High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses v2

This process instruction describes the steps for pre-analytical processing of primary settled solids from wastewater treatment plants for downstream nucleic acid purification and quantification. Previous research has demonstrated that enveloped viral particles, such as SARS-CoV-2, associate with the solids in wastewater. Therefore, concentrating the solids in the sample and removing the water concentrates the viral particles as well and increases the sensitivity of the assay. Bovine coronavirus vaccine (BCoV) is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. The dry weight of the sample is determined to account for sample variability between different treatment plants and between samples collected on different days and allows for the final quantification to be normalized to the precise quantity of solids in the sample. A dry weight conversion factor to adjust for the final amount of solids in the quantified sample is determined by measuring the difference in mass between the “wet” dewatered solid and after the sample is dried at 110°C overnight. For long term storage, up to 50mL of the original primary settled solid sample is stored at 4°C and small aliquot of the dewatered solids is stored at -80°C. This process instruction applies to sample dewatering, BCoV control spike in, homogenization, dry weight measurement, aliquoting and storage.

Electrochemical Impedance Spectroscopy of Zinc Oxide Nanoparticles After Deposition on Screen Printed Electrode

A small aliquot (10–14 μL) of ZnO nanoparticles dispersed in deionized water was deposited by evaporation to produce a dry residue on the working area of a screen-printed electrode. An electrochemical test solution containing K3Fe(CN)6 and KCl was added to the electrode surface for analysis by electrochemical impendence spectroscopy (EIS). Using this deposition analysis technique, a new relationship between the charge transfer resistance (Rct) and the amount of ZnO nanoparticles has been explored. Based on the trend of increasing Rct value with an increase of ZnO nanoparticles, a quantitative analysis method can be established to determine the mass of nanoparticles (0.01–1.00 μg) deposited from an unknown dispersion. To study the matrix effect, addition of Nafion solution to the aqueous dispersion resulted in a change of the linear range to 0.3–0.5 μg nanoparticles. Addition of methanol (10% by volume) to the aqueous dispersion changes the analysis range to 0.2–0.6 μg nanoparticles, while additional methanol (50% by volume) changes the analysis range to 0.06–1.00 μg nanoparticles. The analytical sensitivity, as indicated by the slope of each standard calibration curve, ranked as: aqueous dispersion > Nafion/aqueous dispersion > 10% methanol/aqueous dispersion > 50% methanol/aqueous dispersion. Altogether these results verify that deionized water is the best dispersion medium for EIS analysis of ZnO nanoparticles.

Pulmonary hypertension: Proteins in the blood

The plasma proteome is rich in information. It comprises proteins that are secreted or lost from cells as they respond to their local environment. Changes in the constitution of the plasma proteome offer a relatively non-invasive report on the health of tissues. This is particularly true of the lung in pulmonary hypertension, given the large surface area of the pulmonary vasculature in direct communication with blood. So far, this is relatively untapped; we have relied on proteins released from the heart, specifically brain natriuretic peptide and troponin, to inform clinical management. New technology allows the measurement of a larger number of proteins that cover a broad range of molecular pathways in a single small aliquot. The emerging data will yield more than just new biomarkers of pulmonary hypertension for clinical use. Integrated with genomics and with the help of new bioinformatic tools, the plasma proteome can provide insight into the causative drivers of pulmonary vascular disease and guide drug development.

The evolution of the terrestrial-terminating Irish Sea glacier during the last glaciation

<p>Comprehensive mapping and the Briticechrono geochronology provides a reconstruction of the last advance and retreat of the only land-terminating ice lobe of the western British Irish Ice Sheet. The Irish Sea Glacier was fed by ice from Lake District, Irish Sea and Wales, and extended to maximum limits in the English Midlands. During ice retreat after 27 kyrs, a series of reverse bedrock slopes rendered proglacial lakes endemic in the land-system. Not resembling the more extensive definitions of the classical ‘Glacial Lake Lapworth’, these ice contact lakes were smaller time transgressive moraine- and bedrock-dammed basins that evolved with ice marginal retreat. Combining, for the first time on glacial sediments, OSL bleaching profiles for cobbles with single grain and small aliquot OSL measurements on sands, has produced a coherent chronology from these heterogeneously bleached samples, and constrained for the Irish Sea Glacier a post 30ka ice maximum advance, 26.5±1.8ka maximum extent, and 25.3±1.6 to 20.6±2.2ka retreat vacating the region. With retreat of the Irish Sea Glacier an opportunistic Welsh re-advance 19.7±2.5ka took advantage of the vacated space and rode over Irish Sea Glacier moraines. Our geomorphological chronosequence shows a glacial system forced by climate, but mediated by piracy of ice sources shared with the larger and marine terminating Irish Sea Ice Stream to the west. The Irish Sea Glacier underwent changes flow regime and fronting environments driven by stagnation and decline as the primary impetus to advance was diverted. Ultimately, the glacier of the English Midlands display complex uncoupling and realignment during deglaciation and ice margin retreat towards upland hinterlands ~17.8 kyrs (Lake District and Pennines) and asynchronous behaviour as individual adjacent ice lobes became increasingly important in driving the landform record.</p>

Resolving the effects of 2-D versus 3-D grain measurements on apatite (U–Th) ∕ He age data and reproducibility

