Generation and characterization of stable, highly concentrated titanium dioxide nanoparticle aerosols for rodent inhalation studies

The intensive use of nano-sized titanium dioxide (TiO2) particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of TiO2 nanoparticles (NP) with biological systems ideally needs to be investigated using physico-chemically uniform and well-characterized NP. In this article, we describe the reproducible production of TiO2 NP aerosols using spark ignition technology. Because currently no data are available on inhaled NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation studies in rodents. For anticipated in vivo dosimetry analyses, TiO2 NP were radiolabeled with 48V by proton irradiation of the titanium electrodes of the spark generator. The dissolution rate of the 48V label was about 1% within the first day. The highly concentrated, polydisperse TiO2 NP aerosol (3–6 × 106 cm−3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation, and number concentration. Extensive characterization of NP chemical composition, physical structure, morphology, and specific surface area was performed. The originally generated amorphous TiO2 NP were converted into crystalline anatase TiO2 NP by thermal annealing at 950 °C. Both crystalline and amorphous 20-nm TiO2 NP were chain agglomerated/aggregated, consisting of primary particles in the range of 5 nm. Disintegration of the deposited TiO2 NP in lung tissue was not detectable within 24 h.

A guide to sparkology: the taxonomy of elementary cellular Ca2+ signaling events

Since the discovery of the Ca(2+) spark as an elementary event of cellular Ca(2+) signaling almost 15 years ago, the family of newly described Ca(2+) signal entities has been ever growing. While scientists working in Ca(2+) signaling may have maintained an overview over the specifics of this nomenclature, those outside the field often make the complaint that they feel hopelessly lost. With the present review we collect and summarize systematic information on the many Ca(2+) signaling events described in a variety of tissues and cells, and we emphasize why and how each of them has its own importance. Most of these signals are taking place in the cytosol of the respective cells, but several events have been recorded from intracellular organelles as well, where they may serve their own specific functions. Finally, we also try to convey an integrated view as to why cellular microdomain signaling is of fundamental biological importance.

NO-dependent CaMKII activation during -adrenergic stimulation of cardiac muscle

AIMS:During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. METHODS AND RESULTS:Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. CONCLUSIONS:β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.

"Eventless" InsP 3 -dependent SR-Ca 2+ Release Affecting Atrial Ca 2+ Sparks

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca2+ release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca2+-induced Ca2+ release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca2+ release event frequency (4.14±0.72 vs. 6.20±0.76 events (100 μm)−1 s−1). These individual photolytically triggered Ca2+ release events were identified as Ca2+ sparks, which originated from RyR openings. This was verified by Ca2+ spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca2+ release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca2+ flux but eventless SR-Ca2+ release through InsP3R were characterized using SR-Ca2+ leak/SR-Ca2+ load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca2+ spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca2+ flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Gold nanoparticle aerosols for rodent inhalation and translocation studies

The intensive use of nano-sized particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of nanoparticles (NP) with biological systems after various routes of exposure needs to be investigated using well-characterized NP. We report here on the generation of gold-NP (Au-NP) aerosols for inhalation studies with the spark ignition technique, and their characterization in terms of chemical composition, physical structure, morphology, and specific surface area, and on interaction with lung tissues and lung cells after 1 h inhalation by mice. The originally generated agglomerated Au-NP were converted into compact spherical Au-NP by thermal annealing at 600 °C, providing particles of similar mass, but different size and specific surface area. Since there are currently no translocation data available on inhaled Au-NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation in rodents. For anticipated in vivo systemic translocation and dosimetry analyses, radiolabeled Au-NP were created by proton irradiating the gold electrodes of the spark generator, thus forming gamma ray emitting 195Au with 186 days half-life, allowing long-term biokinetic studies. The dissolution rate of 195Au from the NP was below detection limits. The highly concentrated, polydisperse Au-NP aerosol (1–2 × 107 NP/cm3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation and number concentration. After collection on filters particles can be re-suspended and used for instillation or ingestion studies.

La figure de la sorcière dans la littérature du XIXe siècle (Jules Michelet, George Sand, Eliza Orzeszkowa)

The nineteenth century uncovered and analysed the tragic episodes of witch-hunting and ‘witch’ trials common in Renaissance Europe. Fascinating not only to historians, this subject also inspired men of letters who popularized the image of the witch as an old, ugly and evil person, who thus deserved her lot. Jules Michelet’s La sorcière of 1862 takes a very different approach. Simultaneously a literary and historical work, the book proved scandalous as it rehabilitated the figure of the witch, shedding favourable light on her image: it was the witch who was able to save a last spark of humanity in moments of despair; it was she who acted as comforter and healer to the people. In the context of nineteenth-century literature, certain works by female authors that focused on ‘witches,’ stand out. Whilst certain male authors (Michelet included) presented the witch as a figure from the past, who had finally perished in the 17th century, texts such as George Sand’s La petite Fadette (1848) or Eliza Orzeszkowa’s Dziurdziowie (1885), suggest that the end of witch trials did not imply an end to accusations, persecutions, and even executions of ‘witches’ – and, that in terms of culture, witchcraft or sorcery had not disappeared from the societies they knew.

Does World Natural Heritage status trigger sustainable regional development efforts?

Today, there are over 200 World Natural Heritage (WNH) sites. Although the original aim of the World Heritage (WH) Convention was to spark off concerted international efforts to preserve sites of outstanding and universal value, today a multitude of expectations rests on WNH sites in terms of conservation, tourism, management and regional development. This paper identifies the effects of WNH status on sustainable regional development and the driving factors behind these effects. The results are based on a global survey of WNH sites and qualitative interviews with key WNH personnel. The paper shows that WNH status can be an important trigger for sustainable regional development, but its effectiveness depends on a number of intricately interwoven ‘soft’ success factors. Clearer policies and management guidelines, as envisaged by UNESCO, are crucial to achieving a balance between conservation and development.

Maximal acceleration of calcium release refractoriness by β-adrenergic stimulation requires dual activation of protein kinase A and CaMKII in mouse ventricular myocytes

Time-dependent refractoriness of calcium (Ca2+) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca2+ spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca2+ spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca2+ sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark- to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca2+/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen) without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca2+ release refractoriness in mouse myocytes.

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca2+ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca2+ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca2+ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca2+ signaling in situ, by analyzing Ca2+ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca2+ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca2+ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca2+ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca2+ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca2+ release events.

Implications for the Discovery of Extraterrestrial Life: A Theological Approach

Recent remarks of Pope Francis spark anew an important discussion: are we alone in the Universe? The article follows traces of the idea of extraterrestrial life throughout philosophy, evaluates the current considerations about the probability of extraterrestrial life and discusses the potential implications for the discovery of such life from a theological point of view. This “thought experiment” covers basic insights on creation, revelation and redemption.