This study is a theoretical investigation of amides derived from hardwickiic acid (HA) as potential inhibitors of human acetyl- and butyryl-cholinesterase (hAChE and hBChE) and as drug candidates against Alzheimer's Disease (AD). Twelve compounds were prepared and geometrically optimized using GaussView 5.0.8 and the DFT method with the B3LYP/6-31G basis set to visualize molecular electrostatic potential (MEP) maps and frontier orbitals (HOMO and LUMO). In addition, pharmacokinetic and toxicological properties were studied using the online servers PreADMET and SwissADME. Molecular docking was performed against crystal structures of hAChE and hBChE prepared with the biopolymer module in SYBYL-X 2.0, previously validated. The results revealed similar profiles in surface maps and molecular orbitals for the amide substituent group. Pharmacokinetic predictions demonstrated that all 12 HA amide derivatives showed significant values for blood-brain barrier (BBB) penetration, classifying them as active in the central nervous system (CNS), a crucial pathway for AD treatment. Intermolecular interactions between the compounds and targets suggest that the benzyl amide derivative I had the highest affinity toward the hAChE binding site (-10.1 kcal/mol), while the hydroxy amide derivative L showed the highest affinity for the hBChE binding site (-9.7 kcal/mol). These findings can inform future enzymatic assays of HA amide derivatives against AChE and BChE.

• Klíčová slova
• Acetylcholinesterase, Amides, Butyrylcholinesterase, Hardwickiic acid, Pharmacokinetic and toxicological properties,
• MeSH
• acetylcholinesterasa metabolismus   chemie   MeSH
• amidy * chemie   metabolismus   farmakokinetika   farmakologie   MeSH
• butyrylcholinesterasa metabolismus   chemie   MeSH
• cholinesterasové inhibitory * chemie   metabolismus   farmakokinetika   farmakologie   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• lidé MeSH
• simulace molekulového dockingu * MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• amidy * MeSH
• butyrylcholinesterasa MeSH
• cholinesterasové inhibitory * MeSH

BACKGROUND: Recent studies have demonstrated that prolonged sperm storage adversely affects offspring through epigenetics, yet its broader effects on other molecular levels such as transcription and proteomics in progeny have been rarely explored. RESULTS: We employed comprehensive multi-omics approaches to uncover storage-induced epigenetic changes in sperm and their effects on embryonic development and offspring health. Sperm from common carp (Cyprinus carpio) was stored in vitro in artificial seminal plasma for 14 days, and the impacts of storage on functional properties of sperm and progeny development were investigated. We combined DNA methylome, transcriptomic and proteomic data to elucidate the potential mechanisms by which sperm storage influences progeny development. Prolonged in vitro storage significantly reduced sperm motility and fertilising ability which coincided with changes in the DNA methylation pattern. Integrated analyses of the offspring DNA methylome, comparative transcriptomics and cardiac performance measurements revealed storage-induced alterations of genes associated with nervous system development, myocardial morphogenesis and cellular responses to stimuli. Proteomic analyses showed that in addition to visual perception and nervous system function, pathways of the immunity system were also enriched. Results provide strong evidence of the epigenetic inheritance of the offspring's performances when short-term stored sperm was used for fertilisation. CONCLUSIONS: Short-term sperm storage induces heritable molecular and phenotypic changes in offspring, raising concerns over the potential intergenerational consequences of assisted reproductive practices in aquaculture and possibly other vertebrates.

• Klíčová slova
• Epigenetic inheritance, Epigenetics, Fish sperm, Offspring development, Sperm ageing,
• MeSH
• embryonální vývoj * MeSH
• epigeneze genetická MeSH
• kapři * genetika   fyziologie   embryologie   MeSH
• metylace DNA MeSH
• multiomika MeSH
• proteomika MeSH
• spermie * fyziologie   metabolismus   MeSH
• transkriptom MeSH
• uchování spermatu * škodlivé účinky   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BAF (SWI/SNF) chromatin remodelers engage binding partners to generate site-specific DNA accessibility. However, the basis for interaction between BAF and divergent binding partners has remained unclear. Here, we tested the hypothesis that scaffold proteins augment BAF's binding repertoire by examining β-catenin (CTNNB1) and steroidogenic factor 1 (SF-1, NR5A1), a transcription factor central to steroid production in human cells. BAF inhibition rapidly opposed SF-1/β-catenin enhancer occupancy, impairing SF-1 target activation and SF-1/β-catenin autoregulation. These effects arise due to β-catenin's role as a molecular adapter between SF-1 and an intrinsically disordered region (IDR) of the canonical BAF (cBAF) subunit ARID1A. In contrast to exclusively IDR-driven mechanisms, adapter function is mediated by direct association of ARID1A with β-catenin's folded Armadillo repeats. β-catenin similarly linked cBAF to YAP1, SOX2, FOXO3, and CBP/p300, reflecting a general IDR-mediated mechanism for modular coordination between factors. Molecular visualization highlights β-catenin's adapter role for interaction of cBAF with binding partners.

