39 research outputs found
Preparation of detergent-solubilized membranes from Escherichia coli
No full textAuthors: Daniel Harder & Dimitrios Fotiadis
### Abstract
This protocol describes a method to prepare detergent-solubilized membranes from Escherichia coli (E. coli), e.g. containing an overexpressed membrane protein. The procedure takes less than one day. Cells are broken by pressure cell and membranes are isolated and washed by differential centrifugation. Finally, the membranes are solubilized with the detergent of choice.
### Reagents
1. Cell pellet of E. coli
- Tris
- EDTA
- NaCl
- n-dodecyl-β-D-maltoside (DDM) or another detergent of choice
glycerol
- NaN3
### Equipment
1. French Press
- ultracentrifuge
### Procedure
1. Resuspend the cell pellet from 1 l of E. coli culture in Lysis Buffer (20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA). Adapt the buffer volume to your French pressure cell.
- Disrupt E. coli cells by passage through a French pressure cell (20,000 psi) and remove unbroken cells by centrifugation at 10,000g (4˚C, 10 min).
- Ultracentrifuge the supernatant at 100,000g (4˚C, 1 h).
- Resuspend and homogenize the pellet containing the E. coli membranes in Lysis Buffer and ultracentrifuge again.
- Remove water-soluble proteins adhering to the membrane by homogenization in 20 mM Tris-HCl, pH 8.0, 300 mM NaCl and ultracentrifugation.
- Breakpoint: Membrane pellet can be resuspended in 1 ml buffer without detergent and stored at -80 °C for months to years prior detergent solubilization.
- Resuspend and solubilize the membrane pellet in 1% DDM, 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 0.01% NaN3 for 2 h at 4˚C under gentle agitation (final volume: 7 ml).
- Ultracentrifuge at 100,000g (4˚C, 50 min).
- The supernatant represents solubilized membranes, which can be used for purification of the His-tagged protein or SPA-binding experiments directly.
### Timing
The procedure takes less than one day.
### References
- Casagrande, F. et al. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. *J. Biol. Chem*. 283, 33240-33248 (2008).
### Associated Publications
1. **Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily**. F. Casagrande, M. Ratera, A. D. Schenk, M. Chami, E. Valencia, J. M. Lopez, D. Torrents, A. Engel, M. Palacin, and D. Fotiadis. *Journal of Biological Chemistry* 283 (48) 33240 - 33248 23/09/2008 doi:10.1074/jbc.M806917200
- **Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay**. Daniel Harder and Dimitrios Fotiadis. *Nature Protocols* 7 (9) 1569 - 1578 doi:10.1038/nprot.2012.090
### Author information
**Daniel Harder & Dimitrios Fotiadis**, Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland
Correspondence to: Dimitrios Fotiadis ([email protected])
*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2395) (2012) doi:10.1038/protex.2012.033. Originally published online 7 August 2012*
Purification of His-tagged membrane proteins from detergent-solubilized membranes
No full textAuthors: Daniel Harder & Dimitrios Fotiadis
### Abstract
This protocol describes a method to purify a His-tagged membrane protein form detergent-solubilized Escherichia coli (E. coli) membranes. Solubilized membranes are incubated with Ni-NTA that binds the His-tagged protein. After washing, His-tagged proteins are eluted with histidine. When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively.
### Reagents
1. Tris
- NaCl
- n-dodecyl-β-D-maltoside (DDM) or another detergent of choice
- histidine
- glycerol
- NaN3
- Ni-NTA Superflow beads (Qiagen)
- Wizard Midicolumn (Promega)
### Equipment
- SDS-PAGE facility
### Procedure
1. Prepare solubilized membranes according to our protocol: Preparation of detergent-solubilized membranes from Escherichia coli or start with frozen membranes from 1 l culture by solubilizing in 1% DDM, 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 0.01% NaN3 for 2 h at 4˚C under gentle agitation (final volume: 7 ml) and subsequent ultracentrifugation at 100,000g (4˚C, 50 min).
