European Journal of Cell Biology
Volume 95, Issue 1,
, pages 26-41
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Gelsolin, a multifunctional actin-binding protein, plays a role that is still not fully understood in tumorigenesis. Therefore, the aim of this study was to identify additional gelsolin molecular partners in human melanoma cells, separately in the cytoplasmic compartment and cell nuclei. To this end, we performed immunoprecipitation experiments based on a modified protocol followed by mass spectrometry. The obtained results were confirmed by Western blot analysis, proximity binding assays and confocal microscopy. As expected, gelsolin interacted with actin, in particular we demonstrated its interaction with cytoplasmic β and γ actins and a newly discovered actin isoform, actbl2. We have identified the ribosomal protein Rpsa, also known as the non-integrin laminin receptor (LamR), and the heterogeneous nuclear ribonucleoprotein hnRNP U as new interaction partners with gelsolin. Our data also indicate that gelsolin interacts with specific components of three cytoskeleton systems: nestin (intermediate filaments), Arp3 ( actin cytoskeleton) and β-tubulin (microtubules). We also report for the first time that gelsolin is a component of intermediate bodies, a tubulin-containing structure formed at the end of cytokinesis.
Gelsolin is a multifunctional actin binding protein and is involved in the dynamic reorganization of the actin cytoskeleton due to its capping, cleavage and, under certain circumstances, nucleation (Mannherz et al., 2010, Nag et al., 2013). Gelsolin is a member of the actin-binding protein (AbP) family of the gelsolin/villin family and is characterized by six homologous segments of approximately 14 kDa (Kwiatkowski et al., 1986). Gelsolin is found on the tips of lamellipodia or podosomes and invadopodia (Attanasio et al., 2011, Cervero et al., 2012, Mazur et al., 2010), i.e. on the structures responsible for the migration of macrophages and cancer cells. Gelsolin has also been localized in the cell nuclei of various types of cancer cells, including in astrocytoma (Ohnishi et al., 2009), in colorectal cancer cell subpopulations (Zhuo et al., 2012), and in melanoma cell lines (our unpublished observations), but its possible nuclear function is still not fully elucidated. Among members of the gelsolin family, only supervillin has a nuclear localization sequence (NLS), while gelsolin has neither an NLS nor a nuclear export signal (NES) (Li et al., 2012). Published data show that intracellular gelsolin exists in various states of activation, including free gelsolin and Ca2+bound gelsolin, but only free gelsolin can translocate to the nucleus (Van den Abbeele et al., 2010). On the other hand, it has been found that gelsolin can enter the cell nucleus in complex with a steroid hormone and its receptor (Ambrosino et al., 2010, Nishimura et al., 2003). Nishimura et al. (2003) postulated that the formation of the androgen:androgen-receptor:gelsolin complex is crucial for the interaction of the nuclear actin-activated receptor with RNA polymerase II. Although these data suggest that steroid hormones may be responsible for the nuclear localization of gelsolin in melanoma cells, we postulate that gelsolin may have additional functions in the cell nucleus. Indeed, recent data describes the interaction of gelsolin with FBXO25, an F-box protein that serves as a specificity factor for ubiquitin ligases found mainly in the nuclear domains associated with FBXO25 (FAND) (Teixeira et al., 2010).
Recent reports suggest that gelsolin plays a role in tumorigenesis as both a tumor suppressor and tumor promoter. These conflicting hypotheses are based on observations of a decrease in gelsolin levels (GSN)for example breast, bladder, colon, stomach, kidney, lung, oral, ovarian, pancreatic and prostate cancers (as reviewed by Li et al. (2012)) or upregulated in non-small cell lung cancer, urothelial carcinoma, pancreas and (Rao et al., 2002, Shieh et al., 2006, Thompson et al., 2007, Yang et al., 2004). Interestingly, biphasic gelsolin expression was observed during the transition from oral precancerous lesions to oral cancer (Shieh et al., 2006).GSNdownregulation was observed in precancerous lesions, whileGSNupregulation has been observed in oral cancer progression. In addition, gelsolin has been shown to positively stimulate the proliferation, migration and invasion of human oral squamous cell carcinoma cells (Deng et al., 2015).
