Biology subjects

Sterba, J.

Publications and source records attributed to Sterba, J..

7 recordsLinked to original sources

bioRxiv2026

Improved Sperm Quality and Reduced Cryo-Induced Epigenetic Changes in Sterlet via Density-Gradient Sorting

BackgroundCryopreservation is a valuable tool in aquaculture and conservation programs, yet it exposes spermatozoa to physiological and molecular stresses that may impair motility, fertilisation capacity, and genomic stability. In fishes, where sperm motility is brief and easily activated, post-thaw separation of high-quality sperm remains technically challenging and poorly understood. This study evaluated whether density-gradient centrifugation can isolate a functionally superior subpopulation of sterlet (Acipenser ruthenus) spermatozoa with enhanced motility, fertilising ability, and reduced cryopreservation-induced epigenetic alterations. We further examined whether the use of this selected fraction influences DNA methylation patterns in resulting embryos. ResultsCryopreservation substantially reduced the proportion of motile spermatozoa, while density-gradient centrifugation consistently enriched motile cells both before and after freezing. Motility enhancement reflected a higher proportion of cells capable of activation rather than changes in kinematic behaviour. Fertilisation trials confirmed that the selected post-thaw fraction produced fewer malformed embryos compared with unselected cryopreserved sperm. Cryopreservation and post-thaw selection induced small but significant methylation changes in sperm, predominantly in intergenic regions and promoter-proximal elements. However, these epigenetic differences were not maintained in embryos. Embryo methylomes showed minimal variation between treatments, no distinct clustering by sperm origin, and negligible numbers of differentially methylated regions. Thus, although cryopreservation and sperm selection influenced sperm DNA methylation, these alterations did not translate into measurable changes in embryo methylation patterns. ConclusionsDensity-gradient centrifugation effectively isolates a motile, functionally improved sterlet sperm fraction after cryopreservation, enhancing fertilisation outcomes and reducing developmental abnormalities. Cryopreservation and sperm selection introduce detectable but limited methylation variation in spermatozoa; however, these changes are not inherited by embryos. The findings highlight the utility of post-thaw sperm selection in aquaculture practice and indicate that cryopreservation-associated epigenetic variation in sterlet sperm does not propagate to early developmental stages.

physiology↗

bioRxiv2026

A newly identified three-domain C-type lectin associated with blood feeding in the tick Ixodes ricinus

Ixodes ricinus ticks are widely distributed throughout Europe and represent major vectors of tick-borne encephalitis virus and the Lyme borreliosis agent Borrelia burgdorferi sensu lato. In invertebrates, C-type lectins are commonly associated with innate immune functions, and several such lectins have been predicted in I. ricinus. Given the limited knowledge of lectin function in ticks, we characterized three carbohydrate-recognition domains (CRDs) of a novel C-type lectin identified in the I. ricinus transcriptome (IrCLec). The tertiary structures of CRD1, CRD2, and CRD3, predicted using the AlphaFold 3 program, corresponded to the typical structure of C-type lectins. Conserved carbohydrate-binding motifs were identified in CRD3, whereas non-canonical motifs were present in CRD1 and CRD2. Recombinant His-tagged CRDs were produced and analysed for carbohydrate-binding activity. Glycan array analysis revealed binding of all three domains to selected glycans, while hemagglutination assays demonstrated pronounced binding activity of CRD1 and CRD2 toward human erythrocyte antigens of blood groups A, B, and O. IrCLec expression was highest in the tick midgut and also detected in hemocytes, with expression levels increasing after blood feeding. RNAi-mediated silencing of IrCLec impaired blood feeding efficiency in tick nymphs. Together, these results indicate that IrCLec plays an important role in blood feeding and may additionally participate in lectin-mediated host-pathogen or host-blood component interactions.

biochemistry↗

bioRxiv2024

Analysis of Mutations in Precision Oncology using The Automated, Accurate, and User-Friendly Web Tool PredictONCO

Next-generation sequencing technology has created many new opportunities for clinical diagnostics, but it faces the challenge of functional annotation of identified mutations. Various algorithms have been developed to predict the impact of missense variants that influence oncogenic drivers. However, computational pipelines that handle biological data must integrate multiple software tools, which can add complexity and hinder non-specialist users from accessing the pipeline. Here, we have developed an online user-friendly web server tool PredictONCO that is fully automated and has a low barrier to access. The tool models the structure of the mutant protein in the first step. Next, it calculates the protein stability change, pocket level information, evolutionary conservation, and changes in ionisation of catalytic amino acid residues, and uses them as the features in the machine-learning predictor. The XGBoost-based predictor was validated on an independent subset of held-out data, demonstrating areas under the receiver operating characteristic curve (ROC) of 0.95 and 0.94, and the average precision from the precision-recall curve 0.98 and 0.94 for structure-based and sequence-based predictions, respectively. Finally, PredictONCO calculates the docking results of small molecules approved by regulatory authorities. We demonstrate the applicability of the tool by presenting its usage for variants in two cancer-associated proteins, cellular tumour antigen p53 and fibroblast growth factor receptor FGFR1. Our free web tool will assist with the interpretation of data from next-generation sequencing and navigate treatment strategies in clinical oncology: https://loschmidt.chemi.muni.cz/predictonco/.