- Dilute the supernatant 2-fold with Washing Buffer (20 mM Tris-HCl, pH 8, 300 mM NaCl, 0.04% DDM, 5 mM histidine, 10% glycerol, 0.01% NaN3) and bind for 2 h at 4 °C to Ni-NTA Superflow beads (Qiagen; 0.5 ml bed volume).
- Load the beads onto a gravity flow column (Wizard Midicolumn, Promega), wash with washing buffer (20 ml), and elute with the same buffer containing 200 mM histidine.
- Collect fractions of the eluate (e.g. 250 µl fractions) and identify the fractions containing the purified target protein by SDS-PAGE.
- Purified protein can be used e.g. for SPA-binding experiments after removing the histidine by desalting columns.
### Timing
When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively.
### References
- Casagrande, F. et al. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. *J. Biol. Chem*. 283, 33240-33248 (2008).
### Associated Publications
1. **Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily**. F. Casagrande, M. Ratera, A. D. Schenk, M. Chami, E. Valencia, J. M. Lopez, D. Torrents, A. Engel, M. Palacin, and D. Fotiadis. *Journal of Biological Chemistry* 283 (48) 33240 - 33248 23/09/2008 [doi:10.1074/jbc.M806917200](http://dx.doi.org/10.1074/jbc.M806917200)
- **Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay**. Daniel Harder and Dimitrios Fotiadis. *Nature Protocols* 7 (9) 1569 - 1578 [doi:10.1038/nprot.2012.090](http://dx.doi.org/10.1038/nprot.2012.090)
### Author information
**Daniel Harder & Dimitrios Fotiadis**, Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland
Correspondence to: Dimitrios Fotiadis ([email protected])
*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2396) (2012) doi:10.1038/protex.2012.034. Originally published online 7 August 2012*
The Greek Theatre in Pontos, Russia and the Soviet Union
No full textDespite its outstanding significance for all of Hellenism, the Greek Theatre of Pontos, Russia and the former Soviet Union still remains almost totally unknown, even to those who are interested in and promote ecumenical Hellenism. This article aims at fulfilling this need by reflecting on the diachronic contribution of Hellenism in these most sensitive and historically critical regions which, like Pontos and Russia, played an important role even in the Greek uprising of 1821. The author analytically examines the vicissitudes which Greek theatre activities, since their inception in Pontos after 1860, in Russia after the founding of the Filiki Etaireia in Odessa and in the USSR after the Bolshevik rise to dominance in the early 1900s, have faced in these regions under the geo-political, cultural and linguistic adversities created by the various local regimes.Despite its outstanding significance for all of Hellenism, the Greek Theatre of Pontos, Russia and the former Soviet Union still remains almost totally unknown, even to those who are interested in and promote ecumenical Hellenism. This article aims at fulfilling this need by reflecting on the diachronic contribution of Hellenism in these most sensitive and historically critical regions which, like Pontos and Russia, played an important role even in the Greek uprising of 1821. The author analytically examines the vicissitudes which Greek theatre activities, since their inception in Pontos after 1860, in Russia after the founding of the Filiki Etaireia in Odessa and in the USSR after the Bolshevik rise to dominance in the early 1900s, have faced in these regions under the geo-political, cultural and linguistic adversities created by the various local regimes.Malgré son immense importance pour l’hellénisme, le théâtre grec du Pont, de la Russie et de l’ancienne Union Soviétique demeure encore aujourd’hui presque totalement inconnu, même à ceux qui y portent un intérêt et qui font la promotion de l’hellénisme oecuménique. Le but ce cet article est de combler cette lacune en proposant une réflexion sur la contribution diachronique de l’hellénisme dans ces régions les plus sensibles et d’une importance capitale du point de vue historique, qui comme le Pont et la Russie ont joué un rôle important même lors de la Révolution grecque de 1821. L’auteur examine de façon analytique les difficultés auxquelles ont fait face les activités théâtrales grecques, depuis leur création au Pont après 1860, en Russie après la fondation de la Filiki Etaireia, à Odessa et en Union Soviétique (USSR) après la révolte bolchevique du début des années 1900, compte tenu des adversités géo-politiques, culturelles et linguistiques créées par les divers régimes locaux dans ces régions
The behaviour of Ailanthus altissima weed and its effects on natural ecosystems Author Details
