Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

BACKGROUND: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. METHODS: Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. RESULTS: Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. CONCLUSIONS: These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.

Prometheus Framework for Fuzzy Information Retrieval in Semantic Spaces

This paper introduces a novel vision for further enhanced Internet of Things services. Based on a variety of data (such as location data, ontology-backed search queries, in- and outdoor conditions) the Prometheus framework is intended to support users with helpful recommendations and information preceding a search for context-aware data. Adapted from artificial intelligence concepts, Prometheus proposes user-readjusted answers on umpteen conditions. A number of potential Prometheus framework applications are illustrated. Added value and possible future studies are discussed in the conclusion.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

A numerical model for inert gas transport in the lung based on fractal airway morphology

Patients suffering from cystic fibrosis (CF) show thick secretions, mucus plugging and bronchiectasis in bronchial and alveolar ducts. This results in substantial structural changes of the airway morphology and heterogeneous ventilation. Disease progression and treatment effects are monitored by so-called gas washout tests, where the change in concentration of an inert gas is measured over a single or multiple breaths. The result of the tests based on the profile of the measured concentration is a marker for the severity of the ventilation inhomogeneity strongly affected by the airway morphology. However, it is hard to localize underlying obstructions to specific parts of the airways, especially if occurring in the lung periphery. In order to support the analysis of lung function tests (e.g. multi-breath washout), we developed a numerical model of the entire airway tree, coupling a lumped parameter model for the lung ventilation with a 4th-order accurate finite difference model of a 1D advection-diffusion equation for the transport of an inert gas. The boundary conditions for the flow problem comprise the pressure and flow profile at the mouth, which is typically known from clinical washout tests. The natural asymmetry of the lung morphology is approximated by a generic, fractal, asymmetric branching scheme which we applied for the conducting airways. A conducting airway ends when its dimension falls below a predefined limit. A model acinus is then connected to each terminal airway. The morphology of an acinus unit comprises a network of expandable cells. A regional, linear constitutive law describes the pressure-volume relation between the pleural gap and the acinus. The cyclic expansion (breathing) of each acinus unit depends on the resistance of the feeding airway and on the flow resistance and stiffness of the cells themselves. Special care was taken in the development of a conservative numerical scheme for the gas transport across bifurcations, handling spatially and temporally varying advective and diffusive fluxes over a wide range of scales. Implicit time integration was applied to account for the numerical stiffness resulting from the discretized transport equation. Local or regional modification of the airway dimension, resistance or tissue stiffness are introduced to mimic pathological airway restrictions typical for CF. This leads to a more heterogeneous ventilation of the model lung. As a result the concentration in some distal parts of the lung model remains increased for a longer duration. The inert gas concentration at the mouth towards the end of the expirations is composed of gas from regions with very different washout efficiency. This results in a steeper slope of the corresponding part of the washout profile.

Hans Jürgen auf dem Berliner Corso : ein Schwank in plattdeutscher Mundart

Welsch (Projektbearbeiter): Gedicht über den Bauern Hans Jürgen, der nach dem Verkauf seines Roggens in Berlin einen Stadtbummel unternimmt, sich die Börse anschaut, in den "Corso" (Straßenumzug) gerät und dort im Gedränge seines Reiseproviants verlustig geht. Nach dem eher unpolitischen Inhalt zu urteilen stammt das Werkchen nicht aus dem Jahre 1848

Brenneke als Steuer-Verweigerer vor den Geschwornen

Welsch (Projektbearbeiter): Justizposse: Gerichtsverhandlung gegen den der Steuerverweigerung bezichtigen Berliner Erdarbeiter (Rehberger) Brenneke. Mit zahlreichen politischen Anspielungen

Humoristischer Rückblick auf das Jahr 1848 : Das ganze Jahr achtzehnhundert vierzig und acht, In wunderschöne Reime gebracht

Welsch (Projektbearbeiter): Humoristisch/satirische Kommentierung der Berliner politischen Geschehnisse des Jahres 1848. In zwölf (Monats-)Gedichten

Sie fragen mir: was issen des? der demokratische Congreß: Die Reaction wird lahm jeritten, sie läßt um stillet Beileid bitten: Eene spaßhaftige Underhaltung zwischen eenen reaktionären Esel un den demokratischen Ullo Bohmhammel, Vize-Jefreiten bei de Börjerwehr: Loof doch zu, oller gribenoscher Pejasus ...

