Untersuchungen zur Charakterisierung und zu biologischen Wirkungen von Exosome-like Nanopartikeln pflanzlichen Ursprungs

Bei der Betrachtung von „Exosome-like Nanopartikeln“ eröffnet sich zunächst die Frage: Was sind Exosomen? Als eine Klasse extrazellulärer Vesikel (EVs) gewannen sie in den vergangenen zwei Dekaden zunehmend an Bedeutung. Die Bildung und Freisetzung von EVs ist ein Prozess, der bei praktisch jedem Lebewesen und jeder Zelle aller Domänen stattfindet. Die physiologische Bedeutung von EVs wurde lange Zeit unterschätzt und bis heute nicht umfassend verstanden. Dass die Bildung und Sekretion auf verschiedenen, zum Teil redundanten Mechanismen basieren kann, deutet auf ihre enorme Wichtigkeit hin. Auch wenn die Fracht, insbesondere kleine nichtkodierende RNA (sRNA), eine zentrale Rolle für die Funktionalität der EVs spielt, so handelt sich doch um komplexe und hochfunktionale Gebilde, bei denen alle Komponenten aufeinander abgestimmt sind. Der Hülle kommen dabei offenbar ebenfalls zentrale Aufgaben zu, denn sie schützt nicht nur die teils fragile Fracht, sondern adressiert auch die Zielzelle, kann mit ihr interagieren und ermöglicht die Aufnahme. Pflanzliche Exosome-like Nanopartikel wurden in praktisch allen Organen und Zellen – egal ob lebend, getrocknet oder im Labor kultiviert – gefunden. Es ist anzunehmen, dass sie vielfältige Funktionen erfüllen, darunter interzellularer Informationsaustausch im Sinne der Homöostase und Abwehr pathogener Mikroorganismen. Das verwendete Ausgangsmaterial bzw. die Isolationstechnik ist entscheidend für die Nomenklatur der Exosome-like Nanopartikel, denn wird das Pflanzengewebe homogenisiert, stammen diese „Nanovesikel“ nicht ausschließlich aus dem extrazellulären Raum. Ob und wie sich pflanzliche Nanovesikel von „echten“ EVs unterscheiden, ist und bleibt ungeklärt. Im Rahmen der vorliegenden Dissertation konnten aus Calluskulturen EVs und Nanovesikel isoliert werden. Das schafft nicht nur eine wichtige Grundlage für zukünftige Untersuchungen, sondern auch für eine Produktion von pflanzlichen Nanovesikeln für den pharmazeutischen Einsatz unter gut definierbaren Bedingungen. Da gezeigt werden konnte, dass Nanovesikel die Trocknung von Pflanzenmaterial überstehen können, stellen pflanzliche Drogen ebenso eine mögliche Quelle für neue Vehikel zum Arzneistofftransport dar. Aus einer Auswahl verschiedener Isolationsmethoden zeigte sich die differentielle Hochgeschwindigkeitszentrifugation für die Anreicherung der untersuchten Vesikel als besonders geeignet, obwohl sie mit weniger aufwändigen Techniken konkurrierte. Die morphologischen Eigenschaften der häufig zwischen 100 und 500 nm kleinen Strukturen wurden in der Regel am Transmissionselektronenmikroskop (TEM) begutachtet und von EVs aus Nicotiana tabacum L. zusätzlich am Kryo-TEM. Die Partikelgrößenbestimmung der pflanzlichen Nanovesikel war nur eingeschränkt möglich und die Aggregation der Vesikel machte eine zusätzliche Aufarbeitung notwendig, führte dann jedoch gemeinsam mit den elektronenmikroskopischen Aufnahmen zu einer nicht unrealistischen Einschätzung der Partikelgrößen und zu dem Schluss, dass jede Pflanzenspezies Vesikel individueller Größe produziert. Ebenso individuell waren die Ausbeuten an isolierten Vesikeln. Sie hingen sowohl von der Spezies, als auch vom verwendeten Ausgangsmaterial ab, wobei aus allen untersuchten Pflanzenteilen Nanovesikel isoliert werden konnten. Zur Einschätzung der Menge an isolierten Vesikeln diente die Proteinkonzentration als Surrogatparameter. Während der Membranfarbstoff 3,3'-Dihexyloxacarbocyaniniodid (DiOC6) die Ausbeute erhöhte, konnte er nicht zur Vesikelquantifizierung nutzbar gemacht werden. Die Proteinanalytik von Nanovesikeln aus Drogen zeigte, dass das Proteom bei einigen (nicht allen) Drogen während der Trocknung erheblichen Veränderungen unterlag. Elektronenmikroskopisch konnten dennoch morphologisch intakte Vesikel beobachtet werden, was auf eine zentrale Rolle der Lipide für die Membranintegrität hindeutete. Die massenspektrometrische Identifizierung ausgewählter Proteine zeigte, dass zusätzlich zu der etablierten Isolation durch differentielle Zentrifugation weitere Reinigungsschritte notwendig sind. Es konnte nachgewiesen werden, dass sich die Agarose-Gelelektrophorese grundsätzlich für diesen Zweck einsetzen lässt und die Nanovesikel mithilfe von DiOC6 gleichzeitig im Gel visualisiert werden können, auch wenn die Effizienz bei der Rückgewinnung der Vesikel aus dem Agarosegel noch Optimierung bedarf. Chromatographische Untersuchungen ergaben ein heterogenes Bild bezüglich des Vorkommens von Sekundärmetaboliten. Dass vorwiegend lipophile Substanzen in den Nanovesikeln angereichert waren, deutete darauf hin, dass diese von der Pflanze nicht aktiv in die Nanovesikel eingebracht worden sind. Mit der Etablierung eines HPTLC-Systems zur Analytik der Phospholipide wurden erste Schritte zur Aufklärung von deren Bedeutung für die Hülle der untersuchten Nanovesikel unternommen. Diese scheint für die intestinale Resorption eine wesentliche Rolle zu spielen. Obwohl erste Daten zeigten, dass pflanzliche Nanovesikel meist nur ein geringes inhibitorisches Potential gegenüber der Pankreaslipase aufwiesen, gelangen diese bei einer vielseitigen Ernährung in der Regel in