in subcutaneous fat is involved in the FD process.

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods-reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)-combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10-20 min, whereas for RT-LAMP, the LOD was 30-300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

The goal of this study was to evaluate the effects of mutations in the FGL1 gene associated with pig productive traits to enrich the genetic marker pool for further selection and to support the studies on FGL1 in the context of the fat deposition (FD) process. The variant calling and χ 2 analyses of liver RNA-seq data were used to indicate genetic markers. FGL1 mutations were genotyped in the Złotnicka White (n = 72), Polish Large White (n = 208), Duroc (n = 72), Polish Landrace (PL) (n = 292), and Puławska (n = 178) pig breeds. An association study was performed using a general linear model (GLM) implemented in SAS ® software. More than 50 crucial mutations were identified in the FGL1 gene. The association study showed a significant effect of the FGL1 on intramuscular fat (IMF), loin eye area, backfat thickness at the lumbar, ham mass ( p = 0.0374), meat percentage ( p = 0.0205), and loin fat ( p = 0.0003). Alternate homozygotes and heterozygotes were found in the PL and Duroc, confirming the selective potential for these populations. Our study supports the theory that liver FGL1 is involved in the FD process. Moreover, since fat is the major determinant of flavor development in meat, the FGL1 rs340465447&#95;A allele can be used as a target in pig selection focused on elevated fat levels.

Laser toners appear as attractive materials for barriers and easily laminated interphases for Lab-on-a-Foil microfluidics, due to the excellent adhesion to paper and various membranes or foils. This work shows for the first time a comprehensive study on the adsorption of antibodies on toner-covered poly(ethylene terephthalate) (PET@toner) substrates, together with assessment of such platforms in rapid prototyping of disposable microdevices and microarrays for immunodiagnostics. In the framework of presented research, the surface properties and antibody binding capacity of PET substrates with varying levels of toner coverage (0-100%) were characterized in detail. It was proven that polystyrene-acrylate copolymer-based toner offers higher antibody adsorption efficiency compared with unmodified polystyrene and PET as well as faster adsorption kinetics. Comparative studies of the influence of pH on the effectiveness of antibodies immobilization as well as measurements of surface ζ-potential of PET, toner, and polystyrene confirmed the dominant role of hydrophobic interactions in adsorption mechanism. The applicability of PET@toner substrates as removable masks for protection of foil against permanent hydrophilization was also shown. It opens up the possibility of precise tuning of wettability and antibody binding capacity. Therefore, PET@toner foils are presented as useful platforms in the construction of immunoarrays or components of microfluidic systems.

In this work we investigated the effect of a DNA oligonucleotide sequence on the activity of a DNAzyme with covalently attached hemin. For this purpose, we synthesized seven DNA-hemin conjugates. All DNA-hemin conjugates as well as DNA/hemin complexes were characterized using circular dichroism, determination of melting temperatures and pKa of hemin. We observed that hemin conjugation in most cases led to the formation of parallel G-quadruplexes in the presence of potassium and increased thermal stability of all studied systems. Although the activity of DNA-hemin conjugates depended on the sequence used, the highest activity was observed for the DNA-hemin conjugate based on a human telomeric sequence. We used this DNAzyme for development of "sandwich" assay for detection of DNA sequence. For this assay, we used electric chip which could conduct electricity after silver deposition catalyzed by DNAzyme. This method was proved to be selective towards DNA oligonucleotides with mismatches and could be used for the detection of the target. To prove the versatility of our DNAzyme probe we also performed experiments with streptavidin-coated microplates. Our research proved that DNAzyme with covalently attached hemin can be used successfully in the development of heterogeneous assays.

The hypothalamus-pituitary axis is involved in digest processing, stress response, energy storage and many other processes. In birds, this control differs from in mammals, such as regulation of appetite and satiety centre. The transcriptomics analyses of both brain structures can explain and identify the molecular processes related to body growth and development and nutritional status. Many reports describe chicken transcriptome in literature, but gene expression studies in the other poultry species are extremely rare. Therefore, the present research undertook the attempt to explain hypothalamus-pituitary processes in domestic geese-Polish White Kołuda®, main Polish line. After 16 weeks of fattening, significant differences in geese weight were observed. Therefore, transcriptome of pituitary and hypothalamus profiles could be compared between low and high growth rate geese groups. Due to the lack of domestic geese genome assembly in the public databases, we used three mapping approaches: de novo analysis, mapping to two other pink-footed and swan geese genomes. The functional examination showed that the most enriched biological process in the geese hypothalamus covered the immune response. Moreover, in the hypothalamus, proteins typical for the pituitary such as PRL and GH were differentially expressed (DE). Our study recommends one gene as a candidate for growth rate in geese-the FOS gene, which encodes Fos proto-oncogene-DE in both analysed tissues. The FOS gene is involved in regulating feeding behaviour, immune regulation, stimulating cellular proliferation and controlling growth hormone synthesis. Moreover, the present investigation indicates DE genes involved in gene expression regulation. The study delivers new information about the changes in the pituitary-hypothalamic axis in geese dependent on growth rate differences., (© 2021 Stichting International Foundation for Animal Genetics.)

Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H 2 O 2 -free DNAzyme system with oxidase activity. Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH 2 ) and carboxylated quantum dots (CdTe@COOH QDs). The obtained products were characterized by spectroscopic methods (UV-Vis, fluorescence, circular dichroizm (CD), and IR) and the transmission electron microscopy (TEM) technique. A QD-DNA system with a low polydispersity and high stability in aqueous solutions was successfully obtained. The catalytic activity of the QD-DNA conjugate was examined with Amplex Red and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) indicators using reactive oxygen species (ROS) generated by visible light irradiation. The synthesized QD-DNAzyme exhibited enhanced catalytic activity compared with the reference system (a mixture of QDs and DNAzyme). This proved the assumption that the covalent attachment of DNAzyme to the surface of QD resulted in a beneficial effect on its catalytic activity. The results proved that the QD-DNAzyme system can be used for generation of the signal by light irradiation. The light-induced oxidase activity of the conjugate was demonstrated, proving that the QD-DNAzyme system can be useful for the development of new cellular bioassays, e.g., for the determination of oxygen radical scavengers.