We have previously shown that gelsolin is critical for the migration and invasion of colon adenocarcinoma and melanoma cell lines (Litwin et al., 2012, Litwin et al., 2009). In colon adenocarcinoma cells, overexpression of gelsolin resulted in an increase, while downregulationGSNexpression in melanoma cells resulted in reduced migration potential. In both cases, we observed dramatic changes in the organization of the actin cytoskeleton, i.e. a reduced polymerization state in gelsolin overexpressing cells and an increase in cytoplasmic stress fibers in gelsolin suppressed cells, reflecting the well-known actin cleavage activity. actin from gelsolin. However, gelsolin was present in A375 cell nuclei even 72h after downregulation was startedGSNexpression, although the amount of gelsolin clearly decreased in the cytoplasmic compartment (Litwin et al., 2012). These intriguing observations inspired us to investigate nuclear gelsolin and its possible interaction partners in more detail. For this study, we selected established cell lines derived from human melanoma and aimed to identify gelsolin's molecular partners, with particular emphasis on possible gelsolin nuclear interaction partners.
To this end, we optimized the procedure for immunoprecipitation of endogenous gelsolin from whole cell lysates (WCL) and nuclear fractions (NF) of melanoma cells. Since our aim was to focus on downstream interactions of endogenous gelsolin, we did not use cells transfected with overexpressed tagged gelsolin. Liquid chromatography/mass spectrometry (LC/MS) analysis of co-immunoprecipitated complexes (Co-IP) led to the identification of novel protein partners interacting with endogenous gelsolin. The specificity of these partners was further confirmed by Western blotting (WB), proximity binding assay (PLA) and colocalization microscopic analyses. We believe that our data may have interesting implications for human melanoma biology and may initiate further research into the role of gelsolin in cancer cells.
Antibodies and dyes
Rabbit anti-emerin (FL-254), mouse anti-GAPDH (H-12), goat anti-gelsolin (C-20), mouse anti-hnRPU U (3G6), rabbit anti-laminin-R (H-141) , goat anti-Laminin-R (F-18), mouse anti-nestin (10c2), rabbit anti-Arp3 (H-110), goat anti-HA (Y-11) and normal goat IgG were obtained from Santa Cruz Biotechnology®Inc. (Heidelberg, Germany). Mouse anti-β-actin (AC-15), mouse anti-γ-actin (2-22.214.171.124), mouse anti-all isoactin (AC40), rabbit anti-all isoactin (C-11), mouse anti-β- tubulin (TUB 2.1) and
Identification of gelsolin in melanoma cell nuclei
We first compared the quality of goat polyclonal and mouse anti-gelsolin monoclonal antibodies by Western blotting (WB) and confocal microscopy, as goat IgG was used for immunoprecipitation and immunostaining experiments, and mouse antibodies were used for Co-IP Western blotting and immunostaining. Evaluation of the results showed that both types of antibodies were specific for gelsolin and recognized a single band of 82 kDa (Supplementary Fig. 1A, B). We also observed
To identify novel molecular partners of gelsolin, we co-immunoprecipitated endogenous gelsolin from whole cell lysates (WCL) and nuclear fractions (NF) of melanoma cells using a modified immunoprecipitation protocol. The identification of actin, SERBP1 (SERPINE1 mRNA-binding protein) (Supplementary Table 3) and drabrin (Supplementary Table 5) as gelsolin interaction partners in the LC/MS analysis of coimmunoprecipitates provided a proof of principle for our approach, as there was a seance
Conflict of interest
The authors declare no competing financial interest.
