Expansins are cell wall-modifying proteins implicated in plant growth and stress responses. In this study, we explored the differential localization of expansins in Arabidopsis thaliana shoots, with a focus on EXPA1, EXPA10, EXPA14, and EXPA15 utilizing pEXPA::EXPA translational fusion lines. Employing the chemically inducible system pOp6/LhGR for EXPA1 overexpression and high-throughput automatic phenotyping we evaluated the drought response and photosynthetic efficiency under stress conditions. We observed distinct expression patterns of expansins, with EXPA1 primarily localized in stomatal guard cells, while EXPA10 and EXPA15 showed strong cell wall (CW) localization in epidermal and other tissues. Overexpression of EXPA1 resulted in pronounced changes in CW-related gene expression, particularly during early stages of induction, including the upregulation of other expansins and CW-modifying enzymes. The induced EXPA1 line also displayed significant morphological changes in shoots, including smaller plant size, delayed senescence, and structural alterations in vascular tissues. Additionally, EXPA1 overexpression conferred drought tolerance, as evidenced by enhanced photosynthetic efficiency (Fv/FM), and low steady-state non-photochemical quenching (NPQ) values under drought stress. These findings highlight the critical role of EXPA1 in regulating plant growth, development, and stress response, with potential applications in improving drought tolerance in crops.

• Klíčová slova
• Arabidopsis, EXPA, abiotic stress, cell wall, highthroughput phenotyping,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Myelodysplastic neoplasms (MDS) are heterogeneous hematopoietic disorders characterized by ineffective hematopoiesis and genome instability. Mobilization of transposable elements (TEs) is an important source of genome instability leading to oncogenesis, whereas small PIWI-interacting RNAs (piRNAs) act as cellular suppressors of TEs. However, the roles of TEs and piRNAs in MDS remain unclear. METHODS: In this study, we examined TE and piRNA expression through parallel RNA and small RNA sequencing of CD34+ hematopoietic stem cells from MDS patients. RESULTS: Comparative analysis of TE and piRNA expression between MDS and control samples revealed several significantly dysregulated molecules. However, significant differences were observed between lower-risk MDS (LR-MDS) and higher-risk MDS (HR-MDS) samples. In HR-MDS, we found an inverse correlation between decreased TE levels and increased piRNA expression and these TE and piRNA levels were significantly associated with patient outcomes. Importantly, the upregulation of PIWIL2, which encodes a key factor in the piRNA pathway, independently predicted poor prognosis in MDS patients, underscoring its potential as a valuable disease marker. Furthermore, pathway analysis of RNA sequencing data revealed that dysregulation of the TE‒piRNA axis is linked to the suppression of processes related to energy metabolism, the cell cycle, and the immune response, suggesting that these disruptions significantly affect cellular activity. CONCLUSIONS: Our findings demonstrate the parallel dysregulation of TEs and piRNAs in HR-MDS patients, highlighting their potential role in MDS progression and indicating that the PIWIL2 level is a promising molecular marker for prognosis.

• Klíčová slova
• Bioinformatics, Biomarkers, Myelodysplastic neoplasms, Next-generation sequencing, Transposable elements, piRNA,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Changes in DNA methylation are common events in the pathogenesis of acute myeloid leukemia (AML) and have been repeatedly reported as associated with prognosis. However, studies integrating these numerous and potentially prognostically relevant DNA methylation changes are lacking. Therefore, we aimed for an overall evaluation of these epigenetic aberrations to provide a comprehensive NGS-based approach of DNA methylation assessment for AML prognostication. RESULTS: We designed a sequencing panel targeting 239 regions (approx. 573 kb of total size) described in the literature as having a prognostic impact or being associated with AML pathogenesis. Diagnostic whole-blood DNA samples of adult AML patients divided into a training (n = 128) and a testing cohort (n = 50) were examined. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Altogether, 1935 CpGs affecting the survival (p < 0.05) were revealed in the training cohort. A summarizing value MethScore was then calculated from these significant CpGs. Patients with lower MethScore had markedly longer overall survival (OS) and event-free survival (EFS) than those with higher MethScore (p < 0.001). The predictive ability of MethScore was verified on the independent testing cohort for OS (p = 0.01). Moreover, the proof-of-principle validation was performed using the TCGA dataset. CONCLUSIONS: We showed that comprehensive NGS-based approach of DNA methylation assessment revealed a robust epigenetic signature relevant to AML outcome. We called this signature MethScore and showed it might serve as a strong prognostic marker able to refine survival probability of AML patients.

• Klíčová slova
• Acute myeloid leukemia, DNA methylation, NGS, Prognosis,
• MeSH
• akutní myeloidní leukemie * diagnóza   genetika   MeSH
• doba přežití bez progrese choroby MeSH
• dospělí MeSH
• epigenomika MeSH
• lidé MeSH
• metylace DNA * MeSH
• prognóza MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo.

