Miloslav Karhanek

The yeast metabolic cycle (YMC) in Saccharomyces cerevisiae provides a tractable model for examining how mitochondrial respiration, redox timing, and metabolic phase shape transcriptome abundance and fidelity. Ribosomal-RNA-depleted whole-transcriptome RNA sequencing (WRS) and RNA-seq-derived mismatch analyses were performed across low-dissolved-oxygen (Low-DO)/high-respiration and high-dissolved-oxygen (High-DO)/lower-respiration phases. Among 1505 phase-differentially expressed genes, Low DO was enriched for ribosome biogenesis, rRNA processing, translation, sulfur metabolism, and protein synthesis, whereas High DO was enriched for oxidant detoxification, oxidoreductase activity, and redox-buffering-related pathways. Generalized linear mixed models identified a substitution-class-dependent Low-DO-associated RNA-seq mismatch response. The strongest mismatch-level increase occurred in the collapsed C > T/G > A-compatible class, whereas C > A/G > T did not increase. This pattern was not consistent with a simple single-lesion model and instead supported a mixed Low-DO-associated RNA-seq sequence-discordance landscape. Variant-rate modeling additionally detected T > C/A > G and T > A/A > T increases, indicating that multiple biological and technical processes may contribute to the observed spectrum. Recurrence analysis showed that most called variants were sample-specific, supporting a transient RNA-seq mismatch landscape rather than stable DNA mutation. These findings establish the YMC as a reductionist eukaryotic framework for studying how metabolic phase and redox state shape transcriptome fidelity.

The increasing incidence of autism spectrum disorder (ASD) increases the urgency of establishing the mechanism of its development for effective prevention and treatment. ASD’s etiology includes genetic predisposition and environmental triggers, both of which can play a role in the changed microbiota. Recent research has proved the impact of maternal microbiota on the neurodevelopment of the child. To investigate the co-play of genetic and microbiota factors in ASD development, we performed fecal microbiota transplantation (FMT) from children with ASD to female Shank3b^+/− mice and studied the autism-like symptoms in the male Shank3b^−/− and wild-type (WT) offspring. WT animals with prenatal exposure to ASD microbiota had delayed neurodevelopment and impaired food intake behavior, but also elevated plasma leptin concentration and body weight. Shank3b^−/− mice after FMT ASD exhibited impaired learning and exacerbated anxiety-like behavior in adulthood. Interestingly, FMT ASD improved learning in adolescent Shank3b^−/− mice. Prenatal exposure to ASD microbiota decreased the activity of hypocretin neurons of the lateral hypothalamic area in both genotypes. The combination of genetic predisposition and FMT ASD led to an increased colon permeability, evaluated by zonula occludens (ZO1, ZO3) and claudin factors. These results suggest the effect of parental FMT exposure on shaping offspring behavior in Shank3b^−/− mice and the potential of microbiota in the modulation of ASD.

RNA–DNA differences (RDDs) challenge the traditional view of RNA as a faithful copy of DNA, arising through RNA editing, transcriptional errors, and oxidative damage. Reactive oxygen species (ROS) play a central role, inducing lesions like 8-oxo-guanine that compromise transcription and translation, leading to dysfunctional proteins. This review explores the biochemical basis of RDDs, their exacerbation under oxidative stress, and their dual roles in cellular adaptation and disease. RDDs contribute to genomic instability and are implicated in cancers, neurodegenerative disorders, and autoimmune diseases, while also driving phenotypic diversity. Drawing on terrestrial and spaceflight studies, we highlight the intersection of oxidative stress, RDD formation, and cellular dysfunction, proposing innovative mitigation approaches. Advancements in RDD detection and quantification, along with ROS management therapies, offer new avenues to restore cellular homeostasis and promote resilience. By positioning RDDs as a hallmark of genomic entropy, this review underscores the limits of biological adaptation. Furthermore, the prevalence of guanine-rich codons in antioxidant genes increases their susceptibility to ROS-induced oxidative lesions, linking redox stress, genomic instability, and constrained adaptation. These insights have profound implications for understanding aging, disease progression, and adaptive mechanisms in both terrestrial and space environments.

