Biocell

A negative answer to the question about the reducibility of genetic processes at the level of macroevolutionary events to microevolutionary ones has been obtained by analyzing the evolutionary transition-transversion bias and estimating the rates of molecular transformations in a number of vertebrates by the example of the CYTB gene. As a result, it has been established that, at a divergence at a level below families, the frequency of transitions sustains “a jump,” due to which the rate of molecular evolutions increases by an order of magnitude, whereas there occurs a slight predominance of transversion frequencies with a synchronous linear increase in the frequency of different nucleotide substitutions at the levels of orders and higher. An obvious reason for distinctions between the genetic processes of micro- and macroevolution is the leading role of spontaneous mutations in the formation of species. Their canalization results in stable morphological distinctions formed during postnatal ontogenesis. At the same time, the stages of macroevolution are associated with the transformation of organogenesis to be fixed with by changes in the sets of genes governing the nature of gene regulation and the interaction of genes in development.

The analysis of nucleotide sequences of the Argemone genus samples by bioinformatics methods was aimed at the study of phylogenetic relationships of species within the genus, at the development of genus-specific primer pairs, and TaqMan-probe and their verification in silico. Molecular genetic studies by the polymerase chain reaction method in the “real-time” mode (RT-PCR) were devoted to checking the “performance” and specificity of the developed system of primers and TaqMan-probe using samples of eight species of Argemone and 11 species of other plants. Phylogenetic analysis using internal transcribed spacer 1 (ITS1) sequences confirmed previous phylogenetic studies and improved understanding of relationships within the genus. Before the in vitro test of the developed system of primers and TaqMan-probe, the ability of the extracted DNA to be amplified by the RT-PCR method using the system of primers and the TaqMan-probe to the 18S rRNA gene of eukaryotes was checked to exclude pseudo-negative results during the further verification of specificity. The conditions for RT-PCR were selected, namely, temperatures and times of denaturation, hybridization, elongation, and the number of cycles. The developed system of primers and TaqMan-probe for the RT-PCR method demonstrated high species specificity and sensitivity. Amplification was noted only in DNA samples of eight Argemone species, while PCR analysis of other plant species and negative controls showed no amplification. The detection limit of the developed system was determined to be 0.01%. It is proposed that this marker system be used to detect falsification in food and other products, particularly Argemone contamination in mustard or coconut oil.

The ITS1-5.8S-ITS2 (ITS1-2) region of the 35S rDNA is widely used for molecular barcoding and in the phylogenetics of plants. It is believed that, due to concerted evolution, all copies of 35S rDNA in eukaryotic genomes should be effectively homogenized. However, the existence of intragenomic polymorphism of the ITS1-2 region in plant genomes has recently been demonstrated, which may be a consequence of hybridization within or between species. In this study, the intragenomic polymorphism of the ITS1-2 region was evaluated using Illumina amplicon sequencing in accessions of two invasive species of the genus Reynoutria, R. japonica and R. sachalinensis, from Ukraine and Romania. Hybridization between these species can lead to the emergence of more aggressive invasive forms. The ITS1-2 sequences of the species studied were found to be represented by some major and minor subclasses/variants, indicating their incomplete homogenization. The number of major variants range from two in R. japonica to six in R. sachalinensis. The ITS1-2 variants that are widespread in the genome of one species may be present at low levels in another species, indicating possible interspecies hybridization. The obtained results show that the ITS1-2 intragenomic polymorphism must be taken into account when performing barcoding, reconstructing the phylogeny of low-level taxa, and for the identification of hybrid forms.