No full textAbstract Ailanthus altissima is an invasive species for the native flora of Greece and it could pose a serious threat to the biodiversity and the functioning of ecosystems. The purpose of this study was to investigate the spreading of Ailanthus altissima in urban and non urban areas of North and Central Greece and also to evaluate the effects of its spreading on species composition and floristic diversity in natural ecosystems. The spreading of Ailanthus altissima in urban areas is very intense, mainly in abandoned places (35.29%). It is commonly found in non urban areas of Greece, especially in hedgerows of arable lands (36%) and adjacent wetlands (17%). It is less common in forests (4%), shrublands (11%) and grasslands (9%). The spread of Ailanthus altissima in urban and natural ecosystems is relatively recent. Although it has been recorded at altitudes of up to 640 m, it usually appears at low altitudes of up to 200 m. Floristic diversity was found to be higher in the stands that it dominated (H´=1.574, H´=1.890) in comparison to stands that were dominated by Quercus pubescens (H´=1.468) or Q. coccifera (H´=1.716). This may be contributed to the fact that in those stands synanthropic species, which are usually found in regions of intense human activity, were present together with typical forest vegetation species
First Workshop on Multimodal e-Coaches
No full textThis preprint follows ACM policy: “Authors who publish with ACM have the freedom to post peer-reviewed pre-print
versions of their papers to personal websites and institutional repositories. They can add a single-click link to their final
published papers, and re-use any portion of their published work with the inclusion of a citation and DOI link. Authors
can also post on any repository legally mandated by the agency funding the research on which the work is based, and on
any non-commercial repository or aggregation that does not duplicate ACM tables of contents/substantially duplicate
an ACM-copyrighted volume or issue” (https://authors.acm.org/author-resources/author-rights.)
2e-Coaches are promising intelligent systems that aims at supporting human everyday life, dispatching
advice through different interfaces, such as apps, conversational interfaces and augmented reality
interfaces. This workshop aims at exploring how e-coaches might benefit from spatially and timemultiplexed
interfaces and from different communication modalities (e.g., text, visual, audio, etc.)
according to the context of the interaction.The NESTORE, SAAM, CAPTAIN, HOLOBALANCE, EMPATHIC, MENHIR, vCare projects are supported by
the European Commission under the Horizon 2020 programmes SC1-PM-15-2017 H2020-MSCA-RISE-2018, and
H2020-1.3.3, respectively through the project grants N.769643, 769661, 769830, 769574, 769872, 823907, 769807.
The authors want to thank their respective Consortia. The opinions expressed in this paper are those of the authors and
are not necessarily those of the project partners or the European Commission
Percutaneous radiofrequency ablation of small renal tumours in patients with a single functioning kidney:long-term results
No full textObjectives To evaluate the long-term results of percutaneous radiofrequency ablation (RFA) of small renal tumours in patients with a single functioning kidney (SFK). Methods This is a single-centre prospective study. Patients with an SFK and a tumour smaller than 3.5 cm, treated with RFA over a 7.5-year period, were included. Nineteen consecutive patients (12 male), aged between 33 and 83 years (mean 61.4), were treated for 23 lesions. Primary endpoints were technical success and tumour recurrence rate. Secondary endpoints were the deterioration of renal function and overall survival rate. Results The mean follow-up was 56.1 months (range 36-102). Primary technical success was 100 %. There were no major peri-procedural complications. In two cases, minor complications occurred. There was no significant difference between the baseline glomerular filtration rate (GFR) and GFR at 3, 12 and 24 months post-procedure. In four lesions (17 %), recurrence was detected and an additional RFA session was performed. None of the patients developed renal failure during their lifetime. Three of the patients died because of other reasons. Conclusions Percutaneous RFA of small renal tumours in patients with an SFK offers very satisfactory long-term results regarding preservation of renal function, local tumour control and overall survival. Key Points Tumour in a single functioning kidney requires minimally invasive treatment. Radiofrequency ablation plays an established role in managing small renal tumours. Long-term results of radiofrequency ablation have shown satisfactory local tumour control. Long-term results have also shown that renal function may be preserved.</p