• Klíčová slova
• IDRs, adrenocortical carcinoma, chromatin remodeling, co-activators, scaffold proteins, steroid hormones, transcription factors, transcription regulators, unstructured protein,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   genetika   MeSH
• beta-katenin * metabolismus   genetika   chemie   MeSH
• DNA vazebné proteiny * metabolismus   genetika   chemie   MeSH
• fosfoproteiny metabolismus   genetika   MeSH
• HEK293 buňky MeSH
• jaderné proteiny * metabolismus   genetika   MeSH
• lidé MeSH
• protein FOXO3 metabolismus   genetika   MeSH
• signální proteiny YAP MeSH
• signální transdukce MeSH
• steroidogenní faktor 1 * metabolismus   genetika   MeSH
• transkripční faktory p300-CBP metabolismus   genetika   MeSH
• transkripční faktory * metabolismus   genetika   chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vnitřně neuspořádané proteiny * metabolismus   genetika   MeSH
• zesilovače transkripce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• ARID1A protein, human MeSH Prohlížeč
• beta-katenin * MeSH
• CTNNB1 protein, human MeSH Prohlížeč
• DNA vazebné proteiny * MeSH
• fosfoproteiny MeSH
• jaderné proteiny * MeSH
• NR5A1 protein, human MeSH Prohlížeč
• protein FOXO3 MeSH
• signální proteiny YAP MeSH
• steroidogenní faktor 1 * MeSH
• transkripční faktory p300-CBP MeSH
• transkripční faktory * MeSH
• vnitřně neuspořádané proteiny * MeSH
• YAP1 protein, human MeSH Prohlížeč

Alpha-synuclein (αSyn) is a 14-kDa intrinsically disordered protein that aggregates into insoluble fibrils in synucleinopathies, including Lewy bodies, multiple system atrophy, and Parkinson's disease, contributing to neurotoxicity and disease progression. The ability of these fibrils to seed further aggregation of native protein is central to αSyn pathology. Here, we examined the broader non-amyloid component (NAC) domain, focusing on how residues flanking the hydrophobic 68-71 (GAVV) motif of αSyn (residues 8-11 in NAC35) modulate nucleation, stability, and pathological seeding. Using full-length NAC peptide and truncated variants, we show that the 68-71 (GAVV) stretch is critical for nucleation and aggregation into prion-like fibrils. Peptide inhibitors targeting this hydrophobic region block the formation of seed-competent fibrils. Molecular dynamics simulations showed that these inhibitors alter peptide-peptide interactions and contact key hydrophobic residues within the NAC domain. Further analysis indicates that residues beyond the 68-71 (GAVV) motif, such as 79-95, are critical for stabilizing fibrils and promoting seeding competency. Peptide B interactions with key hydrophobic motifs within the NAC domain were visualized in silico, offering mechanistic insights into how it disrupts aggregation.

• Klíčová slova
• Parkinson's disease, alpha‐synuclein, amyloid fibril, hydrophobic region, non‐amyloid component, peptide inhibitor, synuclein seeding,
• Publikační typ
• časopisecké články MeSH

Electron microscopy represents a powerful visualizing technique, capable of a million times magnification. Prior to imaging, biological samples must undergo complex preparation to withstand the exposition to electrons in the vacuum inside the electron microscope. Here, we describe a preparation technique allowing preservation of scarce and delicate human oocytes for ultrastructural investigation.

• Klíčová slova
• Electron microscopy, Focused ion beam scanning electron microscopy, Human oocytes, Transmission electron microscopy, Ultrastructure,
• MeSH
• elektronová mikroskopie * metody   MeSH
• lidé MeSH
• oocyty * ultrastruktura   MeSH
• transmisní elektronová mikroskopie metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

RNA metabolism plays an essential role in the development of both oocytes and embryos. Their dependence on stored maternal transcripts is due to a long period of silenced transcription in which the oocyte undergoes meiotic maturation and fertilization. Although maternal RNAs are unusually stable, they should be replaced by "zygotic" transcripts during the transition from oocyte to zygote. Analysis of ncRNA and mRNA distribution in the oocyte can provide clues to the fate of RNA in the single-cell environment.Our work focuses on the visualization of the subcellular distribution of specific RNAs in mammalian oocytes and early embryos. The localization of many RNAs in the oocyte and embryo is still not fully understood. In this chapter, we describe an optimized protocol for RNAscope, a type of RNA fluorescence in situ hybridization (RNA FISH), which is a valuable technique for exploring mammalian oocytes and embryos. The method is based on a specific probe design strategy that enables signal amplification together with simultaneous background suppression. Additionally, it is suitable for quantitative spatio-temporal analysis of specific RNA transcripts. RNAscope, a simple and a reliable protocol, contributes to advancing our understanding of RNA biology in the context of germ cell development.