Atrial fibrillation (AF) significantly increases stroke risk, but the risk factors and predictors of AF-stroke are rarely discovered. Hence, it is necessary to identify novel biomarker and therapeutic targets. Our study aimed to find effective pathogenic targets and elucidate the underlying molecular mechanism. First, we conducted weighted co-expression network analysis (WGCNA) in stroke-related dataset and AF-related dataset. The pink module that was highly associated with stroke (r = 0.78, p = 2 × 10–20) and the red module that was correlated with AF (r = 0.71, p = 4 × 10–5) was identified. The male-specific lethal homolog 3 (MSL3) was selected by taking the intersection between top 5 genes in stroke-related dataset and top 5 genes in AF-related dataset. Next, the expression of key gene and the receiver operating characteristic curve (ROC) analysis were also validated in other two datasets. Single sample gene set enrichment analysis (ssGSEA), single sample gene set variation analysis (ssGSVA) and correlation between genes in the key modules were exploited to investigate the function of MSL3. In addition, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and immune cells infiltration were conducted in key modules. Eventually, 10 small molecular drugs that have the potential to treat AF-stroke were filtered. In conclusion, our research find MSL3 can be as a novel biomarker and several candidate molecular drugs for treating AF-stroke.

Introduction of genes conferring resistance to Puccinia graminis is considered as the best approach to protect common wheat against stem rust. To facilitate marker-assisted selection of common wheat breeding lines with the stem rust resistance genes Sr39 and Sr40, the testing of molecular markers for these genes was carried out. The markers used for the research were the following: BE500705, Xmag2090, Xmag464, Xcnl158, Xwmc25, Sr39#50, Sr39#22, BCD260, and Xwmc344. Among the simple sequence repeat markers, only Xmag2090, Xwmc25, and Xwmc344 proved to be polymorphic upon analysis of amplicons by polyacrylamide gel electrophoresis followed by silver staining. The markers Sr39#50 and Sr39#22 produced similar amplicons in the control lines RL5711 with Sr39 and RL6089 with Sr40, while amplified fragments were absent in the cultivars. Sr39#50 and Sr39#22 were used for marker-assisted selection of F2 lines from the cross Khutorianka × RL6089 (Sr40) and F4 lines from the cross Solomiia × RL5711 (Sr39). Using Sr39#50, the Sr40 resistance marker was found in 46% of the F2 offspring of the cross Khutorianka × RL6089. Among the F4 offspring of the cross Solomiia x RL5711, the frequency of genotypes with the combination of the Sr39#50 and Sr39#22 marker amplicons was only 11%. Additionally, 33% of the F4 lines showed the Sr39#22 amplicon of approximately 800 bp but lacked the Sr39#50 resistance markers. The reduced frequency of lines with the Sr39 and Sr40 genes may be due to the decreased survival of genotypes with the 2B chromosome introgression after fall planting. The winter wheat lines with the Sr39 or Sr40 gene may be used as the initial material in breeding programs.

The aim of the study is to identify changes in genetic diversity at the BoLA-DRB3 gene exon 2 of Ukrainian Black-and-White dairy cows on the example of a breeding herd where, over the past decade, targeted selection for increasing Holstein bloodlines has been carried out to improve milk production. Changes in genetic diversity were determined by comparing the biodiversity of the experimental herd with Holstein populations. In three stages, blood samples were obtained from 391 cows of the Ukrainian Black-and-White dairy breed from the Kozatska Dolyna 2006 breeding farm, Khmelnytsky oblast, Ukraine. The allelic spectrum of the BoLA-DRB3 gene exon 2 was determined by PCR–RFLP. The study was carried out in stages: in 2008–2010 in the first population (maternal herd, n = 162), in 2014–2017 in the second population (daughter herd, n = 123), and in 2019–2022 in the third population (granddaughter herd, n = 106). For comparison, the study includes previously published data on Holsteins: United States (n = 1100), Canada (n = 835), Chile (n = 113), Argentina (n = 424), Bolivia (n = 159), Paraguay (n = 139), and Peru (n = 133). The high polymorphism of the BoLA-DRB3 exon 2 gene makes it convenient for analyzing the genetic diversity of cattle. The assessment by Simpson and Berger-Parker indices shows that the measured herd of Ukrainian Black-and-White dairy breed has more genetic diversity at the BoLA-DRB3 gene than the Holstein populations. The allelic structure of the studied herds, evaluated by the Pielu index and Buzas and Gibson evenness, is more uniform. Significant genetic diversity of the Ukrainian Black-and-White dairy breed is confirmed by the Shannon index and the effective number of alleles. Their values are significantly higher than those for all the considered Holstein cattle populations. In the time trend, a decrease in genetic diversity at the BoLA-DRB3 gene was found in Ukrainian Black-and-White dairy cows compared to Holstein cows. The linear forecast for the future period showed that the level of its genetic biodiversity will decrease at the current level of use of Holstein genetic material.

