"CZ.02.1.01/0.0/0.0/18_046/0016045"
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Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.
BACKGROUND AND PURPOSE: The primary objective was to compare diffusion tensor imaging (DTI) scalar parameters of peripheral nerves between subjects with type 2 diabetes mellitus (T2DM) and those without diabetes. Secondarily, we aimed to correlate DTI scalar parameters with nerve morphometric properties. METHODS: Median, tibial, and sural nerves were harvested from 34 male cadavers (17 T2DM, 17 nondiabetic). Each nerve was divided into three segments. The initial segment was scanned using 9.4 Tesla MRI system (three-dimensional pulsed-gradient spin-echo sequence). DTI scalars were calculated from region-average diffusion-weighted signals. Second segment was optically cleared, acquired with optical projection tomography (OPT), and analyzed for morphometrical properties. Toluidine-stained sections were prepared from last segment, and axon- and myelin-related properties were evaluated. RESULTS: DTI scalar parameters of median and tibial nerves were comparable between the groups, while sural nerves of T2DM exhibited on average 41% higher mean diffusivity (MD) (p = 0.03), 38% higher radial diffusivity (RD) (p = 0.03), and 27% lower fractional anisotropy (FA) (p = 0.005). Significant differences in toluidine-evaluated parameters of sural nerves were observed between the groups, with a positive correlation between FA with fiber density (p = 0.0001) and with myelin proportion (p < 0.0001) and an inverse correlation between RD and myelin proportion (p = 0.003). OPT-measured morphometric properties did not correlate with DTI scalar parameters. CONCLUSIONS: High-field DTI shows promise as an imaging technique for detecting axonal and myelin-related changes in small sural nerves ex vivo. The reduced fiber density and decreased myelin content, which can be observed in T2DM, likely contribute to observed FA reduction and increased MD/RD.
- MeSH
- diabetes mellitus 2. typu * diagnostické zobrazování patologie MeSH
- diabetické neuropatie diagnostické zobrazování patologie MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mrtvola * MeSH
- nervus medianus diagnostické zobrazování patologie MeSH
- nervus suralis * diagnostické zobrazování patologie MeSH
- nervus tibialis diagnostické zobrazování patologie MeSH
- reprodukovatelnost výsledků MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- zobrazování difuzních tenzorů * metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- časopisecké články 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 buňky MeSH
- lidé MeSH
- malý jaderný ribonukleoprotein U4-U6 metabolismus genetika MeSH
- mutace * MeSH
- oční proteiny genetika metabolismus MeSH
- prekurzory RNA * metabolismus genetika MeSH
- proteiny vázající RNA metabolismus genetika MeSH
- retinální pigmentový epitel metabolismus patologie MeSH
- retinopathia pigmentosa * genetika metabolismus patologie MeSH
- ribonukleoproteiny malé jaderné metabolismus genetika MeSH
- RNA malá jaderná genetika metabolismus MeSH
- sestřih RNA * genetika MeSH
- spliceozomy metabolismus genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
This study aimed to compare the fascicular anatomy of upper limb nerves visualized using in situ high-resolution ultrasound (HRUS) with ex vivo imaging modalities, namely, magnetic resonance microscopy (MRM), histological cross-sections (HCS), and optical projection tomography (OPT). The median, ulnar, and superficial branch of radial nerve (n = 41) were visualized in 14 cadaveric upper limbs using 22-MHz HRUS. Subsequently, the nerves were excised, imaged with different microscopic techniques, and their morphometric properties were compared. HRUS accurately differentiated 51-74% of fascicles, while MRM detected 87-92% of fascicles when compared to the referential HCS. Among the compared modalities, HRUS demonstrated the smallest fascicular ratios and fascicular cross-sectional areas, but the largest nerve cross-sectional areas. The probability of a fascicle depicted on HRUS representing a cluster of multiple fascicles on the referential HCS increased with the fascicular size, with some differences observed between the larger median and ulnar nerves and the smaller radial nerves. Accordingly, HRUS fascicle differentiation necessitates cautious interpretation, as larger fascicles are more likely to represent clusters. Although HCS is considered the reference modality, alterations in nerve cross-sectional areas or roundness during sample processing should be acknowledged.
- MeSH
- horní končetina * inervace diagnostické zobrazování MeSH
- lidé středního věku MeSH
- lidé MeSH
- magnetická rezonanční tomografie metody MeSH
- mikroskopie metody MeSH
- mrtvola MeSH
- nervus medianus diagnostické zobrazování MeSH
- nervus radialis * diagnostické zobrazování anatomie a histologie MeSH
- nervus ulnaris * diagnostické zobrazování anatomie a histologie MeSH
- senioři MeSH
- ultrasonografie * metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.
Glial cells expressing neuron-glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2-expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single-cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch-clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein-4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN-positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron-like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.
