The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.
- Klíčová slova
- C. elegans, Freeze substitution, High-pressure freezing, Immunogold, Plant, Thin section transmission electron microscopy, Yeast,
- MeSH
- jaderný pór MeSH
- mrazová substituce * metody MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- savci MeSH
- sušené kvasnice * MeSH
- transmisní elektronová mikroskopie MeSH
- zmrazování MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nuclear pore complex (NPC) facilitates the trafficking of proteins and RNA between the nucleus and cytoplasm. The role of nucleoporins (Nups) in transport in the context of the NPC is well established, yet their function in tRNA export has not been fully explored. We selected several nucleoporins from different parts of the NPC to investigate their potential role in tRNA trafficking in Trypanosoma brucei. We show that while all of the nucleoporins studied are essential for cell viability, only TbNup62 and TbNup53a function in tRNA export. In contrast to homologs in yeast TbNup144 and TbNup158, which are part of the inner and outer ring of the NPC, have no role in nuclear tRNA trafficking. Instead, TbNup144 plays a critical role in nuclear division, highlighting the role of nucleoporins beyond nucleocytoplasmic transport. These results suggest that the location of nucleoporins within the NPC is crucial to maintaining various cellular processes.
- Klíčová slova
- FG-Nups, NPC, Trypanosoma brucei, nuclear division, nucleoporins, tRNA trafficking,
- MeSH
- aktivní transport - buněčné jádro MeSH
- buněčné jádro metabolismus MeSH
- jaderný pór * genetika metabolismus MeSH
- komplex proteinů jaderného póru * genetika MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- komplex proteinů jaderného póru * MeSH
Niemann-Pick Type C (NPC) is a progressive and life limiting autosomal recessive disorder caused by mutations in either the NPC1 or NPC2 gene. Mutations in these genes are associated with abnormal endosomal-lysosomal trafficking, resulting in the accumulation of multiple tissue specific lipids in the lysosomes. The clinical spectrum of NPC disease ranges from a neonatal rapidly progressive fatal disorder to an adult-onset chronic neurodegenerative disease. The age of onset of the first (beyond 3 months of life) neurological symptom may predict the severity of the disease and determines life expectancy.NPC has an estimated incidence of ~ 1: 100,000 and the rarity of the disease translate into misdiagnosis, delayed diagnosis and barriers to good care. For these reasons, we have developed clinical guidelines that define standard of care for NPC patients, foster shared care arrangements between expert centres and family physicians, and empower patients. The information contained in these guidelines was obtained through a systematic review of the literature and the experiences of the authors in their care of patients with NPC. We adopted the Appraisal of Guidelines for Research & Evaluation (AGREE II) system as method of choice for the guideline development process. We made a series of conclusive statements and scored them according to level of evidence, strengths of recommendations and expert opinions. These guidelines can inform care providers, care funders, patients and their carers of best practice of care for patients with NPC. In addition, these guidelines have identified gaps in the knowledge that must be filled by future research. It is anticipated that the implementation of these guidelines will lead to a step change in the quality of care for patients with NPC irrespective of their geographical location.
- Klíčová slova
- Diagnosis, Guidelines, Management, NPC, Niemann-Pick Type C,
- MeSH
- lidé MeSH
- Niemannova-Pickova nemoc typu C terapie MeSH
- směrnice pro lékařskou praxi jako téma * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
This paper presents a novel, state-of-the-art predictive control architecture that addresses the computational complexity and limitations of conventional predictive control methodologies while enhancing the performance efficacy of predictive control techniques applied to three-level voltage source converters (NPC inverters). This framework's main goal is to decrease the number of filtered voltage lifespan vectors in each sector, which will increase the overall efficiency of the control system and allow for common mode voltage reduction in three-level voltage source converters. Two particular tactics are described in order to accomplish this. First, a statistical approach is presented for the proactive detection of potential voltage vectors, with an emphasis on selecting and including the vectors that are most frequently used. This method lowers the computational load by limiting the search space needed to find the best voltage vectors. Then, using statistical analysis, a plan is presented to split the sectors into two separate parts, so greatly limiting the number of voltage vectors. The goal of this improved predictive control methodology is to reduce computing demands and mitigate common mode voltage. The suggested strategy's resilience is confirmed in a range of operational scenarios using simulations and empirical evaluation. The findings indicate a pronounced enhancement in computational efficiency and a notable diminution in common mode voltage, thereby underscoring the efficacy of the proposed methodology. This increases their ability to incorporate renewable energy sources into the electrical grid.
