The application of massively parallel sequencing technologies in diagnostics

Massively parallel sequencing (MPS) is rapidly evolving and is starting to be utilized by the clinical field as well as diagnostics. We describe major recent advances that have come about as a result of the application of MPS in the biomedical field and the first approaches in medical genetics that have made use of MPS. Without any doubt, MPS has proven to be a very powerful technique. To unravel the capabilities of MPS for patient care, the most important aspect for the acceptance of MPS within clinics and diagnostics is to guarantee that the large amount of data undergoes vitally important analyses and interpretation and is securely managed

Ion implantation of 226Ra for a primary 222Rn emanation standard

Laser resonance ionization at the RISIKO 30 kV mass separator has been used to produce isotopically and isobarically pure and well quantified 222Rn emanation standards. Based upon laser-spectroscopic preparation studies, ion implantation into aluminum and tungsten targets has been carried out, providing overall implantation efficiencies of 40% up to 60%. The absolute implanted activity of 226Ra was determined by the technique of defined solid-angle α-particle spectrometry, where excellent energy resolution was observed. The 222Rn emanation coefficient of the produced targets was studied using α-particle and γ-ray spectrometry, and yielded results between 0.23 and 0.34, with relative uncertainty on the order of 1%. No dependence exceeding a 1% change of the emanation on humidity could be identified in the range of 15 %rH to 75 %rH, whereas there were hints of a slight correlation between the emanation and temperature. Additionally, and as expected, the emanation coefficient was found to be dependent on the target material as well as the implanted dose. © 202

Optimizing the detection, ablation, and ion extraction efficiency of a single-particle laser ablation mass spectrometer for application in environments with low aerosol particle concentrations

The aim of this study is to show how a newly developed aerodynamic lens system (ALS), a delayed ion extraction (DIE), and better electric shielding improve the efficiency of the Aircraft-based Laser ABlation Aerosol MAss spectrometer (ALABAMA). These improvements are applicable to single-particle laser ablation mass spectrometers in general. To characterize the modifications, extensive sizeresolved measurements with spherical polystyrene latex particles (PSL; 150-6000 nm) and cubic sodium chloride particles (NaCl; 400-1700 nm) were performed. Measurements at a fixed ALS position show an improved detectable particle size range of the new ALS compared to the previously used Liu-type ALS, especially for supermicron particles. At a lens pressure of 2.4 hPa, the new ALS achieves a PSL particle size range from 230 to 3240 nm with 50% detection efficiency and between 350 and 2000 nm with 95% detection efficiency. The particle beam divergence was determined by measuring the detection efficiency at variable ALS positions along the laser cross sections and found to be minimal for PSL at about 800 nm. Compared to measurements by singleparticle mass spectrometry (SPMS) instruments using Liutype ALSs, the minimum particle beam divergence is shifted towards larger particle sizes. However, there are no disadvantages compared to the Liu-type lenses for particle sizes down to 200 nm. Improvements achieved by using the DIE and an additional electric shielding could be evaluated by size-resolved measurements of the hit rate, which is the ratio of laser pulses yielding a detectable amount of ions to the total number of emitted laser pulses. In particular, the hit rate for multiply charged particles smaller than 500 nm is significantly improved by preventing an undesired deflection of these particles in the ion extraction field. Moreover, it was found that by using the DIE the ion yield of the ablation, ionization, and ion extraction process could be increased, resulting in up to 7 times higher signal intensities of the cation spectra. The enhanced ion yield results in a larger effective width of the ablation laser beam, which in turn leads to a hit rate of almost 100% for PSL particles in the size range from 350 to 2000 nm. Regarding cubic NaCl particles the modifications of the ALABAMA result in an up to 2 times increased detection efficiency and an up to 5 times increased hit rate. The need for such instrument modifications arises in particular for measurements of particles that are present in low number concentrations such as ice-nucleating particles (INPs) in general, but also aerosol particles at high altitudes or in pristine environments. Especially for these low particle number concentrations, improved efficiencies help to overcome the statistical limitations of single-particle mass spectrometer measurements. As an example, laboratory INP measurements carried out in this study show that the appli- cation of the DIE alone increases the number of INP mass spectra per time unit by a factor of 2 to 3 for the sampled substances. Overall, the combination of instrument modifications presented here resulted in an increased measurement efficiency of the ALABAMA for different particle types and particles shape as well as for highly charged particles. © 2020 Copernicus GmbH. All rights reserved

New metrology for radon at the environmental level

International audienc

Proteomic Shifts in Embryonic Stem Cells with Gene Dose Modifications Suggest the Presence of Balancer Proteins in Protein Regulatory Networks

Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of “balancer” proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the “elasticity” of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Primary Rn-222 emanation standards for low-level applications