• Klíčová slova
• Embryo, Oocyte, RNA FISH, Single-molecule imaging, mRNA,
• MeSH
• embryo savčí * metabolismus   MeSH
• hybridizace in situ fluorescenční * metody   MeSH
• messenger RNA genetika   MeSH
• myši MeSH
• oocyty * metabolismus   MeSH
• RNA * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• RNA * MeSH

BACKGROUND: Football matches induce acute and residual fatigue, impairing neuromuscular, metabolic, and perceptual performance. Hydrogen-rich water (HRW) is a novel intervention with antifatigue and antioxidative properties. The intermittent high-intensity nature of football, which includes frequent accelerations, decelerations, sprints, changes of direction, and physical contacts, imposes substantial demands on both central and peripheral physiological systems. This results in acute fatigue, observable during or immediately after a match, and residual fatigue, which can persist for 24-72 hours post match, depending on the intensity, match context, and recovery strategies. OBJECTIVE: This study will investigate the effects of pre-exercise HRW administration versus a placebo on neuromuscular performance, biochemical markers, and perceptual measures of fatigue during a 72-hour recovery after a simulated football match. METHODS: Using a randomized, double-blinded, placebo-controlled, parallel design, elite junior football players will undergo neuromuscular performance assessments (repeated sprint ability and countermovement jump test). Metabolic fatigue will be measured by creatine kinase level and muscle soreness, rated using a visual analog scale. These assessments will occur at critical time points: immediately post warm-up; directly following the simulated football match to detect acute fatigue; and 24, 48, and 72 hours after training sessions to detect residual fatigue. RESULTS: Data collection has been scheduled with the clubs to coincide with the beginning of the players' transition period (ie, at the start of August 2025). The expected duration of data collection, including the initial medical examination, is planned to be 1 month. We anticipate publishing the results in late 2025 or during the first half of 2026. CONCLUSIONS: This study will assess the influence of molecular hydrogen on acute fatigue manifestation and recovery quality during a 72-hour period after a simulated football match. The potential positive effects of molecular hydrogen, such as attenuation of oxidative stress, reduction in muscle damage markers, and accelerated neuromuscular recovery, may contribute to faster restoration of functional capacities. If confirmed, these effects could enhance players' readiness to return to high-intensity training and optimize the structure of microcycles in competitive periods. Additionally, understanding the recovery dynamics facilitated by HRW may inform evidence-based recovery strategies and support individualized player monitoring frameworks. The possible positive effect of molecular hydrogen would speed up the players' readiness to train after the match and help protect players against illness and noncontact injuries.

• Klíčová slova
• antifatigue effect, molecular hydrogen, oxidative stress, readiness to play, recovery, repeated sprint ability,
• MeSH
• dvojitá slepá metoda MeSH
• fotbal * fyziologie   MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• randomizované kontrolované studie jako téma MeSH
• sportovní výkon fyziologie   MeSH
• svalová únava MeSH
• únava * MeSH
• voda * MeSH
• vodík * aplikace a dávkování   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• protokol klinické studie MeSH
• Názvy látek
• voda * MeSH
• vodík * MeSH

