Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling

Objective

To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection.

Methods

Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow.

Results

An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell.

Conclusion

Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.     

Evaluation of the measurement uncertainty in automated long-term sampling of PCDD/PCDFs

Since the publication of the first version of European standard EN-1948 in 1996, long-term sampling equipment has been improved to a high standard for the sampling and analysis of polychlorodibenzo-p-dioxin (PCDD)/polychlorodibenzofuran (PCDF) emissions from industrial sources. The current automated PCDD/PCDF sampling systems enable to extend the measurement time from 6–8 h to 15–30 days in order to have data values better representative of the real pollutant emission of the plant in the long period. EN-1948:2006 is still the European technical reference standard for the determination of PCDD/PCDF from stationary source emissions. In this paper, a methodology to estimate the measurement uncertainty of long-term automated sampling is presented. The methodology has been tested on a set of high concentration sampling data resulting from a specific experience; it is proposed with the intent that it is to be applied on further similar studies and generalized. A comparison between short-term sampling data resulting from manual and automated parallel measurements has been considered also in order to verify the feasibility and usefulness of automated systems and to establish correlations between results of the two methods to use a manual method for calibration of automatic long-term one. The uncertainty components of the manual method are analyzed, following the requirements of EN-1948-3:2006, allowing to have a preliminary evaluation of the corresponding uncertainty components of the automated system. Then, a comparison between experimental data coming from parallel sampling campaigns carried out in short- and long-term sampling periods is realized. Long-term sampling is more reliable to monitor PCDD/PCDF emissions than occasional short-term sampling. Automated sampling systems can assure very useful emission data both in short and long sampling periods. Despite this, due to the different application of the long-term sampling systems, the automated results could not be directly compared with manual results, not even in terms of measurement uncertainty. This investigation focuses on both uncertainty and repeatability of the automated sampling method. The standard 20988, developed by Internarional Organization of Standardization (ISO) can be used to estimate the measurement uncertainty. The results confirm that the uncertainties of manual and automated methods are comparable. At the same time, it is not appropriate to consider the manual method as a reference for the evaluation of the uncertainty of the automated sampling system, due to the high variability of both systems.     

Assessment of Iodine Direct Titration in Sea Water by Constant Current Potentiometry

Dissolved iodine in sea water, about 60 μg L^-1, usually exists in equal proportions as iodate and iodide ions. For their determination several techniques have been proposed (Riley, 1975); among them the volumetric one which depends upon the release of elementary iodine by the addition of acid and iodide ions to the iodate present in the sample. The liberated iodine is titrated with thiosulphate the equivalence point being detected photometrically (Matthews and Riley, 1970; Wong and Brewer, 1974) or it can also be reduced with an excess of thiosulphate and the residual thiosulphate back-titrated with iodate amperometrically (Barkley and Thompson, 1960; Truesdale and Spencer, 1974; Tsonkova and Kulev, 1981). To titrate total iodine in the sample the iodide must be previously oxidized to iodate with bromine (Barkley and Thompson, 1960; Tsonkova and Kulev, 1981) or permanganate (Schnepfe, 1972). In the amperometric procedure Truesdale and Spencer (1974) observed that platinum electrodes often do not respond reliably.     

Genotoxicity of gold nanoparticles functionalized with indolicidin towards Saccharomyces cerevisiae

The toxic effects of gold nanoparticles surface-functionalized with the antimicrobial peptide indolicidin(Au NPs-indolicidin) towards the yeast Saccharomyces cerevisiae, one of the major eukaryotic model organisms, have been evaluated. Growth and survival,genotoxicity, as measured by comet assay, and expression of the YCA1, an apoptosis indicating gene, following 72 hr exposure of yeast to Au NPs-indolicidin, and to Au NPs and indolicidin alone have been examined. The gold nanoparticles exerted toxicity with DNA damage, accompanied by reactive oxygen species production(ROS), but they do not inhibit yeast growth and viability. Genotoxicity was less pronounced for surface-functionalized nanoparticles, showing that S. cerevisiae is quite resistant to the complex Au NPs-indolicidin.A progressive reduction of the genotoxic effect was observed along 72 hr exposure,presumably due to the activation of DNA repair mechanisms. These findings suggest the occurrence of a physiological protective response of S. cerevisiae towards nanoparticles,thereby providing useful information to the assessment of the environmental impact of metal nanoparticles.     

Spatially-balanced adaptive web sampling

The paper deals with sampling from a finite population that is distributed over space and has a highly uneven spatial distribution. It suggests a sampling design that allocates a portion of the sample units that are well spread over the population and sequentially selects the remaining units in sub-areas that appear to be of more interest according to the study variable values observed during the survey. In order to estimate the population mean while using this sampling design, a computationally intense estimator, obtained via the Rao–Blackwell approach, is proposed and a resampling method is used that makes the inference computationally feasible. The whole sampling strategy is evaluated through several Monte Carlo experiments.     