Background. BRCA1-associated protein-1 (BAP1), which is an epigenetic factor, plays an important role in regulating gene expression. BAP1 germline mutation is related to and plays different roles in a variety of human tumorigenesis. Calypso is a homologous gene of BAP1 in Drosophila melanogaster. The physiological and genetic role of Calypso in ocular development during metamorphosis was still less studied. Methods and Results. In this work, we found that the knockdown of calypso in D. melanogaster eyes causes a tumor-like malformation. We first dissected the eye imaginal discs of D. melanogaster and then conducted RNA sequencing analysis, which showed that calypso knockdown could affect the activity of the insulin signaling pathway. Both the expression of insulin signaling pathway reporter genes and the Akt phosphorylation level revealed that insulin signaling pathway had been intensively activated in the eyes of D. melanogaster by calypso knockdown. We further knocked down certain target genes in insulin signaling pathway in the eyes of D. melanogaster whose calypso had been already knocked down, and found that the akt, rheb, s6k, or upd3 knockdown could rescue tumor-like phenotype to different degrees. Among them, the eyes of the D. melanogaster with akt or rheb knockdown were restored to their normal shape. Conclusion. The results suggested that the tumor-like phenotype caused by calypso knockdown in D. melanogaster eyes can be mediated by the hyperactivation of PI3K/Akt/mTOR pathway downstream insulin signaling pathway.

Spinal muscular atrophy (SMA) is an autosomal recessive heritable disorder leading to abnormalities and dysfunction of alpha motor neurons, paralysis, and eventual death due to respiratory failure. However, the gene regulatory mechanism related to SMA is still not completely clear. Here, we constructed the gene regulatory network of SMA, in which several SMA-related genes and transcription factors played important roles. In the process, 6544 differentially expressed genes (DEGs) associated with SMA were used and the enrichment analysis and gene regulatory network construction using machine learning was performed. The result showed that, firstly, p53 signaling and DNA replication are closely related to SMA. Then, there is a huge and complicated regulatory network guided by SMA, in which transcription factor SNAPC2, MZF1, and ZNF711 interacted closely with SMA-related genes (SMN1, SMN2) and played key roles in regulating genes of p53 signaling, DNA replication and other SMA-related GO (gene ontology) terms. The transcriptome data was well verified through real-time fluorescence quantitative PCR (RT-qPCR) using the peripheral blood of spinal muscular atrophy patients. Our study revealed the complicated gene regulation network of SMA, and uncover several important SMA-related genes, which it deepens our understanding of SMA-related regulatory mechanisms.

In recent years, the introduction of genetic methods to create a more disease-resistant livestock of farm animals in breeding practice increased. The studies of the genes responsible for the immune status of the organism (particularly, mannose-binding lectin 1 (MBL1) gene) are of a special importance. The frequencies of alleles and genotypes of the mannose-binding lectin 1 (MBL1) gene in Ukrainian small and transboundary cattle breeds (Lebedyn, Brown Carpathian, Jersey, Ayrshire, and Swiss) were determined by a method of PCR-RFLP testing using the HaeIII restriction enzyme (c.2569 T>C polymorphism). The determination of T- and C-alleles of the MBL1 is of a practical importance since the mannose-binding lectin gene is a candidate for markers of resistance to mastitis. The frequencies of the MBL1 T-allele in the studied breeds vary from 0.27 to 0.47, those of TC-genotypes from 0.13 to 0.4, and those of TT-genotypes from 0.17 to 0.33. In four breeds, a shift in the genetic equilibrium due to an excess of homozygotes was detected. The values of effective alleles in the MBL1 locus in the studied breeds are less than the threshold value, which indicates that the number of effective alleles in the populations for this locus is less than possible. The cow population of the endangered Brown Carpathian breed was the most polymorphic (Na = 1.993). The largest degree of realization of genetic variability was also detected in the Brown Carpathian breed (V = 41.379) (which was grown and kept in the private sector of the mountain area), and the lowest was in the Ayrshire breed (V = 13.236), respectively. The calculation of genetic distances revealed that the Jersey and Lebedyn breeds were the closest, while a maximal genetic distance was registered between animals of the Jersey and Brown Carpathian breeds. The analysis of genetic structure of the studied cattle breeds in Ukraine revealed changes in the frequency of one or another genotype depending on the breed. The results obtained in this study contain important information, which can be used in cattle breeding for resistance to mastitis.