Kombination mit potenten Inhibitoren verschiedener Verdauungsenzyme in das Duodenum. Damit ist es wahrscheinlich, dass ein relevanter Anteil der ingestierten Vesikel aus Nahrungsmitteln resorbiert werden und deren Fracht so ihre Wirkung auf den humanen Metabolismus entfalten kann. Insgesamt handelt es sich bei Untersuchungen zur Charakterisierung und zu biologischen Wirkungen von Exosome-like Nanopartikeln pflanzlichen Ursprungs um ein vielschichtiges Gebiet mit dem Potential neue Ansätze für die Pharmakotherapie zu entwickeln, dessen Facettenreichtum noch einige offene Fragen bereithält.The first question coming up, dealing with „exosome-like nanoparticles“ would appear: What are exosomes? As a class of extracellular vesicles (EVs), they attracted increasing attention during the past two decades. Formation and release of EVs is a process that apparently comes to pass in every living organism or cell of all domains of life. The physiological role of EVs has been underestimated for a long term and it has not yet been comprehensively understood. Formation and release of EVs is based on different, partially redundant mechanisms, indicating their great importance. Even though cargo, especially small noncoding RNA (sRNA), plays a key role in EVs´ functionality, these vesicles are highly complex structures with well-coordinated mixtures of bio-molecules. The envelope appears to be responsible for crucial tasks like protecting and addressing the cargo, interaction with and uptake to targeted cells. Plant derived exosome-like nanoparticles have been found in practically every organ and cell – no matter if living, dried or lab-cultivated. It is assumed, that they accomplish a diverse set of tasks like inter-cellular information exchange in the manner of homeostasis and defense against pathogenic microorganisms. Nomenclature of exosome-like nanoparticles depends on the used raw material and isolation technique. If „nanovesicles“ are isolated from homogenates, they do not necessarily originate from extracellular space but if and how they are different from „genuine“ EVs remains puzzling. During the thesis project at hand, EVs and nanovesicles have been firstly isolated from callus cultures, building a sound foundation not only for further characterization but for the production of plant nanovesicles for pharmaceutical purposes under controlled conditions. Additionally, it has been proven that nanovesicles overcome the drying process of plant material. Herbal drugs can therefore stand as sources of vehicles for drug transport and targeting. Among the repertory of possible isolation techniques, differential high performance centrifugation was especially suitable, although less laborious alternatives were available. Transmission electron microscopy (TEM) was the means of choice for assessing morphological properties of the usually 100 to 500 nm small structures. EVs from Nicotiana tabacum L. also have been visualized using a Kryo-TEM device. Although vesicle aggregates troubled particle size analysis, combining data with TEM results, realistic estimations revealed individual sizing of nanovesicles from different plant species. Likewise, yields of isolated vesicles were individual, depending on species as well as on raw material used. Thereby, nanovesicles could be isolated from all investigated plant organs. Protein concentration stood surrogate for vesicle amounts. 3,3'-Dihexyloxacarbocyaniniodid (DiOC6) could not be utilized to quantify vesicles, but helped increasing yields. Proteomics of nanovesicles from herbal drugs demonstrated severe protein alteration in some, not all, dried plants. Nevertheless, TEM images displayed morphologically undamaged nanovesicles indicating a crucial role of lipids for the integrity of vesicle membranes. Mass spectrometric protein identification showed the necessity of further purification after differential centrifugation. Agarose gel electrophoresis can be exploited for this purpose and adding DiOC6 enables a simultaneous visualization of nanovesicles within the gel. Merely the efficiency of vesicle recovery needs some further improvement. Chromatographic explorations revealed a heterogenic array regarding the occurrence of secondary metabolites. Since mainly lipophilic molecules were found to be enriched within the vesicle fraction, it appears plants do not actively package secondary metabolites into nanovesicles. With the establishment of a HPTLC procedure for phospholipid profiling, first steps towards the elucidation of the vesicle envelope´s importance have been made. The envelope is assumed to be essential for intestinal resorption. Although first data showed only slight inhibitory potential for plant derived nanovesicles on pancreatic lipase, in a diversified diet food-derived nanovesicles are usually taken up together with potent inhibitors of distinct digestive enzymes, making it likely that a relevant portion of the ingested vesicles may be absorbed and allow their cargo to impact human metabolism. Conclusively, investigations on characterization and biological effects of exosome-like nanoparticles of plant origin comprise complex issues with the potential of developing new approaches for pharmacotherapy. This topic´s multifaceted nature holds ready a couple of unresolved questions