(U–Th) ∕ He thermochronometry relies on the accurate and precise quantification of individual grain volume and surface area, which are used to calculate mass, alpha ejection (FT) correction, equivalent sphere radius (ESR), and ultimately isotope concentrations and age. The vast majority of studies use 2-D or 3-D microscope dimension measurements and an idealized grain shape to calculate these parameters, and a long-standing question is how much uncertainty these assumptions contribute to observed intra-sample age dispersion and accuracy. Here we compare the results for volume, surface area, grain mass, ESR, and FT correction derived from 2-D microscope and 3-D X-ray computed tomography (CT) length and width data for > 100 apatite grains. We analyzed apatite grains from two samples that exhibited a variety of crystal habits, some with inclusions. We also present 83 new apatite (U–Th) ∕ He ages to assess the influence of 2-D versus 3-D FT correction on sample age precision and effective uranium (eU). The data illustrate that the 2-D approach systematically overestimates grain volumes and surface areas by 20 %–25 %, impacting the estimates for mass, eU, and ESR – important parameters with implications for interpreting age scatter and inverse modeling. FT factors calculated from 2-D and 3-D measurements differ by ∼2 %. This variation, however, has effectively no impact on reducing intra-sample age reproducibility, even on small aliquot samples (e.g., four grains). We also present a grain-mounting procedure for X-ray CT scanning that can allow hundreds of grains to be scanned in a single session and new software capabilities for 3-D FT and FT-based ESR calculations that are robust for relatively low-resolution CT data, which together enable efficient and cost-effective CT-based characterization.

In vivo storage of oocytes leads to lower survival, increased abnormalities and may affect the ploidy status in the yellowtail tetra Astyanax altiparanae

SummaryIn this study we analyzed whether the in vivo storage of oocytes (time after ovulation until fertilization) affects the survival and the ploidy status of the yellowtail tetra Astyanax altiparanae. Fish were induced to spawn and, after ovulation, a small aliquot was stripped and immediately fertilized (positive control group). Subsequently, aliquots (~150 oocytes) were stripped and fertilized at various time points of 60, 120, 180 or 240 min. Developmental stages, abnormalities, survival and the ploidy status of the hatched larvae were examined. As expected, in the control group, 100% of the larvae were diploid. Conversely, triploid individuals were observed just at the 60 min treatment time point (0.6%). In vivo storage of oocytes also influenced the survival rates (P < 0.05); the 180 and 240 min samples, respectively, presented lower survival rates at gastrula (50.10±6.26% and 40.92±5.32%), and somite (17.80±5.14% and 4.41±2.76%) stages and lower hatching rates (12.01±4.04% and 4.41±2.76%). A higher percentage (99.27±0.40%) of normal larvae and only a few abnormal larvae (0.73±0.40%) were observed in the control group (P = 0.0000). This observation did not differ from that observed at the 60 min treatment point (P = 0.9976). A significant increase in the percentage of abnormalities was observed in the other treatments, and, after 240 min, the highest percentage of abnormal larvae was seen (P=0.0024; 83.33±16.67%). In conclusion, we showed that oocyte ageing had a significant effect on survival and may affect the ploidy status in A. atiparanae.

Coarse versus fine-grain quartz optical dating of the sediments related to the 1985 Ms7.1 Wuqia earthquake, northeastern margin of the Pamir salient, China

Optical dating of earthquake related sediments were investigated including one modern sample and three samples from a trench excavated across the 1985 Ms7.4 Wuqia Earthquake surface rupture. The results indicated that equivalent dose (De) values vary with grain size and the method used for De determination. The residual dose of the modern sample is 0.1 ka ( $\begin{array}{} 0.2_{-0.1}^{+0.2} \end{array} $ Gy) for the quartz single grain measurements. Only 1.5–3.6% of the grains have a detectable OSL signal. Single grain quartz ages are similar to the expected ages. Fine grain quartz results overestimate the De values and are much older than single grain quartz and coarse grain quartz small aliquot standardized growth curve (SA-SGC) ages. Single grain quartz OSL dating may be optimal for dating earthquake related deposits, but SA-SGC can save measurement time and has potential for dating some poorly bleaching samples.

Testing the suitability of dim sedimentary quartz from northern Switzerland for OSL burial dose estimation

We investigate the suitability of sedimentary quartz associated with former glacial advances in northern Switzerland to provide reliable burial dose estimates using Optically Stimulated Luminescence (OSL). Previous studies on northern alpine quartz show that its signal characteristics can be poor and potentially problematic. We analyse quartz signals of small aliquots, which reveal the presence of a prominent medium or slow component in the initial part of some signals. Nonetheless, rejection of aliquots with unfavourable signal composition does not alter the burial dose estimates, but significantly reduces the data set for De determination. Signal lifetimes from isothermal decay measurements cover a wide range of values, yet the lowest lifetimes are high enough to guarantee a reliable burial dose estimate for samples of < 400 ka. Comparison of small aliquot and single grain burial dose distributions reveals that signal averaging masks partial bleaching in some of the samples. We therefore strongly recommend single grain measurements for samples from this setting and area, in order to exclude age overestimation due to partial bleaching.

Whole blood thrombin generation in Bmal1-deficient mice

SummaryThe Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437 ± 7 nM.min; mean ± SD, n=7) when compared with WT mice (ETP=220 ± 45 nM.min; mean ± SD, n=5). The peak heights also differed significantly (p=0.027). By applying SEM we found that Bmal1 deficient mice display a denser fibrin network with smaller pores compared to WT mice. In conclusion, the whole blood TG assay in mice revealed to be reproducible. As a proof-of-principle we have shown that the whole blood TG assay is capable of detecting a prothrombotic phenotype in Bmal1-KO mice.