An Erratum to this paper has been published: https://doi.org/10.3103/S0095452724060124

The use of tetracyclines in medicine, veterinary medicine, and stock raising has led to the spread of bacterial resistance to tetracycline, particularly among dangerous representatives of Staphylococcus aureus. Therefore, the analysis of resistance to tetracycline and the creation of approaches to overcome it are extremely relevant. Studies of 64 clinical isolates of S. aureus, which were characterized by moderate biofilm formation, showed that 33 of them contained plasmid DNA. A significant spread (in 96% of studied isolates) of the known transmissible tetracycline resistance genes tet(K) and tet(M) was shown in the examined plasmid-containing doxycycline-resistant clinical isolates of S. aureus. Plasmid-containing doxycycline-resistant clinical isolates with tet(K) and tet(M) genes lost resistance to the tetracycline antibiotic doxycycline after treatment of their cells with gold nanoparticles of 30 nm in a concentration of 3.2–9.6 μg/mL or medium-sized silver of 30 nm in a concentration of 20–40 μg/mL. Elimination of tet genes responsible for acquired resistance was confirmed by PCR.

Background. Emerging research has identified ferroptosis as a novel form of programmed cell death, and Arachidonic acid 15-lipoxygenase-1 (ALOX15) stands out as a pivotal gene in mediating this process. Nonetheless, the role of ALOX15 in human tumors remains elusive. Methods. We utilized TIMER 2.0 to investigate the differential expression profiles of ALOX15 between pan-cancer and normal tissues. Further data from the TCGA, GEPIA, UALCAN, HPA, and CPTAC databases were analyzed to verify the levels of mRNA, protein expression, and promoter methylation across various cancer types. The survival prognosis, clinical features, and genetic alterations of ALOX15 were also evaluated. GO/KEGG enrichment analyses and single-cell transcriptome sequencing were employed for functional enrichment analysis. The gene mutation of ALOX15 and its prognostic value were analyzed using the cBioPortal platform. Finally, the relationship between ALOX15 and immune cell infiltration, Immune Checkpoints (ICKs), genomic instability, and drug sensitivity was further explored using GSCA. Results. Our findings revealed that the transcription and protein expression of ALOX15 were significantly reduced in HNSC, LUAD, LUSC, SKCM, KICH, and THCA, while they were up-regulated in ESCA, LIHC, PRAD, and UCEC. Notably, the expression of ALOX15 had prognostic value for certain cancers, including LUAD, LUSC, LIHC, KIRC, HNSC, THCA, and LGG. Additionally, ALOX15 expression was markedly correlated with clinical characteristics, immune cell infiltration, ICKs, genomic instability, and antitumor drug sensitivity in various tumors. Gene mutations of ALOX15 and their prognostic value were discovered in pan-cancers. Furthermore, GO/KEGG analysis and single-cell transcriptome sequencing indicated that ALOX15 was significantly associated with cancer-related pathways. Conclusion. Our comprehensive pan-cancer analysis shed light on the role and significance of ALOX15, suggesting its potential as a prognostic and immunotherapeutic marker for pan-cancer. These findings may provide new directions and evidence for cancer therapeutics.