Phytotoxicity and antioxidative enzymes of green microalga (Desmodesmus subspicatus) and duckweed (Lemna minor) exposed to herbicides MCPA, chloridazon and their mixtures

In this study, we evaluate the toxicity of MCPA (auxin-like growth inhibitor), chloridazon (CHD) (PSII-inhibitor) and their mixtures to floating plants and planktonic algae. Toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) and CHD (5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone) was first assessed in two growth inhibition tests with Lemna minor (ISO/DIS 20079) and Desmodesmus subspicatus (ISO 8692). Next, herbicide mixtures at concentrations corresponding to the EC values were used to assess their interactive effects, and the biomarkers were: for duckweed fresh weight, frond area, chlorophyll content and number of fronds, and for algae cell count and cell volume. The 3d EC?? and EC?? values using cell counts of D. subspicatus were 142.7 and 529.1 mg/L for MCPA and 1.7 and 5.1 mg/L for CHD. The 7d EC?? and EC?? values using frond number of L. minor amounted to 0.8 and 5.4 mg/L for MCPA and 0.7 and 10.4 mg/L for CHD. Higher sensitivity of reproductive (number of cells/fronds) than growth processes (cell volume/frond area) to herbicides applied individually and in mixtures was especially pronounced in the responses of Desmodesmus. Herbicide interactions were assessed by the two-way ANOVA and Abbott's formula. Generally, an antagonistic interaction with Lemna was revealed by MCPA and chloridazon, whereas additive effect of both herbicides was observed for Desmodesmus. A significant stimulation of SOD and APX activity by binary mixtures was noted in algal cells mainly after 24 and 48 hours of exposure. The extremely high stimulation of the activity of both enzymes was induced by the combination EC??CHD + EC??MCPA (48 h). Presumably due to oxidative stress, the treatment with CHD at concentration EC?? after 72 h was lethal for algae grown in aerated cultures, in contrast to standardized test conditions. Taking into account the consequences of risk assessment for herbicide mixtures we can state that a relatively low toxicity, as well as the lack of significant synergy between MCPA and CHD to non-target plants appears to be the most important result.     

Chemoreception of botanical nematicides by Meloidogyne incognita and Caenorhabditis elegans

Plant-parasitic nematodes, such as Meloidogyne incognita, cause serious damage to various agricultural crops worldwide, and their control necessitates environmentally safe measures. We have studied the effects of plant secondary metabolites on M. incognita locomotion, as it is an important factor affecting host inoculation inside the soil. We compared the effects to the respective behavioral responses of the model saprophytic nematode Caenorhabditis elegans. The tested botanical nematicides, all reported to be active against Meloidogyne sp. in our previous works, are small molecular weight molecules (acids, alcohols, aldehydes, and ketones). Here, we specifically report on the attractant or repellent properties of trans-anethole, (E,E)-2,4-decadienal, (E)-2-decenal, fosthiazate, and 2-undecanone. The treatments for both nematode species were made at sublethal concentration levels, namely, 1 mM (<EC₅₀), and the chemical controls used for the experiments were the commercial nematicides fosthiazate and oxamyl. According to our results, trans-anethole, decenal, and oxamyl attract C. elegans, while 2-undecanone strongly attracts M. incognita. These findings can be of use in the development of nematicidal formulations, contributing to the disruption of nematode chemotaxis to root systems.     

Monitoring of glyphosate and AMPA in soil samples from two olive cultivation areas in Greece: aspects related to spray operators activities

The persistence of glyphosate and its primary metabolite AMPA (aminomethylphosphonic acid) was monitored in two areas in Southern Greece (Peza, Crete and Chora Trifilias, Peloponnese) with a known history of glyphosate use, and the levels of residues were linked to spray operators’ activities in the respective areas. A total of 170 samples were collected and analysed from both areas during a 3-year monitoring study. A new method (Impact Assessment Procedure - IAP) designed to assess potential impacts to the environment caused by growers’ activities, was utilised in the explanation of the results. The level of residues was compared to the predicted environmental concentrations in soil. The ratio of the measured concentrations to the predicted environmental concentrations (MCs/PECs) was >?1 in Chora the first 2 years of sampling and <?1 in the third year, whilst the MCs/PECs ratio was <?1 in Peza, throughout the whole monitoring period. The compliance to the instructions for best handling practices, which operators received during the monitoring period, was reflected in the amount of residues and the MCs/PECs ratio in the second and especially the third sampling year. Differences in the level of residues between areas as well as sampling sites of the same area were identified. AMPA persisted longer than the parent compound glyphosate in both areas.