Author Correction: Role of thalamus in human conscious perception revealed by low-intensity focused ultrasound neuromodulation
No full textA machine learning-based risk stratification model for ventricular tachycardia and heart failure in hypertrophic cardiomyopathy
No full text\ua9 2021 The Author(s). Background: Machine learning (ML) and artificial intelligence are emerging as important components of precision medicine that enhance diagnosis and risk stratification. Risk stratification tools for hypertrophic cardiomyopathy (HCM) exist, but they are based on traditional statistical methods. The aim was to develop a novel machine learning risk stratification tool for the prediction of 5-year risk in HCM. The goal was to determine if its predictive accuracy is higher than the accuracy of the state-of-the-art tools. Method: Data from a total of 2302 patients were used. The data were comprised of demographic characteristics, genetic data, clinical investigations, medications, and disease-related events. Four classification models were applied to model the risk level, and their decisions were explained using the SHAP (SHapley Additive exPlanations) method. Unwanted cardiac events were defined as sustained ventricular tachycardia occurrence (VT), heart failure (HF), ICD activation, sudden cardiac death (SCD), cardiac death, and all-cause death. Results: The proposed machine learning approach outperformed the similar existing risk-stratification models for SCD, cardiac death, and all-cause death risk-stratification: it achieved higher AUC by 17%, 9%, and 1%, respectively. The boosted trees achieved the best performing AUC of 0.82. The resulting model most accurately predicts VT, HF, and ICD with AUCs of 0.90, 0.88, and 0.87, respectively. Conclusions: The proposed risk-stratification model demonstrates high accuracy in predicting events in patients with hypertrophic cardiomyopathy. The use of a machine-learning risk stratification model may improve patient management, clinical practice, and outcomes in general
Underlying motivating factors for movie-induced tourism among Emiratis and Indian expatriates in the United Arab Emirates
No full text© The Author(s) 2020. This study explores the underlying motivating factors of Emiratis and Indian expatriates to visit destinations featured in movies. The results revealed a five-factor structure for Indians: novelty, fantasy-driven, personal connection, prestige and movie connection, vis-à-vis a three-factor structure for Emiratis: novelty, fantasy-driven and personal connection with the movie location. An analyses of variance analysis (ANOVA) showed significant differences in the individual mean scores of items, with the exception of novelty. Regarding gender, while no significant differences were found between the male and female Indian expatriate populations across all factors and underlying items, differences for some items were observed between male and female Emiratis, with male participants demonstrating higher motivation than female participants. These findings support the notion that the underlying factors influencing movie-induced tourism differ between cultures, while the influence of gender was found to be limited. Overall, the study enhances the understanding of practitioners and policymakers tasked with attracting tourists to movie destinations
FHBF: Federated hybrid boosted forests with dropout rates for supervised learning tasks across highly imbalanced clinical datasets
No full textAlthough several studies have deployed gradient boosting trees (GBT) as a robust classifier for federated learning tasks (federated GBT [FGBT]), even with dropout rates (federated gradient boosting trees with dropout rate [FDART]), none of them have investigated the overfitting effects of FGBT across heterogeneous and highly imbalanced datasets within federated environments nor the effect of dropouts in the loss function. In this work, we present the federated hybrid boosted forests (FHBF) algorithm, which incorporates a hybrid weight update approach to overcome ill-posed problems that arise from overfitting effects during the training across highly imbalanced datasets in the cloud. Eight case studies were conducted to stress the performance of FHBF against existing algorithms toward the development of robust AI models for lymphoma development across 18 European federated databases. Our results highlight the robustness of FHBF, yielding an average loss of 0.527 compared with FGBT (0.611) and FDART (0.584) with increased classification performance (0.938 sensitivity, 0.732 specificity). © 2023 The Author(s