bioinformatics↗

bioRxiv2024

A Computational Workflow for Analysis of Missense Mutations in Precision Oncology

Every year, more than 19 million cancer cases are diagnosed, and this number continues to increase annually. Since standard treatment options have varying success rates for different types of cancer, understanding the biology of an individuals tumour becomes crucial, especially for cases that are difficult to treat. Personalised high-throughput profiling, using next-generation sequencing, allows for a comprehensive examination of biopsy specimens. Furthermore, the widespread use of this technology has generated a wealth of information on cancer-specific gene alterations. However, there exists a significant gap between identified alterations and their proven impact on protein function. Here, we present a bioinformatics pipeline that enables fast analysis of a missense mutations effect on stability and function in known oncogenic proteins. This pipeline is coupled with a predictor that summarises the outputs of different tools used throughout the pipeline, providing a single probability score, achieving the balanced accuracy above 86%. The pipeline incorporates a virtual screening method to suggest potential FDA/EMA-approved drugs to be considered for treatment. We showcase three case studies to demonstrate the timely utility of this pipeline. To facilitate access and analysis of cancer-related mutations, we have packaged the pipeline as a web server, which is freely available at https://loschmidt.chemi.muni.cz/predictonco/.

cancer biology↗

bioRxiv2024

Fertilization by short-term stored sperm alters DNA methylation patterns in common carp (Cyprinus carpio) embryos at single-base resolution

Short-term storage of sperm in vitro is widely used for artificial fertilization in aquaculture. It has been shown that short-term storage affects sperm motility characteristics, resulting in diminished fertility. However, the detrimental effects of short-term sperm storage on embryos development have remained largely unexplored in single-base methylome resolution. The main aim of the present study was to investigate DNA methylation in the offspring of common carp (Cyprinus carpio) derived from short-term stored sperm. Sperm were stored in artificial seminal plasma on ice (0-2{degrees}C) for 0, 3 and 6 days in vitro, fertilization was performed using oocytes from a single female, and embryos were collected at the mid-blastula stage. Sperm and embryo DNA was extracted, analysed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) and whole genome bisulfite sequencing (WGBS). DNA methylation was then assessed. Sperm storage showed negative effects on motility, viability and DNA integrity, but had no effect on global DNA methylation of spermatozoa and resulting embryos. Results from the WGBS showed that methylation of 3313 genes was affected in the embryos fertilized with the 6-day-stored sperm, and the differentially methylated regions (DMRs) identified were mainly involved in cell adhesion, calcium, mitogen-activated protein kinase and adrenergic signalling, melanogenesis, metabolism and RNA transport. Such results suggest that prolongation of storage time may have certain impacts on embryonic development. These initial results provide valuable information for future consideration of the DNA methylome in embryos generated from short-term stored sperm, which is widely used for genetic management of broodstock in aquaculture.

molecular biology↗

bioRxiv2024

Dysregulation of the p53 pathway provides a therapeutic target in aggressive pediatric sarcomas with stem-like traits

Pediatric sarcomas are bone and soft tissue tumors that often exhibit high metastatic potential and refractory stem-like phenotypes, resulting in poor outcomes. Aggressive sarcomas frequently harbor a disrupted p53 pathway. However, whether sarcoma stemness is associated with abrogated p53 function and might be attenuated via p53 reactivation remains unclear. Here, we show that highly tumorigenic stem-like sarcoma cells exhibit dysregulated p53, making them vulnerable to drugs that restore wild-type p53 activity. Immunohistochemistry of mouse xenografts and human tumor tissues revealed that p53 dysregulations together with enhanced expression of the stemness-related transcription factors SOX2 or KLF4 are crucial features in pediatric osteosarcoma, rhabdomyosarcoma, and Ewings sarcoma development. p53 dysregulation appears to be an important step for sarcoma cells to acquire a fully stem-like phenotype, and p53-positive pediatric sarcomas exhibit a high frequency of early metastasis. Importantly, p53 signaling reactivation via MDM2/MDMX inhibition selectively induces apoptosis in aggressive stem-like Ewings sarcoma cells while sparing healthy fibroblasts. Collectively, our results suggest that restoration of canonical p53 activity provides a promising strategy for improving the treatment of pediatric sarcomas with unfavorable stem-like traits. HIGHLIGHTSO_LISOX2 and KLF4 are crucial factors in pediatric sarcoma tumorigenesis C_LIO_LIDysregulated p53 pathway predisposes sarcoma cells to acquire stem-like features C_LIO_LIp53 positivity is associated with early metastasis in pediatric sarcoma patients C_LIO_LIRestoring wild-type p53 signaling selectively kills stem-like Ewings sarcoma cells C_LI

cancer biology↗

bioRxiv2021

Shotgun proteomic profiling of dormant, 'non-culturable' Mycobacterium tuberculosis

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by non-culturability - a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, non-culturable M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions. This approach revealed the accumulation of a significant number of proteins (1379) in cells after 4 months of storage in dormancy; among them, 468 proteins were significantly different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system phoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages, especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins with antigenic properties makes it possible to distinguish immunoreactive proteins (40) among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

microbiology↗