AJM, TR and AM thank youFoundation for Polish Sciencefor financial support under the HOMING Plus Program (HOMING Plus/2010-2/8) under the Innovative Economy Program of the European Union. Special thanks go to Ilona Styczeń for her important administrative contribution to this work. The costs of publication were financed from the program of the Wrocław Center for Biotechnology "National Center for Leading Research (KNOW)" for the years 2014-2018.
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Identification of the hormone-regulated dynamic nuclear actin network associated with the estrogen receptor alpha in the nucleus of human breast cancer cells
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Novel invadopodia components revealed by differential proteomics analysis
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Proteomic analysis of macrophage podosome fraction reveals similarities with expanding initiation centers
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The glucocorticoid receptor is associated with the nuclear matrix RNA-binding protein hnRNP U
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γ-Tubulin localizes to actin-based membrane protuberances and inhibits the formation of stress fibers
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Unconventional actin conformations are located on intermediate filaments in mitosis
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Keratin expression and its importance in five cultured melanoma cell lines derived from primary, relapsed and metastatic melanoma
To install Karta FEBS.
CNS stem cells express a new class of intermediate filament proteins
Gelsolin affects the migratory capacity of human colonic adenocarcinoma and melanoma cells
The natural sciences.
Integrative analysis of an isolated ubiquitin proteome using tandem ubiquitin binding units (TUBEs)
UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) transporters form glycosylation related complexes with mannosine acetylglucosaminyltransferases (Mgats)
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Modulation of actin filament dynamics by actin-binding proteins found in lamellipodia
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Functional characterization of the Wiskott-Aldrich Syndrome (WASH) protein and scar homolog, a bimodular nucleation-promoting factor capable of interacting with the biogenesis of lysosome-associated organelle subunit 2 (BLOS2) and β-tubulin
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Nuclear localization analysis of p40 laminin binding protein precursor (LBP/p40)
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Genetic and biochemical interactions between the Arp2/3 complex, Cmd1p, casein kinase II and Tub4p in yeast
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The multifunctionality of the 37/67 kd laminin receptor makes it a suitable target for novel cancer gene therapy
Conformation-specific antibodies reveal different actin structures in the nucleus and cytoplasm
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Tissue expression of gelsolin in oral cancer progression and its clinicopathological implications
Expression of the 67 kD laminin receptor has been associated with tumor progression and poor prognosis in epithelial ovarian cancer
Post-translational modification and regulation of actin
current opinion. cell biol.
How common are the extraribosomal functions of ribosomal proteins?
Prognostic significance of gelsolin expression level and variability in non-small cell lung cancer
Nestin is highly expressed in late-stage melanoma and neurotic nevi
Actin as a target of modification by bacterial protein toxins
Comparative proteomic analysis of relevant primary and metastatic melanoma cell lines
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A 33 kD polypeptide with homology to the laminin receptor: a component of the translation mechanism
percent US National Science Academy.
The vimentin tail interacts in vivo with actin-containing structures
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Effect of estradiol on human breast cancer cells in culture
Ribosomal proteins as new actors in oncogenesis
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Gelsolin regulates the proliferation, apoptosis, migration and invasion of human oral cancer cells
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A look at the laminin receptor: a critical discussion of the non-integrin 37/67-kDa laminin receptor/RPSA protein
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Precise transcription initiation by RNA polymerase II in a soluble extract of isolated mammalian testes
answer nucleic acid
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RACK1 scaffold protein: multiple roles in human cancer
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Human herpesvirus U94A 6A latency transcript expression impairs cytoskeletal function in human nerve cells
2022, Molecular and Cellular Neuroscience
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For example, cytoplasmic gelsolin is required for OPC differentiation (Shao et al., 2017) and may play a role in stabilizing the microtubule network during morphological maturation (Liu et al., 2003). Although the mechanism by which this is achieved is unclear, studies in cancer cells have shown that gelsolin can directly interact with β-tubulin and other microtubule-binding proteins such as NAT10 and NuMA-1 (Mazur et al., 2016). Reduced WASF1 function in OPC also results in inhibition of oligodendrocyte maturation and reduction in the number of myelinated axons in vivo.