Introduction. Chemotherapeutics are widely recognized for their adverse side-effects during anti-cancer regiments. One of the complementary approaches to circumvent this dilemma could be the exploitation of natural compounds, which could optimally counteract the cellular damages during chemotherapy. The present study ventures to evaluate the natural flavonoid, chrysin (5,7-dihydroxyflavone) for its therapeutic immunomodulatory properties along with the chemotherapeutic drug, cyclophosphamide (CP). Methods. Male Wistar albino rats were utilized for this study. Assays were conducted for acute toxicity, Hemolysis, Phagocytosis, Natural Killer (NK) cell cytotoxicity, and oxidative stress. RT-PCR, ELISA and Western Blot were performed to assess the expression of inflammatory markers. Results. Assay results such as Phagocytosis Index (0.009 ± 0.001), NK cell cytotoxicity (61.10 ± 4.99%), expression of Perforin (0.45 ± 0.05 fold) and Granzyme (0.86 ± 0.01 fold), hepatic antioxidative enzymes GSH (27.75 ± 1.54 μg/mg ), SOD (7.10 ± 0.35 U/mg ) and CAT (249.06 ± 31.30 μM/min/mg) and splenic hepatic antioxidative enzymes GSH (20.88 ± 0.74 μg/mg), SOD (7.10 ± 0.35 U/mg) and CAT (249.06 ± 31.30 μM/min/mg) among the CP-treated groups were compared with those for the CP+chrysin treated groups which were evaluated to be significantly increased with values of 0.016 ± 0.001, 82.73 ± 2.87%, 0.77 ± 0.08 fold, 1.11 ± 0.02 fold, 47.60 ± 3.02 μg/mg, 08.97 ± 0.42 U/mg, 467.19 ± 15.92 μM/Min/mg, 29.02 ± 1.59 μg/mg, 5.17 ± 0.94 U/mg, 310.29 ± 9.1330 μM/Min/mg, respectively. Histopathological examination indicated that CP+chrysin treated groups could recover from cellular damage triggered during the CP treatment. Conclusion. Results indicate the cytoprotective role of chrysin, which in turn, could be reliably administered as a complementary therapy along with CP during chemotherapy.

Tay-Sachs disease or GM2 gangliosidosis, is caused by a deficiency of beta-hexosaminidase A (HEXA), resulting in lysosomal accumulation of GM2 ganglioside. However, deficiencies or reduced activities of HEXA and HEXB result in Sandhoff disease. The patients manifest with the macular cherry-red spots due to lipid-laden ganglion cells, hypotonia, low muscle tone, intractable seizures, developmental arrest, blindness, and neurological deterioration. The aim of this study was to identify the TSD-causing variant in a large Pakistani family showing typical symptoms of Tay-Sachs disease. Here, we studied a large Pakistani family with six TSD patients for the identification of the pathogenic variant by targeted DNA sequencing. As a result, we identified a missense substitution (p.Arg499His) in exon 13 of HEXA that was completely cosegregated among affected and normal individuals. In conclusion, we identified a missense substitution (p.Arg499His) in HEXA gene in a large consanguineous Pakistani family and further enriched the mutational spectrum of HEXA through Pakistani patients for the early diagnosis of the disease.

Bletilla striata ((Thunberg) H. G. Reichenbach) a member of the Orchidaceae Juss. family is grown in greenhouse culture as an ornamental plant, and it is also valuable as a raw material for medicinal products. In this regard, the development of B. striata cultivation and propagation technologies is relevant. The aim of the work was to find out the peculiarities of the leaf stomata of B. striata plants based on the micromorphological structure of their surface during adaptation to the ex vitro conditions. The leaf epidermis structure was used as a biological marker of plant adaptation to assess the influence of in vitro and ex vitro growing conditions. Plants were propagated in vitro and transferred ex vitro to outdoors conditions (Kyiv, Ukraine). The structure of the leaf epidermis formed in vitro and ex vitro was studied using optical microscopy. We found that the leaves of B. striata are amphistomatic, but stomata rarely occur on the adaxial surface. The density of stomata on the abaxial surface is on average 70–85 pcs/mm2, their dimensions according to the guard cells are 36.16 × 29.61 μm, the stomatal pores are 22.83 × 10.89 μm. Morphometric parameters of the stomatal apparatus revealed statistically significant differences in the shape parameters of the stomata between plants cultivated in vitro and grown outdoors. A statistically significant increase of the stomata density on the abaxial leaf surfaces and a decrease in the number of epidermal cells on the adaxial surface of ex vitro grown plants were also established. So, the changes in growing conditions were reflected in the structure of the leaf epidermis. This indicated the success of plant adaptation and favorable cultivation conditions.