Sloshing of a bubbly magma reservoir as a mechanism of triggered eruptions

open4siLarge earthquakes sometimes activate volcanoes both in the near field as well as in the far field. One possible explanation is that shaking may increase the mobility of the volcanic gases stored in magma reservoirs and conduits. Here experimentally and theoretically we investigate how sloshing, the oscillatory motion of fluids contained in a shaking tank, may affect the presence and stability of bubbles and foams, with important implications for magma conduits and reservoirs. We adopt this concept from engineering: severe earthquakes are known to induce sloshing and damage petroleum tanks. Sloshing occurs in a partially filled tank or a fully filled tank with density-stratified fluids. These conditions are met at open summit conduits or at sealed magma reservoirs where a bubbly magma layer overlays a newly injected denser magma layer. We conducted sloshing experiments by shaking a rectangular tank partially filled with liquids, bubbly fluids (foams) and fully filled with density-stratified fluids; i.e., a foam layer overlying a liquid layer. In experiments with foams, we find that foam collapse occurs for oscillations near the resonance frequency of the fluid layer. Low viscosity and large bubble size favor foam collapse during sloshing. In the layered case, the collapsed foam mixes with the underlying liquid layer. Based on scaling considerations, we constrain the conditions for the occurrence of foam collapse in natural magma reservoirs. We find that seismic waves with lower frequencies < 1 Hz, usually excited by large earthquakes, can resonate with magma reservoirs whose width is > 0.5 m. Strong ground motion > 0.1 m s− 1 can excite sloshing with sufficient amplitude to collapse a magma foam in an open conduit or a foam overlying basaltic magma in a closed magma reservoir. The gas released from the collapsed foam may infiltrate the rock or diffuse through pores, enhancing heat transfer, or may generate a gas slug to cause a magmatic eruption. The overturn in the magma reservoir provides new nucleation sites which may help to prepare a following/delayed eruption. Mt. Fuji erupted 49 days after the large Hoei earthquake (1707) both dacitic and basaltic magmas. The eruption might have been triggered by magma mixing through sloshing.Acknowledgments We thank Ralf Bauz, Stefan Mikulla, and Peter Neuendorf for their help in the GFZ bubble-lab, and Matthias Rosenau for providing the glucose syrup. Helpful and careful reviews by David P. Hill and an anonymous reviewer, and the journal editor, Lionel Wilson, and Maggie Mangan are deeply appreciated. This work was supported by KAKENHI 24681035, the European Union through the ERC StG project CCMP-POMPEI, grant number 230583, and the European Supersite project MED-SUV, grant number 308665.openNamiki, Atsuko; Rivalta, Eleonora; Woith, Heiko; Walter, Thomas R.Namiki, Atsuko; Rivalta, Eleonora; Woith, Heiko; Walter, Thomas R