- MeSH
- antigeny metabolismus MeSH
- astrocyty metabolismus MeSH
- ischemie mozku * metabolismus MeSH
- mozek metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- nervové kmenové buňky * metabolismus MeSH
- neuroglie metabolismus MeSH
- oligodendroglie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Amplification of HER2, a receptor tyrosine kinase and a breast cancer-linked oncogene, is associated with aggressive disease. HER2 protein is localised mostly at the cell membrane, but a fraction translocates to mitochondria. Whether and how mitochondrial HER2 contributes to tumorigenicity is currently unknown. METHODS: We enriched the mitochondrial (mt-)HER2 fraction in breast cancer cells using an N-terminal mitochondrial targeting sequence and analysed how this manipulation impacts bioenergetics and tumorigenic properties. The role of the tyrosine kinase activity of mt-HER2 was assessed in wild type, kinase-dead (K753M) and kinase-enhanced (V659E) mtHER2 constructs. RESULTS: We document that mt-HER2 associates with the oxidative phosphorylation system, stimulates bioenergetics and promotes larger respiratory supercomplexes. mt-HER2 enhances proliferation and invasiveness in vitro and tumour growth and metastatic potential in vivo, in a kinase activity-dependent manner. On the other hand, constitutively active mt-HER2 provokes excessive mitochondria ROS generation, sensitises to cell death, and restricts growth of primary tumours, suggesting that regulation of HER2 activity in mitochondria is required for the maximal pro-tumorigenic effect. CONCLUSIONS: mt-HER2 promotes tumorigenicity by supporting bioenergetics and optimal redox balance.
- MeSH
- buněčné dýchání fyziologie MeSH
- energetický metabolismus MeSH
- karcinogeneze metabolismus MeSH
- lidé MeSH
- mitochondrie * metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prsu * metabolismus genetika MeSH
- oxidativní fosforylace MeSH
- proliferace buněk MeSH
- reaktivní formy kyslíku metabolismus MeSH
- receptor erbB-2 * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Water removal is one of the computational bottlenecks in the processing of high-resolution MRSI data. The purpose of this work is to propose an approach to reduce the computing time required for water removal in large MRS data. METHODS: In this work, we describe a singular value decomposition-based approach that uses the partial position-time separability and the time-domain linear predictability of MRSI data to reduce the computational time required for water removal. Our approach arranges MRS signals in a Casorati matrix form, applies low-rank approximations utilizing singular value decomposition, removes residual water from the most prominent left-singular vectors, and finally reconstructs the water-free matrix using the processed left-singular vectors. RESULTS: We have demonstrated the effectiveness of our proposed algorithm for water removal using both simulated and in vivo data. The proposed algorithm encompasses a pip-installable tool ( https://pypi.org/project/CSVD/), available on GitHub ( https://github.com/amirshamaei/CSVD), empowering researchers to use it in future studies. Additionally, to further promote transparency and reproducibility, we provide comprehensive code for result replication. CONCLUSIONS: The findings of this study suggest that the proposed method is a promising alternative to existing water removal methods due to its low processing time and good performance in removing water signals.
The incidence of cutaneous malignant melanoma is increasing worldwide. While the treatment of initial stages of the disease is simple, the advanced disease frequently remains fatal despite novel therapeutic options . This requires identification of novel therapeutic targets in melanoma. Similarly to other types of tumours, the cancer microenvironment plays a prominent role and determines the biological properties of melanoma. Importantly, melanoma cell-produced exosomes represent an important tool of intercellular communication within this cancer ecosystem. We have focused on potential differences in the activity of exosomes produced by melanoma cells towards melanoma-associated fibroblasts and normal dermal fibroblasts. Cancer-associated fibroblasts were activated by the melanoma cell-produced exosomes significantly more than their normal counterparts, as assessed by increased transcription of genes for inflammation-supporting cytokines and chemokines, namely IL-6 or IL-8. We have observed that the response is dependent on the duration of the stimulus via exosomes and also on the quantity of exosomes. Our study demonstrates that melanoma-produced exosomes significantly stimulate the tumour-promoting proinflammatory activity of cancer-associated fibroblasts. This may represent a potential new target of oncologic therapy .
- MeSH
- exozómy metabolismus MeSH
- fibroblasty metabolismus patologie MeSH
- lidé MeSH
- melanom experimentální metabolismus patologie MeSH
- nádorové buňky kultivované MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.
- MeSH
- fluorescenční mikroskopie * metody MeSH
- genetická transkripce * MeSH
- lidé MeSH
- regulace genové exprese * MeSH
- RNA-polymerasa II metabolismus MeSH
- transkripční faktory metabolismus MeSH
- vazba proteinů MeSH
- zobrazení jednotlivé molekuly metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
