Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.

• MeSH
• Alexander Disease * genetics   pathology   metabolism   MeSH
• Astrocytes * metabolism   pathology   MeSH
• Cell Differentiation * physiology   MeSH
• Glial Fibrillary Acidic Protein * metabolism   genetics   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Coculture Techniques MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mutation MeSH
• Neural Stem Cells metabolism   MeSH
• Neurons metabolism   pathology   MeSH
• Organoids metabolism   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The honeybee (Apis mellifera) is a key pollinator critical to global agriculture, facing threats from various stressors, including the ectoparasitic Varroa mite (Varroa destructor). Previous studies have identified shared bacteria between Varroa mites and honeybees, yet it remains unclear if these bacteria assemble similarly in both species. This study builds on existing knowledge by investigating co-occurrence patterns in the microbiomes of both Varroa mites and honeybees, shedding light on potential interactions. Leveraging 16S rRNA datasets, we conducted co-occurrence network analyses, explored Core Association Networks (CAN) and assess network robustness. Comparative network analyses revealed structural differences between honeybee and mite microbiomes, along with shared core features and microbial motifs. The mite network exhibited lower robustness, suggesting less resistance to taxa extension compared to honeybees. Furthermore, analyses of predicted functional profiling and taxa contribution revealed that common central pathways in the metabolic networks have different taxa contributing to Varroa mites and honeybee microbiomes. The results show that while both microbial systems exhibit functional redundancy, in which different taxa contribute to the functional stability and resilience of the ecosystem, there is evidence for niche specialization resulting in unique contributions to specific pathways in each part of this host-parasite system. The specificity of taxa contribution to key pathways offers targeted approaches to Varroa microbiome management and preserving honeybee microbiome. Our findings provide valuable insights into microbial interactions, aiding farmers and beekeepers in maintaining healthy and resilient bee colonies amid increasing Varroa mite infestations.

• MeSH
• Bacteria * classification   genetics   isolation & purification   MeSH
• Microbiota * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Varroidae * microbiology   MeSH
• Bees microbiology   parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

G-quadruplexes (G4s) are functional elements of the human genome, some of which inhibit DNA replication. We investigated replication of G4s within highly abundant microsatellite (GGGA, GGGT) and transposable element (L1 and SVA) sequences. We found that genome-wide, numerous motifs are located preferentially on the replication leading strand and the transcribed strand templates. We directly tested replicative polymerase ε and δ holoenzyme inhibition at these G4s, compared to low abundant motifs. For all G4s, DNA synthesis inhibition was higher on the G-rich than C-rich strand or control sequence. No single G4 was an absolute block for either holoenzyme; however, the inhibitory potential varied over an order of magnitude. Biophysical analyses showed the motifs form varying topologies, but replicative polymerase inhibition did not correlate with a specific G4 structure. Addition of the G4 stabilizer pyridostatin severely inhibited forward polymerase synthesis specifically on the G-rich strand, enhancing G/C strand asynchrony. Our results reveal that replicative polymerase inhibition at every G4 examined is distinct, causing complementary strand synthesis to become asynchronous, which could contribute to slowed fork elongation. Altogether, we provide critical information regarding how replicative eukaryotic holoenzymes navigate synthesis through G4s naturally occurring thousands of times in functional regions of the human genome.

• MeSH
• Aminoquinolines MeSH
• DNA Polymerase II * antagonists & inhibitors   metabolism   MeSH
• DNA Polymerase III * antagonists & inhibitors   metabolism   MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Genome, Human * MeSH
• Holoenzymes metabolism   MeSH
• Picolinic Acids pharmacology   MeSH
• Humans MeSH
• Microsatellite Repeats MeSH
• Poly-ADP-Ribose Binding Proteins MeSH
• DNA Replication * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Publikace se zaměřuje na kardiovaskulární komplikace nádorových nemocí a protinádorové léčby. Určeno odborné veřejnosti.; Klinický obor kardioonkologie se zaměřuje se na celoživotní péči o pacienty po onkologické a hematoonkologické léčbě s kardiovaskulárními komplikacemi a spojuje tak práci onkologa či hematoonkologa, kardiologa, angiologa, internisty i praktického lékaře. V péči oboru jsou i pacienti s primárními srdečními nádory. Na počátku 70. let minulého století se objevily první informace o fatálním kardiotoxickém účinku chemoterapie obsahující antracyklinové deriváty. Během dalších deseti let byly publikovány velké randomizované studie, které vyhodnotily kardiotoxicitu těchto léků jako akutní, subakutní, chronickou a pozdní závažnou komplikaci jinak velmi efektivní protinádorové terapie, zejména v hematoonkologii. V současné době snad neexistuje onkologický léčebný režim, který by nevedl k nějaké formě kardiovaskulárního poškození. Od roku 1990 je zaznamenán strmý pokles onkologické mortality s nárůstem přežívajících po terapii, ale zároveň s rizikem rozvoje kardiovaskulární toxicity. Většina kardiovaskulárních komplikací souvisí přímo s aktuálním podáním onkologického léčebného protokolu nebo k nim dochází v průběhu celé terapie. Mezi nejzávažnější projev kardiotoxicity patří poškození funkce srdce. Je častý zejména jako pozdní následek terapie řadu let po jejím ukončení u pacientů v remisi onkologického onemocnění a představuje závažný problém především u pacientů léčených v dětském a adolescentním věku. Onkologická léčba ale výrazně zvyšuje riziko dalších kardiovaskulárních onemocnění. Zájem odborníků se proto obrací do oblasti arytmií, metabolického syndromu, ischemické choroby srdeční, srdečního selhání, cévních mozkových příhod apod. Nová monografie předních odborníků oboru kardioonkologie se zaměřuje na pregraduální a postgraduální výchovu. Publikace cílí nejen na specialisty ve specializovaných kardiologických a onkologických centrech, ale především na velkou síť ambulantních lékařů, kteří musí pacienty převzít do dalšího mnohdy celoživotního sledování.