Comment on “Potential short‐term earthquake forecasting by farm animal monitoring” by Wikelski, Mueller, Scocco, Catorci, Desinov, Belyaev, Keim, Pohlmeier, Fechteler, and Mai

Based on an analysis of continuous monitoring of farm animal behavior in the region of the 2016 M6.6 Norcia earthquake in Italy, Wikelski et al., 2020; (Seismol Res Lett, 89, 2020, 1238) conclude that animal activity can be anticipated with subsequent seismic activity and that this finding might help to design a "short-term earthquake forecasting method." We show that this result is based on an incomplete analysis and misleading interpretations. Applying state-of-the-art methods of statistics, we demonstrate that the proposed anticipatory patterns cannot be distinguished from random patterns, and consequently, the observed anomalies in animal activity do not have any forecasting power

A Holistic Exploration of Fatigue Experienced by Women before a Myocardial Infarction

Most women report fatigue in the weeks and months leading to a myocardial infarction (MI). However, fatigue is a complex phenomenon. Dimensions of this MI related fatigue, such as timing, distress, intensity, quality, patterns, and associated characteristics have not been established through focused study of this symptom. A lack of a holistic understanding of this symptom makes clinical decision making difficult, given that fatigue is a frequently reported symptom overall

Physico-chemical behaviour of underground waters after the october 1, 1995 Dinar earthquake, SW Turkey

On the evening of October 1, 1995, a MS46.1 earthquake destroyed the city of Dinar, SW Turkey. Within 48 hours after the main shock, a team of the German Earthquake Task Force arrived in the area to investigate possible earthquake-related changes in the physico-chemical composition of shallow and deep groundwaters. A mapping was performed to characterise different groundwater types and a continuously monitoring station was installed within the geothermal field of Afyon. Repeated measurements, performed 1, 6, 12 and 18 months after the event, reveal post-seismic changes in water discharge, water temperature, and conductivity. We will focus on the changes of spring water discharge observed in the vicinity of the epicentre. In the first month after the earthquake the groundwater discharge increased at springs located within the down-thrown block, whereas a slight decrease was observed at sites on the hanging wall

Women’s Prodromal Myocardial Infarction Symptom Perception, Attribution, and Care Seeking

Most women experience several prodromal symptoms prior to myocardial infarction (MI). While investigators have focused on acuteMI symptom perception, attribution, and care seeking, few have studied how women experience and process prodromalMI symptoms and ultimately decide to seek care

Seismological and Geophysical Signatures of the Deep Crustal Magma Systems of the Cenozoic Volcanic Fields Beneath the Eifel, Germany