Many neurodegenerative diseases have a multifactorial etiology and variable course that cannot be explained by current models. Neurotrophic viruses have long been suggested to play a role in these diseases, although their exact involvement remains unclear. Human herpesvirus 6A (HHV-6A) is one of the most commonly detected viruses in the adult brain and has been clinically linked to multiple sclerosis (MS) and more recently to Alzheimer's disease (AD). HHV-6A is a ubiquitous viral pathogen capable of infecting glial cells and neurons. Primary childhood infection is followed by latency induction, characterized by expression of the U94A viral transcript in the absence of viral replication. Here we investigate the effects of U94A on central nervous system cells. We found that U94A expression inhibits migration and impairs cytoplasmic maturation of human oligodendrocyte precursor cells (OPCs) without affecting their viability, a phenotype that may contribute to the failure of remyelination observed in many patients with multiple sclerosis. Subsequent proteomic analysis of U94A-expressing OPCs revealed altered expression of genes involved in tubulin-related regulation of the cytoskeleton. Since HHV-6A appears to be significantly associated with early AD pathology, we extended our initial analysis of the effects of U94A on human-derived neurons. We found that U94A expression suppresses neurite outgrowth in primary human cortical neurons and impairs synaptic maturation. Based on these data, we suggest that the expression of U94A by latent HHV-6A in glial cells and neurons makes them susceptible to dysfunction and degeneration. This is why,latentViral infections of the brain represent a unique pathological risk factor that may contribute to disease processes.
Intracellular partners of fibroblast growth factors 1 and 2 - implications for function
2021, Cytokines and Growth Factor Reviews
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Upregulation of its expression has been correlated with several types of cancer [268-270]. The cytoplasmic gelation-solation transformation of ACTBL2 has been reported to be controlled by gelsolin , but Mazur et al. have shown that gelsolin can also be found in the testis . Interestingly, as previously mentioned, we report that gelsolin also interacts with FGF1, indicating the possibility of forming a ternary FGF1-gelsolin-ACTBL2 complex involved in cell growth, cell migration and tumorigenesis.
Fibroblast growth factors 1 and 2 (FGF1 and FGF2) are considered primarily as ligands of surface receptors through which they regulate a wide spectrum of biological processes. They are secreted non-canonically and, unlike other growth factors, are able to move from the endosome to the interior of the cell. These unique features, as well as the role of the intracellular pool of FGF1 and FGF2, are far from fully understood. An increasing number of reports address this issue, focusing on the intracellular interactions of FGF1 and 2. Here, we summarize the current state of knowledge about FGF1 and FGF2 binding partners in the cell and the possible role of these interactions. Partner proteins are grouped according to their function, including proteins involved in secretion, cell signaling, nucleocytoplasmic transport, nucleic acid binding and processing, ATP binding, and cytoskeletal assembly. In-depth network analysis of these binding partners may pinpoint novel, non-classical functions of FGF1 and FGF2 and reveal an additional level of fine-grained control of well-known FGF-regulated cellular processes.
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2017, Journal of Proteomics
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Importantly, increased expression of ACTBL2 has recently been observed in human pancreatic ductal adenocarcinoma (PDAC)  and has been associated with the growth properties of hepatoma cells, contributing to poor postoperative prognosis . Mazur et al.  showed that gelsolin (GSN), which is the binding partner of ACTBL2, is localized in the testis. Gelsolin has been shown to play a role in tumorigenesis in human melanoma cells as well as increasing the activity of the androgen receptor (AR) .