• MeSH
• Chemotherapy, Adjuvant MeSH
• Cardio-Oncology MeSH
• Cardiotoxicity MeSH
• Cardiovascular Diseases MeSH
• Neoplasms MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• kardiologie
• onkologie
• NML Publication type
• kolektivní monografie

The selection of proper reference genes and materials is critical in the design of PCR experiments, especially for differential expression studies. In this study, we propose a method to identify robust endogenous control miRNAs in the visceral adipose tissue of C57BL/6J mice with non-alcoholic fatty liver disease induced by alternating Western and control diets. This study outlines a comprehensive methodology for the analysis of microRNA endogenous controls using microfluidic cards in conjunction with miRNA profiling through small RNA sequencing and subsequent validation by quantitative PCR and the RefFinder algorithm. Criteria included were fold change, p-value, reads per million, and gene stability assessment. A set of six putative endogenous microRNAs was identified (miR-331-3p, let-7a-5p, miR-1839-5p, miR-151a-5p, let-7d-5p, and let-7c-5p). Subsequent validation and analysis using the RefFinder algorithm assessed the stability of the selected genes, and a combination of the three most stable endogenous miRNA controls (miR-331-3p, let-7a- 5p, and miR-1839-5p) exhibiting consistent expression patterns with minimal variability was set. Given the absence of universal endogenous controls, individual evaluation of normalizers for each experiment is imperative for accurate miRNA expression measurements. This approach, which combines multiple techniques and assessments, provides a reliable strategy for identifying and validating endogenous controls in miRNA studies.

• MeSH
• Algorithms MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Disease Models, Animal * MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Non-alcoholic Fatty Liver Disease * genetics   metabolism   pathology   MeSH
• Intra-Abdominal Fat * metabolism   MeSH
• Gene Expression Regulation MeSH
• Gene Expression Profiling methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.

• MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   genetics   MeSH
• Mutation * MeSH
• Eye Proteins genetics   metabolism   MeSH
• RNA Precursors * metabolism   genetics   MeSH
• RNA-Binding Proteins metabolism   genetics   MeSH
• Retinal Pigment Epithelium metabolism   pathology   MeSH
• Retinitis Pigmentosa * genetics   metabolism   pathology   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   genetics   MeSH
• RNA, Small Nuclear genetics   metabolism   MeSH
• RNA Splicing * genetics   MeSH
• Spliceosomes metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Dual reporters encoding two distinct proteins within the same mRNA have had a crucial role in identifying and characterizing unconventional mechanisms of eukaryotic translation. These mechanisms include initiation via internal ribosomal entry sites (IRESs), ribosomal frameshifting, stop codon readthrough and reinitiation. This design enables the expression of one reporter to be influenced by the specific mechanism under investigation, while the other reporter serves as an internal control. However, challenges arise when intervening test sequences are placed between these two reporters. Such sequences can inadvertently impact the expression or function of either reporter, independent of translation-related changes, potentially biasing the results. These effects may occur due to cryptic regulatory elements inducing or affecting transcription initiation, splicing, polyadenylation and antisense transcription as well as unpredictable effects of the translated test sequences on the stability and activity of the reporters. Unfortunately, these unintended effects may lead to misinterpretation of data and the publication of incorrect conclusions in the scientific literature. To address this issue and to assist the scientific community in accurately interpreting dual-reporter experiments, we have developed comprehensive guidelines. These guidelines cover experimental design, interpretation and the minimal requirements for reporting results. They are designed to aid researchers conducting these experiments as well as reviewers, editors and other investigators who seek to evaluate published data.