The Quaternary volcanic fields of the Eifel (Rhineland-Palatinate, Germany) had their last eruptions less than 13,000 years ago. Recently, deep low-frequency (DLF) earthquakes were detected beneath one of the volcanic fields showing evidence of ongoing magmatic activity in the lower crust and upper mantle. In this work, seismic wide- and steep-angle experiments from 1978/1979 and 1987/1988 are compiled, partially reprocessed and interpreted, together with other data to better determine the location, size, shape, and state of magmatic reservoirs in the Eifel region near the crust-mantle boundary. We discuss seismic evidence for a low-velocity gradient layer from 30–36 km depth, which has developed over a large region under all Quaternary volcanic fields of the Rhenish Massif and can be explained by the presence of partial melts. We show that the DLF earthquakes connect the postulated upper mantle reservoir with the upper crust at a depth of about 8 km, directly below one of the youngest phonolitic volcanic centers in the Eifel, where CO2 originating from the mantle is massively outgassing. A bright spot in the West Eifel between 6 and 10 km depth represents a Tertiary magma reservoir and is seen as a model for a differentiated reservoir beneath the young phonolitic center today. We find that the distribution of volcanic fields is controlled by the Variscan lithospheric structures and terrane boundaries as a whole, which is reflected by an offset of the Moho depth, a wedge-shaped transparent zone in the lower crust and the system of thrusts over about 120 km length. ©2020. The Authors

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites, Proteins and Lipids with Perspectives on Their Potential and Sources

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles

Investigating the Origin of Seismic Swarms

According to the U.S. Geological Survey’s Earthquake Hazards Program, a seismic swarm is “a localized surge of earth- quakes, with no one shock being conspicuously larger than all other shocks of the swarm. They might occur in a variety of geologic environments and are not known to be indicative of any change in the long- term seismic risk of the region in which they occur” (http://vulcan.wr.usgs.gov/Glossary/ Seismicitydescription_earthquakes.html). The definition reveals how little is actually known about seismic swarms. For example, could certain seismic settings be more prone to swarms? Could a fault zone prone to large energetic earthquakes release part of its stress through seismic swarms? Do swarms keep hazards in balance, or could their onset increase hazards? To gain insight into the nature of seismic swarms in nonvolcanic areas and to better understand their influence on seismic hazards, the Istituto Nazionale di Geofisica e Vulcanologia (INGV) and the German Research Centre for Geoscience (GFZ) began a combined research project within the framework of the Network of European Research Infrastructures for Earthquake Risk Assessment and Mitigation (NERA; see http:// www.nera-eu.org/). The project focused on monitoring swarm activity occurring in the Pollino range in Southern Apennines, Italy.Published361-3721.1. TTC - Monitoraggio sismico del territorio nazionaleN/A or not JCRrestricte

Targeted suicide gene transfections reveal promising results in nu/nu mice with aggressive neuroblastoma

Neuroblastoma represents the third most common malign neoplasm occurring in children and the most common in newborn. Although mortality in childhood cancer declined in the last decade, high-risk patients have poor prospects, due to the aggressiveness of the cancer. In the recent past, we underlined the potential of sapofectosid as novel and efficient transfection enhancer, demonstrating non-toxic gene delivery, but its value in tumor therapies has yet to be elucidated. A suicide gene, coding for saporin, a ribosome-inactivating protein type I, was incorporated into targeted, peptide-based nanoplexes. The nanoplexes were characterized for their size, zeta potential and appearance by electron microscopy. Gene delivery was observed via confocal imaging. In vitro transfections were conducted to monitor the real-time cell viability. After initial tolerability studies, NMRI nu/nu-mice bearing tumors from Neuro-2A-Luc-cells (murine neuroblastoma cells, transduced with a luciferase gene), were treated with targeted nanoplexes (30 μg saporin-DNA i.v./treatment) and sapofectosid (30 μg s.c. treatment). The treatment was compared to a vehicle (PBS) control and treatment without sapofectosid in terms of body weight, tumor growth and integrated density of tumor luminescence. The study revealed an anti-tumoral effect of the sapofectosid mediated gene therapy in the Neuro-2A-tumor model. The treatments were well tolerated by the animals indicating the applicability of this approach. With these results, we were able to proof the efficacy of a therapy, consisting of targeted suicide gene nanoplexes and sapofectosid, a novel and potent transfection enhancer. This study points out the enormous value for future targeted cancer and gene therapies