Early diagnosis of colorectal cancer (CRC) can be valuable in increasing patient survival. Recently, proteomics strategies have been applied to identify markers for early-stage cancer diagnosis with remarkable results. To extend these studies, we used two-dimensional gel electrophoresis and mass spectrometry to profile the expression of proteins extracted from fresh-frozen samples of human colorectal cancer tissue and comparable areas of adjacent normal mucosa (serving as controls). The four gel spots were found to be consistently differentially stained between tumor and control samples. After mass spectrometric analysis of these spots, six proteins were identified; five of these have previously been reported to be associated with colorectal cancer. Actin beta-like protein 2 (ACTBL2), unrelated to colorectal cancer in previous reports, was found in greater abundance in colorectal tumor samples by both proteomic and immunohistochemical analysis. Thus, the association of ACTBL2 and differential upregulation in colorectal cancer is novel and as such may contribute to our understanding of colorectal carcinogenesis and potentially play a role in the development of colorectal cancer markers.
Colorectal cancer (CRC) is one of the leading causes of death worldwide and good early detection markers are lacking. In this study, we performed a proteomic analysis of tumor tissue versus normal tissue. We confirmed the discovery of several previously identified CRC-related proteins and, most importantly, identified a new protein, ACTBL2, which we have shown is upregulated in CRC. Once additional CRC-related proteins have been identified, the potential for developing marker panels with better results in early cancer detection could be realised.
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Proteomic analysis of potential targets for non-response to infliximab in patients with ulcerative colitis
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Stratigraphy and Pleistocene climatic environments of the Terek-Kuma Plain (northwestern Caspian region) inferred from palynological, paleomagnetic and rodent records of the long sequence of Otkaznoye sediments
International Quaternary, Volume 409, Part A, 2016, pp. 16-32
The article presents the results of high-resolution palynological, paleomagnetic and microtheriological studies of Pleistocene sequences of sediments at Otkaznoje on the eastern promontory of Ciscaucasia; a sequence that is unique in its completeness and stratigraphic thickness (∼160 m). The sequence includes loess-paleosol, alluvial and alluvial-proluvial layers. The detailed climatostratigraphic division of the Otkaznoje sequence allows for the correlation of the established paleoclimate record with the global marine isotopic stages. The climate-induced variability of the landscape of the Terek-Kuma Plain has been reconstructed for all interglacial and glacial periods of the Late Pleistocene (i.e. Middle and Late Pleistocene) and for some intervals in the later stages of the Late Pleistocene.
Hybrid surgery of intralobular sequestration of the lung with aortic aneurysm
The Annals of Thoracic Surgery, tom 98, wydanie 1, 2014, s. e11-e13
A 62-year-old man with chronic atrial fibrillation, congestive heart failure, and a history of pneumonia was referred to our hospital for further investigation of pulmonary sequestration. Enhanced CT revealed intralobar sequestration of the left lower pulmonary lobe (IPS) and dilation of the aortic aneurysm at the origin of the abnormal supplying artery. We performed a hybrid operation consisting of endovascular repair of the thoracic aorta and excision of the IPS and left lower lobe by video thoracic surgery. The patient was discharged after 5 days without complications.
New drugable sites for insulin-degrading enzymes identified by applied structural bioinformatics analysis
Procedia Computer Science, tom 80, 2016, s. 2292-2296
The insulin-degrading enzyme (IDE) plays a key role in the proteolysis of several substrates such as insulin and β-amyloid. Pathologically, IDE has been linked to type 2 diabetes and Alzheimer's disease, but potent and selective regulators of IDE remain elusive. We apply structural bioinformatics techniques to the largest set of defined IDE structuresuntil nowidentified structural clusters associated with different conformational states and their respective clasteroids. The IDE uses its large-scale internal structural movements to assume various conformational states and perform molecular functions. The conformational space occupied by IDE structures can be displaced by mutations and intermolecular interactions with other proteins, small molecules or peptide substrates. We note that IDE-N is generally more dynamic than IDE-C and suggest that there are likely other open conformational states of IDE whose structures remain unknown. We also identified novel drug-taking sites that are specific to specific IDE conformational states, these sites could potentially be used to design research probes or therapeutic agents for specific spatiotemporal contexts.