Welsch (Projektbearbeiter): Launige, mit einer Vielzahl politischer Anspielungen gespickte Kommentierung der politischen Geschehnisse aus demokratischer Sicht. Wahrscheinlich anläßlich des ersten Kongresses der Demokraten Deutschlands vom 14. - 17. Juni 1848 in Frankfurt/Main, auf dem der Beschluß gefaßt wurde, in Berlin einen 'Central-Ausschuß' zu installieren [Hachtmann; Berlin 1848, S. 642]

Eene drömerige Viehsion: Det neue Stück, die Revlution, Is jetzt beim dritten Akte schon, Der zweete ward damit geschlossen, det man zwölf Menschen doot geschossen: Det war'n proprer Zug, der Leichenzug an'n 20. Oktober! ...

Welsch (Projektbearbeiter): Bitter-sarkastische Kommentierung der Beisetzung der am 16. Oktober 1848 in einer gewalttätigen Auseinandersetzung mit der Bürgerwehr getöteten Arbeiter: "Die zehn Särge voll Errungenschaften ... waren ... ufgeputzt mit Blumen un Kränze wie der 19. März mit Versprechungen; aber mit den eigentlichen Inhalt sind et hier wie da reene Leichen."

Etsch! Etsch! Et is ja widder nich losgegangen: Ihr habt euch schonst widder blamirt! Strafpredigt von Madam Bohmhammeln gehalten den 25. Septbr. uffen Abend an ihren Jemahl Ullo Bohmhammel, Vice-Gefreiten bei de Börgerwehr: Nu sage mich mal Ullo, bist Du blos alleene besoffen oder is de ganze Welt verrückt? ...

Welsch (Projektbearbeiter): Heftige Kritik an der aus Sicht des Verfassers zaudernden und zu wenig entschiedenen Politik der Linken in der Preußischen Nationalversammlung in Form einer Strafpredigt der Madame Bohmhammel an ihren Gatten Ullo. Hintergrund: die Debatte über die sogenannte 'Interpellation Pax', betreffend den 'reaktionären Geist' in der preußischen Armee (25. September 1848): " ... det seh ick doch in, des sich die Linkschen in de Kammer widder mitten Löffel über't Maul haben wischen lassen." Warnung vor General Wrangel, der 50.000 Mann rings um Berlin zusammengezogen hat

Ganz Berlin muß nach Wien, Jegen die Croaten ziehn! Dann is Windischjrätz verloren! Karbe hat es uns jeschworen: Eene Rede, jered't von Herr Karben, am 31. October, uf de Kummödientreppe, un wortjetrei abjekuppirt: Freinde! Brüder! Mitbürger! Berliner! Souveränet Volk! ...

Welsch (Projektbearbeiter): Satire auf den Werdegang der sehr weitgehenden und radikalen Forderungen der Berliner Volksredner, der immer nach demselben Muster abläuft: die Forderungen werden auf einer Volksversammlung vom Redner formuliert und von der Versammlung gutgeheißen. Darauf wird eine Deputation gebildet, die die fraglichen Forderungen der Nationalversammlung als Petition übergibt. Nach dem Empfang der Deputation durch die Nationalversammlung macht sich der Abgeordnete D'Ester die Petition zu eigen und übergibt sie dem Präsidenten, der sie in die Kommissionen verweist. Die Kommissionen wiederum beraten darüber und erstatten am nächsten Tag Bericht. "Un so war et, un so is et, un so wird et bleiben." Geschildert vor dem Hintergrund der Niederschlagung der Wiener Revolution am Beispiel einer - fiktiven - Rede von Adolph Friedrich Karbe