Congenital disorders of glycosylation (CDG) and Niemann-Pick type C (NPC) disease are inborn errors of metabolism that can both present with infantile-onset severe liver disease and other multisystemic manifestations. Plasma bile acid and N-palmitoyl-O-phosphocholineserine (PPCS) are screening biomarkers with proposed improved sensitivity and specificity for NPC. We report an infant with ATP6AP1-CDG who presented with cholestatic liver failure and elevated plasma oxysterols and bile acid, mimicking NPC clinically and biochemically. On further investigation, PPCS, but not the bile acid derivative N-(3β,5α,6β-trihydroxy-cholan-24-oyl) glycine (TCG), were elevated in plasma samples from individuals with ATP6AP1-, ALG1-, ALG8-, and PMM2-CDG. These findings highlight the importance of keeping CDG within the diagnostic differential when evaluating children with early onset severe liver disease and elevated bile acid or PPCS to prevent delayed diagnosis and treatment.
- Klíčová slova
- ATP6AP1, N-palmitoyl-O-phosphocholineserine (PPCS), Niemann-pick type C (NPC), bile acids, congenital disorders of glycosylation (CDG), oxysterols,
- MeSH
- dítě MeSH
- glykosylace MeSH
- hydrolasy MeSH
- kojenec MeSH
- lidé MeSH
- Niemannova-Pickova nemoc typu C * MeSH
- oxysteroly * MeSH
- vakuolární protonové ATPasy * MeSH
- vrozené poruchy glykosylace * MeSH
- žlučové kyseliny a soli MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- ATP6AP1 protein, human MeSH Prohlížeč
- hydrolasy MeSH
- oxysteroly * MeSH
- vakuolární protonové ATPasy * MeSH
- žlučové kyseliny a soli MeSH
The first indication of the aluminum (Al) toxicity in plants growing in acidic soils is the cessation of root growth, but the detailed mechanism of Al effect is unknown. Here we examined the impact of Al stress on the activity of non-specific phospholipase C (NPC) in the connection with the processes related to the plasma membrane using fluorescently labeled phosphatidylcholine. We observed a rapid and significant decrease of labeled diacylglycerol (DAG), product of NPC activity, in Arabidopsis seedlings treated with AlCl₃. Interestingly, an application of the membrane fluidizer, benzyl alcohol, restored the level of DAG during Al treatment. Our observations suggest that the activity of NPC is affected by Al-induced changes in plasma membrane physical properties.
- Klíčová slova
- Arabidopsis thaliana, BA, benzyl alcohol, BODIPY, BODIPY, 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene, BY-2, Bright Yellow 2, DAG, diacylglycerol, HP-TLC, high-performance thin-layer chromatography, MS, Murashige-Skoog, NPC, non-specific phospholipase C, PA, phosphatidic acid, PC, phosphatidylcholine, PC-PLC, phosphatidylcholine-specific phospholipase C, PI-PLC, phosphatidylinositol-specific phospholipase C, PIP2, phosphatidylinositol 4, 5-bisphosphate, PLD, phospholipase D, PM, plasma membrane., aluminum toxicity, benzyl alcohol, diacylglycerol, membrane fluidity, non-specific phospholipase C,
- MeSH
- Arabidopsis účinky léků enzymologie MeSH
- benzylalkohol farmakologie MeSH
- buněčná membrána účinky léků metabolismus MeSH
- diglyceridy metabolismus MeSH
- fosfolipasy typu C metabolismus MeSH
- hliník farmakologie MeSH
- ionty MeSH
- kořeny rostlin účinky léků metabolismus MeSH
- semenáček účinky léků metabolismus MeSH
- sloučeniny boru metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1,2-diacylglycerol MeSH Prohlížeč
- 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene MeSH Prohlížeč
- benzylalkohol MeSH
- diglyceridy MeSH
- fosfolipasy typu C MeSH
- hliník MeSH
- ionty MeSH
- sloučeniny boru MeSH