One Reply to “Banking Crisis, Labor Market Reform and Unemployment: Commentary”
Journal of Comparative Economics, tom 43, wydanie 4, 2015, s. 1142-1147
Aleksynka (2015) points to several significant methodological errors in the labor market indicator data used in the work of Bernal-Verdugo, Furceri and Guillaume (2013) [BFG]. This article reviews the empirical results presented in the BFG and shows that these methodological flaws have little influence on the results and conclusions. In particular, we find that: (i) while in countries with more flexible labor markets the impact of banking crises is more acute but short-lived, in countries with more rigid labor markets the effect is initially more moderate but very persistent; (ii) comprehensive labor market reforms have a positive impact on unemployment, albeit only in the medium term.
Clinico-pathological and prognostic value of ZEB1-AS1 long non-coding RNA in solid tumors: a meta-analysis
Clinica Chimica Acta, tom 484, 2018, s. 91-98
Studies have shown that the antisense 1 E-box 1 (ZEB1-AS1) binding the zinc finger homeobox is overexpressed in a number of malignancies. However, the sample size of these studies was limited, so the clinicopathological and prognostic value of ZEB1-AS1 in solid tumors remains undetermined. Clinico-pathological features and prognosis in patients with solid tumors.
Combined odds ratios (OR) and hazard ratios (HR) were estimated with a 95% confidence interval (CI) to assess the relationship between ZEB1-AS1 and clinicopathological characteristics and prognosis of cancer patients.
A total of 10 studies involving 861 patients were included in this meta-analysis. Combined results suggest that high ZEB1-AS1 expression was associated with poor differentiation (low vs. high + moderate: OR = 2.99, 95% CI = [2.03, 4.39]), increased lymph node metastases ( YES vs. NO: OR = 4.62, 95% CI=[2.90, 7.37]) and advanced TNM stage (I+II vs. III+IV: OR=0.41, 95% CI=[ 0.23; 0.75]), but not to sex and tumor size. In addition, high ZEB1-AS1 expression was associated with poor overall survival (OS; HR=1.86, 95% CI=[1.57, 2.14]) and disease-free survival (DFS; HR=2.03, 95 %CI=[1.28, 2.77]). Thus, ZEB1-AS1 may be an independent predictor of OS (HR=2.07, 95% CI=[1.57, 2.56]) in cancer patients.
High ZEB1-AS1 expression is associated with advanced clinicopathological features, and ZEB1-AS1 overexpression may be a potential prognostic biomarker in human cancer. However, more research is needed involving multiple types of cancer and large samples.
Computing and data infrastructure to connect EEE stations
Nuclear Instruments and Methods in Physical Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Vol. 824, 2016, pp. 329-330
The Extreme Energy Event (EEE) experiment is dedicated to the search for high-energy cosmic rays using a network of telescopes installed in about 50 secondary schools in Italy. This project requires a specific data management infrastructure to collect data recorded at widely separated stations and enable coordinated analysis. This infrastructure is hosted by INFN-CNAF, which operates a cloud based on the OpenStack open source platform and provides infrastructure as a service (IaaS) to its users. In 2014, EEE started using it to collect, monitor and retrieve data acquired at all EEE stations. For synchronization between stations and the INFN-CNAF infrastructure, BitTorrent Sync, a free peer-to-peer software developed to optimize data synchronization between distributed nodes, was used. All data folders are synchronized with the central repository in real time, which enables immediate reconstruction of data and their publication on the monitoring website. We present the architecture and functionalities of this data management system, which provides a flexible environment for the specific needs of an EEE project.
Current address: Faculty of Science, Department of Experimental Biology, Masaryk University, Czech Republic.
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