• MeSH
• Eukaryota genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Protein Biosynthesis genetics   MeSH
• Genes, Reporter * MeSH
• Guidelines as Topic MeSH
• Research Design standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Intracellular trafficking involves an intricate machinery of motor complexes, including the dynein complex, to shuttle cargo for autophagolysosomal degradation. Deficiency in dynein axonemal chains, as well as cytoplasmic light and intermediate chains, have been linked with ciliary dyskinesia and skeletal dysplasia. The cytoplasmic dynein 1 heavy chain protein (DYNC1H1) serves as a core complex for retrograde trafficking in neuronal axons. Dominant pathogenic variants in DYNC1H1 have been previously implicated in peripheral neuromuscular disorders (NMD) and neurodevelopmental disorders (NDD). As heavy-chain dynein is ubiquitously expressed, the apparent selectivity of heavy chain dyneinopathy for motor neuronal phenotypes remains currently unaccounted for. Here, we aimed to evaluate the full DYNC1H1-related clinical, molecular and imaging spectrum, including multisystem features and novel phenotypes presenting throughout life. We identified 47 cases from 43 families with pathogenic heterozygous variants in DYNC1H1 (aged 0-59 years) and collected phenotypic data via a comprehensive standardized survey and clinical follow-up appointments. Most patients presented with divergent and previously unrecognized neurological and multisystem features, leading to significant delays in genetic testing and establishing the correct diagnosis. Neurological phenotypes include novel autonomic features, previously rarely described behavioral disorders, movement disorders and periventricular lesions. Sensory neuropathy was identified in nine patients (median age of onset 10.6 years), of which five were only diagnosed after the second decade of life, and three had a progressive age-dependent sensory neuropathy. Novel multisystem features included primary immunodeficiency, bilateral sensorineural hearing loss, organ anomalies and skeletal manifestations, resembling the phenotypic spectrum of other dyneinopathies. We also identified an age-dependent biphasic disease course with developmental regression in the first decade and, following a period of stability, neurodegenerative progression after the second decade of life. Of note, we observed several cases in whom neurodegeneration appeared to be prompted by intercurrent systemic infections with double-stranded DNA viruses (Herpesviridae) or single-stranded RNA viruses (Ross River fever, SARS-CoV-2). Moreover, the disease course appeared to be exacerbated by viral infections regardless of age and/or severity of neurodevelopmental disorder manifestations, indicating a role of dynein in anti-viral immunity and neuronal health. In summary, our findings expand the clinical, imaging and molecular spectrum of pathogenic DYNC1H1 variants beyond motor neuropathy disorders and suggest a life-long continuum and age-related progression due to deficient intracellular trafficking. This study will facilitate early diagnosis and improve counselling and health surveillance of affected patients.

• MeSH
• Cytoplasmic Dyneins * genetics   MeSH
• Child MeSH
• Adult MeSH
• Phenotype MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Neurodevelopmental Disorders genetics   MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Telomeres, essential for maintaining genomic stability, are typically preserved through the action of telomerase, a ribonucleoprotein complex that synthesizes telomeric DNA. One of its two core components, telomerase RNA (TR), serves as the template for this synthesis, and its evolution across different species is both complex and diverse. This review discusses recent advancements in understanding TR evolution, with a focus on plants (Viridiplantae). Utilizing novel bioinformatic tools and accumulating genomic and transcriptomic data, combined with corresponding experimental validation, researchers have begun to unravel the intricate pathways of TR evolution and telomere maintenance mechanisms. Contrary to previous beliefs, a monophyletic origin of TR has been demonstrated first in land plants and subsequently across the broader phylogenetic megagroup Diaphoretickes. Conversely, the discovery of plant-type TRs in insects challenges assumptions about the monophyletic origin of TRs in animals, suggesting evolutionary innovations coinciding with arthropod divergence. The review also highlights key challenges in TR identification and provides examples of how these have been addressed. Overall, this work underscores the importance of expanding beyond model organisms to comprehend the full complexity of telomerase evolution, with potential applications in agriculture and biotechnology.

• MeSH
• Phylogeny MeSH
• Evolution, Molecular * MeSH
• RNA * genetics   metabolism   MeSH
• Plants genetics   MeSH
• Telomerase * genetics   metabolism   MeSH
• Telomere * metabolism   genetics   MeSH
• Viridiplantae genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.

• MeSH
• RNA, Bacterial * metabolism   genetics   chemistry   MeSH
• Nitrogen metabolism   MeSH
• Escherichia coli * genetics   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions * MeSH
• Conserved Sequence MeSH
• RNA, Small Untranslated * metabolism   genetics   chemistry   MeSH
• Mutation MeSH
• Host Factor 1 Protein * metabolism   genetics   chemistry   MeSH
• Escherichia coli Proteins * metabolism   genetics   chemistry   MeSH
• Gene Expression Regulation, Bacterial MeSH
• RNA Stability * genetics   MeSH
• Gene Expression Profiling MeSH
• Transcriptome genetics   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH