Sensitivity of Saturation Transfer Electron Spin Resonance Extended to Extremely Slow Mobility in Glassy Materials

A novel extension of the saturation transfer (ST) ESR technique that enables the determination of extremely long rotational correlation times of nitroxide spin labels up to values around 10⁴s is proposed. The method is based on the observation that the integral of ST-ESR spectra is sensitive to the spin–lattice relaxation time of the electron of the spin label, which in turn is directly dependent upon the rotational correlation time. The method is applied to the spin label TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) in glycerol. From the known viscosity data and the related rotational correlation times of the TEMPOL spin label in glycerol, the rotational correlation times of unknown samples can be determined. The method is especially applicable to systems with a very high viscosity, such as glassy materials. The method is applied to a 20 wt% glucose–water mixture in the glassy state, giving a value for the highest limiting rotational correlation time of about 10³s at a temperature of 45 K below the glass transition temperature of this system. This is an extension by six decades for the rotational correlation time, as compared to the current application of ST-ESR.     

Radon loss from encapsulated sediments in Ge gamma-ray spectrometry for the annual radiation dose determination in luminescence dating

In Ge gamma-ray spectrometry for the annual radiation dose determination in the luminescence dating of sediments, the picture of ²²⁶Ra enrichment or depletion (in the ²³⁸U decay series) obtained via measurement of its ²¹⁴Pb and ²¹⁴Bi daughters may be disturbed by the ²²²Rn-content of the sample being decreased due to manipulations such as drying and pulverizing. Therefore, it is common practice to start the measurement only about 1 month after encapsulating the material, after which the ²²⁶Ra(1600 a)- ²²²Rn(3.82 d) mother-daughter equilibrium is re-established. Evidently, this only holds on condition that no significant escape of Rn occurs out of the sediment after making it up for counting. In order to experimentally investigate this effect, in the present work measurements were carried out with various types of dried and pulverized sediments that were either encapsulated in screw-cap polystyrene vials or in sealed glass containers, or that were mixed with molten wax followed by solidification in a cylindrical geometry. From the results obtained, it could be concluded that preparation and counting of the sediment-wax mixture is the method of choice.     

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry,targeted sequence,type of DNA input and PCR efficiency

The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of ?1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. Figure

The qPCR monitoring chemistries form six groups with distinct fluorescence kinetics. The displacement of the amplification curve depends on the chemistry, DNA input and probe-targeting. The observed shift in C_q values can be corrected and PCR efficiencies can be derived.     

Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

Robust calibrations on reduced sample sets for API content prediction in tablets: Definition of a cost-effective NIR model development strategy

Owing to spectral variations from other sources than the component of interest, large investments in the NIR model development may be required to obtain satisfactory and robust prediction performance. To make the NIR model development for routine active pharmaceutical ingredient (API) prediction in tablets more cost-effective, alternative modelling strategies were proposed. They used a massive amount of prior spectral information on intra- and inter-batch variation and the pure component spectra to define a clutter, i.e., the detrimental spectral information. This was subsequently used for artificial data augmentation and/or orthogonal projections. The model performance improved statistically significantly, with a 34–40% reduction in RMSEP while needing fewer model latent variables, by applying the following procedure before PLS regression: (1) augmentation of the calibration spectra with the spectral shapes from the clutter, and (2) net analyte pre-processing (NAP). The improved prediction performance was not compromised when reducing the variability in the calibration set, making exhaustive calibration unnecessary. Strong water content variations in the tablets caused frequency shifts of the API absorption signals that could not be included in the clutter. Updating the model for this kind of variation demonstrated that the completeness of the clutter is critical for the performance of these models and that the model will only be more robust for spectral variation that is not co-linear with the one from the property of interest.     

Development of an in vitro confinement test to predict the clinical handling of polymer-based injectable bone substitutes

The objective of the current study was to present a simple and standardized system as a preliminary attempt to assess the confinement of polymer-based injectable bone substitutes (IBSs) in vitro. Four different types of polymer-based IBSs were selected as model compounds, a thermosensitive collagen gel, a colloidal gelatin gel, a covalently crosslinked oligo(poly(ethylene glycol)fumarate (OPF) gel and a OPF-calcium phosphate composite. A ceramic-based IBS (i.e. a self-setting calcium phosphate cement) was used as reference. The confinement of all IBSs was tested under three different conditions: (1) no flow (as control), (2) dynamic flow after injection and (3) dynamic flow during injection. The results presented herein confirmed that the proposed test can be used to quantify the confinement of various IBSs within artificial defects under static or dynamic flow conditions, thereby offering a potential tool for predictive quantitative determination of the confinement of IBSs in vivo.     

Multiplex genotyping assays for fine‐resolution subtyping of the major human Y‐chromosome haplogroups E,G, I,J, and R in anthropological,genealogical, and forensic investigations

Inherited DNA polymorphisms located within the nonrecombing portion of the human Y chromosome provide a powerful means of tracking the patrilineal ancestry of male individuals. Recently, we introduced an efficient genotyping method for the detection of the basal Y‐chromosome haplogroups A to T, as well as an additional method for the dissection of haplogroup O into its sublineages. To further extend the use of the Y chromosome as an evolutionary marker, we here introduce a set of genotyping assays for fine‐resolution subtyping of haplogroups E, G, I, J, and R, which make up the bulk of Western Eurasian and African Y chromosomes. The marker selection includes a total of 107 carefully selected bi‐allelic polymorphisms that were divided into eight hierarchically organized multiplex assays (two for haplogroup E, one for I, one for J, one for G, and three for R) based on the single‐base primer extension (SNaPshot) technology. Not only does our method allow for enhanced Y‐chromosome lineage discrimination, the more restricted geographic distribution of the subhaplogroups covered also enables more fine‐scaled estimations of patrilineal bio‐geographic origin. Supplementing our previous method for basal Y‐haplogroup detection, the currently introduced assays are thus expected to be of major relevance for future DNA studies targeting male‐specific ancestry for forensic, anthropological, and genealogical purposes.