• Klíčová slova
• brain, heterochromatin, immunofluorescence staining, liver, proteomics, quantitative mass spectrometry,
• MeSH
• heterochromatin * MeSH
• membránové proteiny MeSH
• mozek MeSH
• myši MeSH
• proteom * MeSH
• proteomika MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• heterochromatin * MeSH
• membránové proteiny MeSH
• proteom * MeSH

Patients with lower-risk myelodysplastic syndromes (LR-MDS) have a generally favorable prognosis; however, a small proportion of cases progress rapidly. This study aimed to define molecular biomarkers predictive of LR-MDS progression and to uncover cellular pathways contributing to malignant transformation. The mutational landscape was analyzed in 214 LR-MDS patients, and at least one mutation was detected in 137 patients (64%). Mutated RUNX1 was identified as the main molecular predictor of rapid progression by statistics and machine learning. To study the effect of mutated RUNX1 on pathway regulation, the expression profiles of CD34 + cells from LR-MDS patients with RUNX1 mutations were compared to those from patients without RUNX1 mutations. The data suggest that RUNX1-unmutated LR-MDS cells are protected by DNA damage response (DDR) mechanisms and cellular senescence as an antitumor cellular barrier, while RUNX1 mutations may be one of the triggers of malignant transformation. Dysregulated DDR and cellular senescence were also observed at the functional level by detecting γH2AX expression and β-galactosidase activity. Notably, the expression profiles of RUNX1-mutated LR-MDS resembled those of higher-risk MDS at diagnosis. This study demonstrates that incorporating molecular data improves LR-MDS risk stratification and that mutated RUNX1 is associated with a suppressed defense against LR-MDS progression.

• MeSH
• akutní myeloidní leukemie * genetika   MeSH
• lidé MeSH
• mutace MeSH
• myelodysplastické syndromy * patologie   MeSH
• nádorová transformace buněk genetika   metabolismus   MeSH
• prognóza MeSH
• protein PEBP2A2 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein PEBP2A2 MeSH
• RUNX1 protein, human MeSH Prohlížeč

The neuroprotective E3-ubiquitin ligase CHIP is linked to healthy aging. Here, we present a protocol using a patient-derived iPSC line with a triplication of the α-synuclein gene to produce gene-edited cells isogenic for CHIP. We describe iPSC differentiation into cortical neurons and their identity validation. We then detail mass spectrometry-based approaches (SWATH-MS) to identify dominant changes in the steady state proteome generated by loss of CHIP function. This protocol can be adapted to other proteins that impact proteostasis in neurons. For complete details on the use and execution of this protocol, please refer to Dias et al. (2021).

• Klíčová slova
• CRISPR, Cell Biology, Cell Differentiation, Cell culture, Mass Spectrometry, Neuroscience, Proteomics, Stem Cells,
• MeSH
• hmotnostní spektrometrie MeSH
• indukované pluripotentní kmenové buňky * MeSH
• lidé MeSH
• neurony MeSH
• proteom genetika   MeSH
• proteomika metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH

CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular "health". Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP.

• Klíčová slova
• bioinformatics, cell biology, omics, organizational aspects of cell biology, proteomics,
• Publikační typ
• časopisecké články MeSH

Studying the long-term impact of continuous-flow left ventricular assist device (CF-LVAD) offers an opportunity for a complex understanding of the pathophysiology of vascular changes in aortic tissue in response to a nonphysiological blood flow pattern. Our study aimed to analyze aortic mRNA/miRNA expression changes in response to long-term LVAD support. Paired aortic samples obtained at the time of LVAD implantation and at the time of heart transplantation were examined for mRNA/miRNA profiling. The number of differentially expressed genes (Pcorr < 0.05) shared between samples before and after LVAD support was 277. The whole miRNome profile revealed 69 differentially expressed miRNAs (Pcorr < 0.05). Gene ontology (GO) analysis identified that LVAD predominantly influenced genes involved in the extracellular matrix and collagen fibril organization. Integrated mRNA/miRNA analysis revealed that potential targets of miRNAs dysregulated in explanted samples are mainly involved in GO biological process terms related to dendritic spine organization, neuron projection organization, and cell junction assembly and organization. We found differentially expressed genes participating in vascular tissue engineering as a consequence of LVAD duration. Changes in aortic miRNA levels demonstrated an effect on molecular processes involved in angiogenesis.

• Klíčová slova
• aorta, left ventricular assist device, mRNA, mechanical circulatory support, microRNA,
• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• longitudinální studie MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• onemocnění aortální chlopně etiologie   metabolismus   patologie   MeSH
• podpůrné srdeční systémy škodlivé účinky   MeSH
• regulace genové exprese * MeSH
• senioři MeSH
• srdeční selhání patologie   chirurgie   MeSH
• stanovení celkové genové exprese MeSH
• transplantace srdce škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• mikro RNA MeSH

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

• MeSH
• blastocysta fyziologie   MeSH
• buněčná diferenciace MeSH
• buněčný rodokmen MeSH
• DNA vazebné proteiny fyziologie   MeSH
• embryonální vývoj MeSH
• endoderm cytologie   MeSH
• mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory   fyziologie   MeSH
• myši MeSH
• proteiny vázající RNA fyziologie   MeSH
• proteosyntéza * MeSH
• TOR serin-threoninkinasy fyziologie   MeSH
• transkripční faktory fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• mTOR protein, mouse MeSH Prohlížeč
• Mybbp1a protein, mouse MeSH Prohlížeč
• proteiny vázající RNA MeSH
• TOR serin-threoninkinasy MeSH
• transkripční faktory MeSH

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

• Klíčová slova
• aging, brain barrier, cerebrospinal fluid, choroid plexus, development, single-cell RNA sequencing, single-nucleus RNA sequencing,
• MeSH
• analýza jednotlivých buněk MeSH
• buněčná diferenciace genetika   MeSH
• buněčný rodokmen genetika   MeSH
• epitelové buňky metabolismus   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• mozek metabolismus   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši embryologie   MeSH
• nemoci mozku genetika   patofyziologie   MeSH
• plexus chorioideus embryologie   metabolismus   fyziologie   MeSH
• signální transdukce MeSH
• stárnutí fyziologie   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši embryologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH