Eine Untersuchung des Zusammenhangs von psychosozialer Gesundheit mit MOBAK- Ergebnissen und ausserschulischer physischer Aktivität bei Erstklässlern

Hintergrund Weltweit leiden etwa 10 bis 20 Prozent der Kinder und Jugendlichen an psychoso-zialen Problemen. Ein aktiver Lebensstil und regelmässige sportliche Betätigung können präventiv und therapeutisch gegen psychosoziale Probleme wirken. Da mo-torische Basiskompetenzen Bedingung für eine Teilhabe an der Bewegungskultur sind, galt es zu untersuchen, ob motorische Basiskompetenzen direkt mit der psy-chosozialen Gesundheit zusammenhängen. Methode In der MOBAK-Test-Reihe wurden die motorischen Basiskompetenzen, aufgeteilt in je vier Test-Items zu Sich-Bewegen und Etwas-Bewegen, von Erstklässlern in Frankfurt getestet. In der parallel laufenden SKIB-Studie wurden ausserdem ihre psychosoziale Gesundheit und physische Aktivität durch Elternbefragung mittels des KINDL-Fragebogens ermittelt. In der vorliegenden Arbeit wurden alle Zusam-menhänge zwischen den Ergebnissen des KINDL-Fragebogens zur psychosozialen Gesundheit, den Ergebnissen der MOBAK-Tests und der ausserschulischen physi-schen Aktivität empirisch untersucht. Ergebnisse Während die Ergebnisse von Sich-Bewegen in signifikantem Zusammenhang so-wohl mit der psychosozialen Gesundheit als Gesamtkonstrukt als auch mit einzel-nen Aspekten der psychosozialen Gesundheit stehen, sind mit den Ergebnissen bei den Test-Items zu Etwas-Bewegen keine signifikanten Zusammenhänge zu erken-nen. Der Umfang und die Art des ausserschulischen Sports korrelieren signifikant positiv mit der psychosozialen Gesundheit. Diskussion Mit dem empirischen Beweis der positiven Zusammenhänge von motorischen Ba-siskompetenzen, psychosozialer Gesundheit und ausserschulischem Sport, wird die Wichtigkeit von Bewegung für Kinder und Jugendliche deutlich. Diese Studie bildet die Grundlage für weitere Untersuchungen bezüglich spezifischer For-schungsfragen hinsichtlich der Kausalitäten der gefundenen Zusammenhänge. Bei-spielsweise zeigen die klaren Unterschiede zwischen Mädchen und Knaben, dass weitere Studien Aufschluss über geschlechterspezifische Förderungsmöglichkeiten geben können oder Schulsport durch gezielte Ausrichtung einen positiven Effekt auf die psychosoziale Gesundheit haben kann

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer

Heat inactivation of fetal bovine serum increases protein contamination of extracellular vesicles

Introduction: Extracellular vesicles (EVs) released in cell cultures are influenced by the cell culture conditions, such as the use of fetal bovine serum (FBS). FBS contains EVs and it is usually depleted of EVs by ultracentrifugation (UC) and/or heat inactivated (HI). Several studies have evaluated the effect of different UC protocols for FBS by evaluating both cells and EVs. However, less is known about the effect of HI on the cells and the released EVs. The aim of this study was therefore to evaluate the effect of HI on EV purity. Methods: To determine the effect of heat inactivation, three different protocols were applied based on different combinations of: 1) UC at 118,000 × g for 18h and 2)HI at 56◦C for 30 min. The three conditions tested were: FBS ultracentrifuged but not heat inactivated (no-HI), FBS heat inactivated before UC (HI-before EV-dep), and FBS heat inactivated after EV depletion (HI-after EV-dep). The FBS was add to themedia of threemelanoma cell lines (MML1, UM22Ctr and UM22BAP1) at a final concentration of 10%. After 72h, large and small EVs were isolated by differential UC. The EV purity was determined by protein quantity, electron microscopy (EM) and nanoparticle tracking analysis (NTA). Results: The protein quantity (μg/μl) of large EVs was similar in the three conditions analyzed. On the contrary for small EVs, the protein amount was higher when the HI was performed after EV depletion as compared to HI before the UC and UC alone. However, significantly more particles were not detected in the HI-after EV-dep which resulted in a lower purity of small EVs in HI-after EV-dep illustrated by calculating the ratio of number of particles/μg proteins. Presence of contaminants (indicated by strong background) was observed in EM pictures of small EVs isolated in HI-after EV-dep condition differently from large EV samples. Summary/Conclusion: The HI of FBS induces release of contaminating elements that end up in small EVpellets if not previously removed

Klinische Korrelate autobiographischer Gedächtnisleistungen bei Patienten mit chronisch schizophrenen Psychosen

Schizophrenen Erkrankungen gelten heute grundsätzlich als neurobiologische Störungen und auch neurokognitive Einschränkungen bei schizophrener Patienten stellen mittlerweile einen hinreichend replizierten und unverrückbaren Befund dar. Recht wenig Beachtung wurde in diesem Zusammenhang bisher jedoch dem autobiographischen Gedächtnis geschenkt. Lange wurde gerade im neurokognitiven Forschungskontext das autobiographische Gedächtnis dem episodischen Gedächtnis gleichgesetzt und auch die funktionellen Aspekte dieses Gedächtnissystems fanden wenig Berücksichtigung innerhalb neurokognitiver Fragestellungen. Aus beeinträchtigten autobiographischen Gedächtnisprozessen lassen sich per definitionem grundsätzlich auch Störungen Selbst-­bezogener Prozesse ableiten. Diesen Selbst-­bezogenen Funktionen des autobiographischen Gedächtnisses muss gerade im Kontext der schizophrenen Erkrankungen eine besondere klinische Bedeutsamkeit beigemessen werden. Ziel der vorliegenden Arbeit ist es, autobiographische Gedächtnisprozesse schizophrener Patienten sowie deren klinische Einbettung in das bekannte psychopathologische und neurokognitive Störungsbild zu untersuchen, neue Erkenntnisse hierzu zu gewinnen und diese in die bereits bestehende Befundlage zu integrieren

Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol

Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. Method: EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®. Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis

Hypersharp Neutrino Lines

Neutrino lines from very long lived nuclei in simple crystals such as metals have hypersharp natural width, motionally narrowed by lattice vibrations in analogy to recoilless emission. A generalized hypersharp line fraction including the recoilless part can be derived in a frequency modulation approach. The nue lines of natural width in 3H to 3He 2-body beta-decay can then be resonantly captured with geometrical cross section. The extreme sharpness DeltaE/E~10-29 of the tritium nue line can probe the Planck length L via its limits on the widths of states, DeltaE/E(L) =L(L/R)beta =10-20(beta ~1) to 10-40 (beta= L/R(fm)). Stringent limits can be set on beta, thus, on models of quantum gravity

Tritium in Metallen: Präparationstechnik (Tritiumlabor): Eigenschaften von Tritium in Vanadium, Niob, Tantal und Palladium

In order to study the behaviour of tritium in metals, an all metal apparatus has been built for the safe handling of 100 mg of tritium (1000 Ci). This amount of tritium, for example, can load 10 g Pd to a concentration of T/Pd = 0.37. Samples can be loaded with tritium in a defined way by changing the gas pressure and the temperature. A PDP-II computer controlled the experiments by measuring pressures, room and sam pie temperatures and by adjusting the power for heating the sample. The main results of our studies in palladium, vanadium, niobium and tantalum are as follows: For the first time the phase diagram of the palladium-tritium system has been determined up to high concentrations. At constant concentration and temperature the following inequali ties hold for the desorption pressure: $P_{T_2}$(x, T) > $P_{D_2}$ (x, T) > $P_{H_2}$ (x, T). This is a consequence of the larger decrease of the energy of the vibrational levels in the molecule in comparison to the ones in the palladium lattice under isotopic exchange. In the two phase region ($\alpha$+$\beta$) the following values of the disintegration enthalpy $\Delta$H and -entropy $\Delta$S using the van't Hoff equation In p = - $\Delta$H/RT + $\Delta$S/R were obtained: [...

Extracellular vesicle DNA from human melanoma tissues contains cancer-specific mutations

Liquid biopsies are promising tools for early diagnosis and residual disease monitoring in patients with cancer, and circulating tumor DNA isolated from plasma has been extensively studied as it has been shown to contain tumor-specific mutations. Extracellular vesicles (EVs) present in tumor tissues carry tumor-derived molecules such as proteins and nucleic acids, and thus EVs can potentially represent a source of cancer-specific DNA. Here we identified the presence of tumor-specific DNA mutations in EVs isolated from six human melanoma metastatic tissues and compared the results with tumor tissue DNA and plasma DNA. Tumor tissue EVs were isolated using enzymatic treatment followed by ultracentrifugation and iodixanol density cushion isolation. A panel of 34 melanoma-related genes was investigated using ultra-sensitive sequencing (SiMSen-seq). We detected mutations in six genes in the EVs (BRAF, NRAS, CDKN2A, STK19, PPP6C, and RAC), and at least one mutation was detected in all melanoma EV samples. Interestingly, the mutant allele frequency was higher in DNA isolated from tumor-derived EVs compared to total DNA extracted directly from plasma DNA, supporting the potential role of tumor EVs as future biomarkers in melanom

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

<p>Abstract</p> <p>Background</p> <p>Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages.</p> <p>Method</p> <p>Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy.</p> <p>Results</p> <p>RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages.</p> <p>Conclusions</p> <p>Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.</p