Miglustat has been indicated for the treatment of Niemann-Pick disease type C (NP-C) since 2009. The aim of this observational study was to assess the effect of miglustat on long-term survival of patients with NP-C. Data for 789 patients from five large national cohorts and from the NPC Registry were collected and combined. Miglustat-treated and untreated patients overall and within sub-groups according to age-at-neurological-onset, that is, early infantile-onset (<2 years), late infantile-onset (2 to <6 years), juvenile-onset (6 to <15 years), and adolescent/adult-onset (≥15 years) were analysed and compared. Survival was analysed from the time of first neurological manifestation (Neurological onset group, comprising 669 patients) and from diagnosis (Diagnosis group, comprising 590 patients) using a Cox proportional hazard model adjusted for various covariates. Overall, 384 (57.4%) patients in the Neurological onset group and 329 (55.8%) in the Diagnosis group were treated with miglustat. Miglustat treatment was associated with a significant reduction in risk of mortality in both groups (entire Neurological onset group, Hazard ratio [HR] = 0.51; entire Diagnosis group, HR = 0.44; both P < .001). The effect was observed consistently in all age-at-neurological-onset sub-groups (HRs = 0.3 to 0.7) and was statistically significant for late infantile-onset patients in both groups (Neurological onset group, HR = 0.36, P < .05; Diagnosis group, HR = 0.32, P < .01), and juvenile-onset patients in the Diagnosis group only (HR = 0.30, P < .05). Despite the limitations of the data that urge cautious interpretation, the findings are consistent with a beneficial effect of miglustat on survival in patients with NP-C.
- Klíčová slova
- NP-C, NPC registry, Niemann-pick disease type C, Zavesca, miglustat, observational national cohorts, survival,
- MeSH
- 1-deoxynojirimycin analogy a deriváty terapeutické užití MeSH
- analýza přežití MeSH
- dítě MeSH
- dospělí MeSH
- inhibitory enzymů MeSH
- internacionalita MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- Niemannova-Pickova nemoc typu C farmakoterapie mortalita MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- registrace MeSH
- retrospektivní studie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- pozorovací studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-deoxynojirimycin MeSH
- inhibitory enzymů MeSH
- miglustat MeSH Prohlížeč
The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleoplasm and one of the more intricate structures of eukaryotic cells. Typically composed of over 300 polypeptides, the NPC shares evolutionary origins with endo-membrane and intraflagellar transport system complexes. The modern NPC was fully established by the time of the last eukaryotic common ancestor and, hence, prior to eukaryote diversification. Despite the complexity, the NPC structure is surprisingly flexible with considerable variation between lineages. Here, we review diversification of the NPC in major taxa in view of recent advances in genomic and structural characterisation of plant, protist and nucleomorph NPCs and discuss the implications for NPC evolution. Furthermore, we highlight these changes in the context of mRNA export and consider how this process may have influenced NPC diversity. We reveal the NPC as a platform for continual evolution and adaptation.
- Klíčová slova
- eukaryogenesis, evolutionary biology, nuclear pores, nuclear protein transport,
- MeSH
- biologická evoluce * MeSH
- biologický transport MeSH
- jaderný pór metabolismus MeSH
- membránové proteiny metabolismus MeSH
- messenger RNA metabolismus MeSH
- mitóza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- membránové proteiny MeSH
- messenger RNA MeSH
UNLABELLED: In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18(+) NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.
- MeSH
- genom * MeSH
- HeLa buňky chemie MeSH
- karcinom MeSH
- kontaminace DNA MeSH
- lidé MeSH
- nádorové buněčné linie chemie MeSH
- nádory nosohltanu genetika MeSH
- nasofaryngeální karcinom MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza RNA MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.
- Klíčová slova
- Cholesterol, Lipid domains, Lysosomal storage diseases, Membrane fluidity, Niemann-Pick disease type C, Sphingosine,
- MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- CHO buňky MeSH
- cholesterol metabolismus MeSH
- Cricetulus * MeSH
- fluidita membrány účinky léků MeSH
- křečci praví MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- metabolismus lipidů účinky léků MeSH
- Niemannova-Pickova nemoc typu C * metabolismus patologie genetika MeSH
- protein NPC1 MeSH
- sfingosin * analogy a deriváty metabolismus MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cholesterol MeSH
- protein NPC1 MeSH
- sfingosin * MeSH