In dieser Dissertation wird die Darstellung von Referenzatmosphären des radioaktiven Edelgases ²²²Rn im Bereich niedriger Aktivitätskonzentrationen, unter 300 Bq m⁻³, beschrieben. Diese sind bespielsweise zur, auf das intenationale Einheitensystem SI rückgeführten, Kalibrierung von Radonmessgeräten notwendig. Dies ist Voraussetzung für die internationale Vergleichbarkeit und Harmonisierung von Radonmessungen. Dieser Konzentrationsbereich macht es notwendig, dass die Herstellung solcher Referenzatmosphären nicht mittels abklingender, gasförmiger ²²²Rn Standards, sondern mit sogenannten Emanationsquellen erfolgt. Dies sind ²²⁶Ra Quellen die derart beschaffen sind, dass ein Teil des entstehenden Tochternuklids ²²²Rn kontinuierlich freigesetzt wird. Dadurch kann beispielsweise in einem geschlossenen Referenzvolumen im Laufe einiger Halbwertszeiten ein Gleichgewichtszustand mit einer zeitlich stabilen Aktivitätskonzentration hergestellt werden. Der Grad dieser Freisetzung ist abhängig vom Herstellungsverfahren der Quelle und den Umgebungsparametern, wie beispielsweise Temperatur und relative Luftfeuchte, was im Laufe dieser Arbeit empirisch belegt wird. Verschiedene Ansätze zur Herstellung solcher Quellen auf dem Prinzip einer Dünnschichtgeometrie des ²²⁶Ra wurden untersucht, nämlich die Elektrodeposition, die Ionenimplantation und die physikalische Gasphasenabscheidung. Die Rückführung der so deponierten ²²⁶Ra Aktivität auf das SI erfolgte, für ²²²Rn Emanationsquellen erstmals, über die Absolutmethode der Alphaspektrometrie unter definiertem Raumwinkel. Die Bestimmung der Emanation erfolgte zunächst im Gleichgewichtszustand der Quellen basierend auf der Gammaspektrometrie der kurzlebigen ²²²Rn Folgeprodukte. Im Laufe der Arbeit konnte gezeigt werden, wie sich solche Messungen durch Methoden der statistischen Inversion auf dynamische Verhältnisse übertragen lassen. Dadurch können erstmals dynamische Änderungen der Emanation, beispielsweise in Folge von Änderungen in den Umgebungsparametern, messtechnisch, zunächst basierend auf der Gammaspektrometrie, erfasst werden. Auf Basis dieser Erkenntnis wurde ein System, der integrated Radon Source/Detector (IRSD), konzeptioniert und implementiert, welches die ²²²Rn Quelle mit einem alphaspektrometrischen Detektor vereint. Dieses System erlaubt es kontinuierlich durch hocheffiziente Alphaspektrometrie die verbleibende ²²²Rn Aktivität in der Quelle zu bestimmen und mittels der statistischen Inversion annähernd in Echtzeit die Emanation von ²²²Rn zu bestimmen, selbst wenn diese nur wenige ²²²Rn Atome pro Sekunde beträgt. Somit ist es erstmals möglich Referenzatmosphären auch im typischen Konzentrationsbereich der Außenluft rückgeführt und unter wechselnden klimatischen Bedingungen darzustellen. Abschließend beinhaltet diese Dissertation praktische, zum Teil neue, Anwendungsmethoden für die Kalibrierung von Radonmessgeräten mit Hilfe der entwickelten Emanationsquellen.In this dissertation, the preparation of reference atmospheres of the radioactive noble gas ²²²Rn in the range of low activity concentrations, under 300 Bq m⁻³, is described. These are necessary, for example, for the calibration of radon measuring instruments traceable to the international system of units, the SI. This is integral for international comparability and harmonization of radon measurements. This concentration range requires that the production of such reference atmospheres is not done by means of decaying, gaseous ²²²Rn standards, but with so-called emanation sources. These are ²²⁶Ra sources which are of such nature, that a part of the daughter nuclide ²²²Rn is continuously released. Thus, for example, in a closed reference volume, an equilibrium is established at a time-stable activity concentration over the course of a few half-lives. The degree of this release depends on the source manufacturing process and details and environmental parameters such as the temperature and relative humidity, which is empirically demonstrated throughout this work. Different approaches to fabricate such sources on the principle of a thin-film geometry of ²²⁶Ra have been investigated, namely electrodeposition, ion implantation and phyiscal vapor deposition. The traceability of the ²²⁶Ra activity deposited in this way to the SI was performed, for the first time for such emanation sources, via the absolute method of defined solid angle alpha-particle spectrometry. The emanation was initially determined only in its equilibrium state based on gamma-ray spectrometry of the short-lived ²²²Rn progeny. In the course of this work, it could be shown how such measurements can be adapted to dynamic conditions by means of statistical inversion, whereby for the first time dynamic changes of the emanation, for example as a consequence of changes in the environmental parameters, can be quantified, initially based on gamma-ray spectrometry. Based on this insight, a system, the so-called integrated Radon Source/Detector (IRSD), was conceptualized and implemented, which combines the ²²²Rn source with an alpha-spectrometric detector. This system allows for continuous, highly efficient alpha-particle spectrometry to determine the remaining ²²²Rn activity in the source and, using statistical inversion, to quantify in near real time the emanation of ²²²Rn, even if it only amounts to some ²²²Rn atoms per second. This makes it possible for the first time to realize reference atmospheres even in the typical concentration range of outdoor air and under changing climatic conditions. Finally, this dissertation contains practical and partly new application methods for the calibration of radon measurement instruments using the developed emanation sources