RNA-DNA differences (RDDs) challenge the traditional view of RNA as a faithful copy of DNA, arising through RNA editing, transcriptional errors, and oxidative damage. Reactive oxygen species (ROS) play a central role, inducing lesions like 8-oxo-guanine that compromise transcription and translation, leading to dysfunctional proteins. This review explores the biochemical basis of RDDs, their exacerbation under oxidative stress, and their dual roles in cellular adaptation and disease. RDDs contribute to genomic instability and are implicated in cancers, neurodegenerative disorders, and autoimmune diseases, while also driving phenotypic diversity. Drawing on terrestrial and spaceflight studies, we highlight the intersection of oxidative stress, RDD formation, and cellular dysfunction, proposing innovative mitigation approaches. Advancements in RDD detection and quantification, along with ROS management therapies, offer new avenues to restore cellular homeostasis and promote resilience. By positioning RDDs as a hallmark of genomic entropy, this review underscores the limits of biological adaptation. Furthermore, the prevalence of guanine-rich codons in antioxidant genes reflects a compensatory strategy against ROS-induced damage, linking redox stress, genomic instability, and constrained adaptation. These insights have profound implications for understanding aging, disease progression, and adaptive mechanisms in both terrestrial and space environments.

Aim: Mutations in the mitochondrial (mt) genome contribute to metabolic dysfunction and their accumulation relates to disease progression and resistance development in cancer cells. This study explores the mutational status of the mt genome of cisplatin-resistant vs. -sensitive testicular germ cell tumor (TGCT) cells and explores its association with their respiration parameters, expression of respiratory genes, and preferences for metabolic pathways to reveal new markers of therapy resistance in TGCTs. Methods: Using Illumina sequencing with Twist Enrichment Panel, the mutations of mt genomes of sensitive 2102EP, H12.1, NTERA-2, T-cam and resistant 2102EP Cis, H12.1 ODM, 1411HP, 1777NRpmet, NTERA-2 Cis and T-cam Cis cell lines were identified. The mt respiration of the cells was assessed using high-resolution respirometry method (O2k-respirometer Oroboros) and the differential expression profiles of mt respiratory genes were determined using RT-qPCR. Associated preferences for metabolic pathways were compared using Glycolysis/OXPHOS assay. Results: In resistant TGCT cells, new mutations in mt genes MT-ND1-6, MT-RNR, MT-CO1-3, MT-ATP6 , and MT-CYB were recognized. The respiratory rates of the 1777NRpmet cell line were the highest, while those of the 1411HP line the lowest; rates of the control and all other TGCT cell lines fell between these two lines. The statistically significant differences in gene expression of the respiratory genes were recorded only in NTERA-2 Cis and T-cam Cis cell lines. Sensitive cell lines NTERA-2 and 2102EP preferred oxidative phosphorylation (OXPHOS), while glycolysis was typical for resistant NTERA-2 Cis, 2102EP Cis and 1411HP cell lines. Metastatic 1777NRpmet cells seem to utilize both. An isogenic pair of cell lines H12.1 and H12.1ODM showed the opposite dependence, sensitive H12.1 preferring glycolysis, while resistant H12.1ODM OXPHOS. Conclusion: In summary, our study identified new mutations in mt genes of resistant TGCT cell lines that are associated with different mt respiration parameters, gene expression patterns and preferences for metabolic pathways, providing potential novel molecular biomarkers that distinguish the resistant TGCT phenotype or specify its histological classification.

Olfactory dysfunction associated with coronavirus 2 (SARS-CoV-2) infection is in most cases transient, recovering spontaneously within a few days. However, in some patients it persists for a long time, affects their everyday life and endangers their health. Hence, we focused on patients with persistent loss of smell. The aim of this study was to evaluate olfactory dysfunction using a standardized test. Due to the pandemic, olfactory testing was performed online. Smell tests (Odorized Markers Test, OMT) were sent home to the patients. Together with the smell self-testing, participants reported and assessed several parameters (age, sex, subjective assessment of smell and taste, nasal patency, etc.) in an online questionnaire. Based on the questionnaire outcomes, the results were sent to the patients along with a list of participating otolaryngologists who provided them with professional care. From March to June 2021, 1025 patients requested smell testing, of these, 824 met the inclusion criteria of this study. The duration of the olfactory dysfunction at the time of testing ranged from 1 month to 1 year. Using the OMT, impaired smell ability—anosmia or hyposmia—was confirmed in 82.6% of participants. A total of 17.4% of participants were determined to be normosmic however, more than 50% of them complained of parosmia and/or phantosmia. Our study demonstrates the relevance of psychophysical smell testing and its suitability for remote use during the pandemic. This study also revealed several correlations between prolonged olfactory dysfunction and the monitored parameters.