Ixodes ricinus ticks act as vectors for numerous pathogens that present substantial health threats. Additionally, they harbor vertically transmitted symbionts, some of which have been linked to diseases. The difficulty of isolating and cultivating these symbionts has hampered our understanding of their biological role, their potential to cause disease, and their modes of transmission. To expand our understanding of the tick symbiont Midichloria mitochondrii and Rickettsia helvetica, which has been linked to disease in humans, we utilized deep sequencing on 16 individual adult female ticks collected from coastal dune and forested areas in the Netherlands. By employing a combination of second- and third-generation sequencing techniques, we successfully reconstructed the complete genomes of M. mitochondrii from 11 individuals, R. helvetica from eight individuals, and the mitochondrial genome from all ticks. Additionally, we visualized the location of R. helvetica in tick organs and constructed genome-scale metabolic models (GEMs) of both symbionts to study their environmental dependencies. Our analysis revealed a strong cophylogeny between M. mitochondrii and mitochondrial genomes, suggesting frequent maternal transmission. In contrast, the absence of cophylogeny between R. helvetica and the mitochondrial genomes, coupled with its presence in the receptaculum seminis of I. ricinus females, raises the possibility of paternal transmission of R. helvetica. Notably, the genetic diversity of R. helvetica was found to be very low, except for the rickA virulence gene, where the presence of up to 13 insertions of a 33 nt-long repeat led to significant variability. However, this variation could not account for the differences in infection prevalence observed across eight distinct locations in the Netherlands. By employing deep sequencing, it becomes feasible to extract complete genomes and genetic data of symbionts directly from their host organisms. This methodology serves as a robust means to gain fresh insights into their interactions. Our observations, which suggest paternal transmission of R. helvetica, a relatively unexplored mode of transmission in ticks, require validation through experimental investigations. The genetic variations identified in the rickA virulence gene of R. helvetica have the potential to influence the infectivity and transmission dynamics of R. helvetica.IMPORTANCETicks are vectors of numerous human pathogens; however, the microbial interactions within ticks and the mechanisms governing pathogen transmission remain poorly understood. This study uses deep sequencing of individual Ixodes ricinus to reconstruct high-quality genomes of endosymbionts and the mitochondrion of the tick, revealing previously undetected microbial dynamics. Notably, we recovered low-abundance Rickettsia and Midichloria genomes from single ticks and present evidence that suggests paternal transmission of R. helvetica. These findings offer novel insights into the ecology and evolution of tick-associated microbes and have implications for understanding the origins and spread of tick-borne diseases.

• Klíčová slova
• Ixodes ricinus, Midichloria mitochondrii, Rickettsia helvetica, cophylogenetic analysis, deep sequencing, genome reconstruction, genome-scale metabolic modeling, paternal transmission, symbionts,
• MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• genom mitochondriální MeSH
• klíště * mikrobiologie   genetika   MeSH
• Rickettsia * genetika   fyziologie   MeSH
• symbióza * genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

PURPOSE: [18F] Poly-ADP-ribose polymerase inhibitors (PARPi), a novel radiotracer, enables visualization of PARP1 upregulation by PET imaging. Here, we aimed to quantify PARPi uptake in tumor lesions of metastatic castration-resistant PCa (mCRPC) patients and perform a comparison with prostate specific membrane antigen (PSMA) expression using PET/CT scans. METHODS: Data from 22 male patients with mCRPC, who underwent [18F]PARPi and [68Ga]Ga-PSMA-11 PET/CT scans, were retrospectively quantified. Lesions with relevant PARPi uptake (higher than background) were delineated and correlated with their [68Ga]PSMA uptake using standardized uptake values (SUV). Additionally, a comparison was performed to investigate the effects of homologous recombination deficiency (HRD) alterations on PARPi tumor uptake. RESULTS: The majority of metastatic PCa lesions that exhibited PARPi uptake were located in the bones (n = 57), with mean SUVmax values of 4.9 ± 1.5 for PARPi and 30.9 ± 28.3 for [68Ga]PSMA. Additionally, 3 local prostate lesions, 14 lymph nodes and 4 further metastatic lesions were detected. Significant correlations were identified between PARPi- and [68Ga]PSMA uptake, as measured by SUVmean (r = 0.48, p < 0.001), SUVpeak (r = 0.48, p < 0.001) and SUVmax (r = 0.43, p < 0.001) of the osseous metastatic lesions and SUVpeak (r = 0.49, p = 0.04) of extraosseous lesions. No significant differences were found between PARPi uptake of metastatic lesions in patients with or without HRD alterations (all p > 0.05). CONCLUSION: Results showed a considerable uptake of [18F]PARPi in mCRPC patients and indicated a correlation between PARPi uptake and PSMA expression, suggesting the potential of using [18F]PARPi as a diagnostic imaging tool in mCRPC patients. More studies are needed to evaluate the clinical benefit of this innovative radiotracer.

• Klíčová slova
• 18F-PARPi, PET/CT, PSMA, Prostate cancer, mCRPC,
• Publikační typ
• časopisecké články MeSH

The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)-DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4' of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key DNA repair enzyme that excises 8-oxoguanine, a mutagenic base lesion, from double-stranded DNA. In this study, we crystallographically visualized an intermediate state of the enzymatic reaction. To achieve this, we employed a specifically designed pH-sensitive mutant of hOGG1 and applied a freeze-trapping technique to capture the reaction intermediate. The resulting crystal structure revealed a previously unknown reaction pathway involving a hemiaminal-type intermediate, captured here for the first time. These findings provide new insights into the catalytic mechanism of hOGG1.

• MeSH
• DNA-glykosylasy * chemie   genetika   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• guanin analogy a deriváty   chemie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 8-hydroxyguanine MeSH Prohlížeč
• DNA-glykosylasy * MeSH
• DNA MeSH
• guanin MeSH
• oxoguanine glycosylase 1, human MeSH Prohlížeč