Cold exposure results in activation of metabolic processes required for fueling thermogenesis, potentially promoting improved metabolic health. However, the metabolic complexity underlying this process is not completely understood. We aimed to analyze changes in plasma metabolites related to acute cold exposure and their relationship to cold-acclimatization level and metabolic health in cold-acclimatized humans. Blood samples were obtained before and acutely after 10–15 min of ice-water swimming (<5 °C) from 14 ice-water swimmers. Using mass spectrometry, 973 plasma metabolites were measured. Ice-water swimming induced acute changes in 70 metabolites. Pathways related to amino acid metabolism were the most cold-affected and cold-induced changes in several amino acids correlated with cold-acclimatization level and/or metabolic health markers, including atherogenic lipid profile or insulin resistance. Metabolites correlating with cold-acclimatization level were enriched in the linoleic/α-linolenic acid metabolic pathway. N-lactoyl-tryptophan correlated with both cold-acclimatization level and cold-induced changes in thyroid and parathyroid hormones. Acute cold stress in cold-acclimatized humans induces changes in plasma metabolome that involve amino acids metabolism, while the linoleic and α-linolenic acid metabolism pathway seems to be affected by regular cold exposure. Metabolites related to metabolic health, thermogenic hormonal regulators and acclimatization level might represent prospective molecular factors important in metabolic adaptations to regular cold exposure.

The current guidelines for diagnosis, prognosis, and treatment of endometrial cancer (EC), based on clinicopathological factors, are insufficient for numerous reasons; therefore, we investigated the relevance of miRNA expression profiles for the discrimination of different EC subtypes. Among the miRNAs previously predicted to allow distinguishing of endometrioid ECs (EECs) according to different grades (G) and from serous subtypes (SECs), we verified the utility of miR-497-5p. In ECs, we observed downregulated miR-497-5p levels that were significantly decreased in SECs, clear cell carcinomas (CCCs), and carcinosarcomas (CaSas) compared to EECs, thereby distinguishing EEC from SEC and rare EC subtypes. Significantly reduced miR-497-5p expression was found in high-grade ECs (EEC G3, SEC, CaSa, and CCC) compared to low-grade carcinomas (EEC G1 and mucinous carcinoma) and ECs classified as being in advanced FIGO (International Federation of Gynecology and Obstetrics) stages, that is, with loco-regional and distant spread compared to cancers located only in the uterus. Based on immunohistochemical features, lower miR-497-5p levels were observed in hormone-receptor-negative, p53-positive, and highly Ki-67-expressing ECs. Using a machine learning method, we showed that consideration of miR-497-5p expression, in addition to the traditional clinical and histopathologic parameters, slightly improves the prediction accuracy of EC diagnosis. Our results demonstrate that changes in miR-497-5p expression influence endometrial tumorigenesis and its evaluation may contribute to more precise diagnoses.

The discrimination of different subtypes of endometrial carcinoma (EC) is frequently problematic when using the current histomorphological classification; therefore, new markers for this differentiation are needed. Here, we examined differences in miRNA expression between well- and poorly-differentiated (grades 1 and 3) endometrioid endometrial carcinoma (EEC) and between EEC and serous endometrial carcinoma (SEC). The expression of 84 tumour-suppressor miRNAs was analysed by real-time polymerase chain reactions in 62 EC and 20 non-neoplastic endometrial specimens. The potential functions of the differentially expressed miRNAs were determined by bioinformatics analyses. The expression of let-7c-5p, miR-125b-5p, miR-23b-3p, and miR-99a-5p in grade 3 EEC was decreased compared to grade 1 EEC. To discriminate between EEC and SEC, let-7g-5p, miR-195-5p, miR-34a-5p, and miR-497-5p expression was significantly downregulated in SEC. In bioinformatic analyses, miRNAs that could discriminate grade 1 from grade 3 mainly targeted genes involved in PI3K-AKT signaling, whereas miRNAs that could discriminate EEC from SEC targeted genes involved in several signaling pathways, but mainly MAPK signaling. Taken collectively, our results indicate that the activation of certain signaling pathways can be useful in the molecular characterization of EEC and SEC.

Autism spectrum disorder (ASD) is a complex of neurodevelopmental conditions with increasing incidence. The microbiota of children with ASD is distinct from neurotypical children, their food habits are also different, and it is known that nutrient intake influences microbiota in a specific way. Thus, this study investigates the food habits of children with ASD and their association with the gut microbiota. Children with ASD had their dietary energy intakes similar to controls, but they more often demonstrated food selectivity, which seemed to result in deficiency of micronutrients such as vitamins K, B6, C, iron, cooper, docosahexaenoic and docosapentanoic acid. Using high-throughput sequencing, a DNA library of intestinal microbiota was performed. Core microbiota was similar in children with and without ASD, but Dichelobacter, Nitriliruptor and Constrictibacter were found to be putative markers of ASD. The changes in gut microbiota that we observed in connection to food selectivity, intake of fats and omega-3 in particular, fermented milk products and animal/plant protein consumption had similar character, independent of diagnosis. However, high fibre intake was connected with a decreased α-diversity only in children with ASD. High carbohydrate and fibre intake influenced β-diversity, changing the abundance of Bacteroides and other genera, many of them members of the Clostidiaceae. Modulating food habits of ASD children can influence their gut microbiota composition.