Non-invasive prenatal testing (NIPT): Combination of copy number variant and gene analyses using an “in-house” target enrichment next generation sequencing—Solution for non-centralized NIPT laboratory?

Objective

Recent studies have integrated copy number variant (CNV) and gene analysis using target enrichment. Here, we transferred this concept to our routine genetics laboratory, which is not linked to centralized non-invasive prenatal testing (NIPT) facilities.

Method

From a cohort of 100 pregnant women, 22 were selected for the analysis of maternal genomic DNA (gDNA) along with fetal cell-free DNA. Using targeted enrichment, 135 genes were analyzed, combined with aberrations of chromosomes 21, 18, 13, X, and Y. The data were subjected to specificity and sensitivity analyses, and correlated with the results from invasive testing methods.

Results

The sensitivity/specificity was determined for the CNV analysis of chromosomes: 21 (80%/75%), 18 (-/82%), 13 (100%/67%), and Y (100%/100%). The gene detection was valid for maternal gDNA. However, for cell-free fetal DNA, it was not possible to determine the boundary between an artifact and a real sequence variant.

Conclusion

The target enrichment method combining CNV and gene detection seems feasible in a regular laboratory. However, this method can only be responsibly optimized with a sufficient number of controls and further validation on a strong bioinformatic background. The present results showed that NIPT should be performed in specialized centers, and that its introduction to isolated laboratories may not provide valid data.     

Modeling non-point source pollution from rice farming in the Thachin River Basin

With the worldwide intensification of agriculture, non-point source pollution of surface waters has become a pressing issue. Conventional river water quality models consider non-point sources as accumulated entries into the rivers and do not investigate into the processes generating the pollution at its source, thus preventing the determination of effective mitigation measures. The models require extensive data inputs, which is a deficiency in many developing and emerging countries with limited data availability. The current study applies a Material Flow Model as a complementary approach to quantify non-point source pollution from agricultural areas. Rice farming in the Thachin River Basin is presented as a case study, with a focus on nutrients. The total nitrogen and phosphorus flows from rice farming to the river system are quantified, the key parameters influencing these flows are determined and potential mitigation measures are discussed. The results show that rice contributes considerable nutrient loads to the Thachin River Basin. Scenario simulations demonstrate that a significant nutrient load reduction could be achieved by following the official recommendations for fertilizer application, thus confirming the local efforts to introduce best management practice. Our results underline the importance of non-point source pollution control in intensive agricultural areas, particularly of tropical lowland delta areas such as the Central Plains of Thailand. The specific benefit of applying a Material Flow Model in this context is that with limited data availability, one can reach an understanding of the system and gain a first overview over its key pollution problems. This can serve as supportive basis for determining consecutive in-depth research requirements.     

Assessment of indoor air concentrations of VOCs and their associated health risks in the library of Jawaharlal Nehru University,New Delhi

The present work investigated the levels of total volatile organic compounds (TVOC) and benzene, toluene, ethylbenzene, m/p-xylene, and o-xylene (BTEX) in different microenvironments in the library of Jawaharlal Nehru University in summer and winter during 2011–2012. Carcinogenic and non-carcinogenic health risks due to organic compounds were also evaluated using US Environmental Protection Agency (USEPA) conventional approaches. Real-time monitoring was done for TVOC using a data-logging photo-ionization detector. For BTEX measurements, the National Institute for Occupational Safety and Health (NIOSH) standard method which consists of active sampling of air through activated charcoal, followed by analysis with gas chromatography, was performed. Simultaneously, outdoor measurements for TVOC and BTEX were carried out. Indoor concentrations of TVOC and BTEX (except benzene) were higher as compared to the outdoor for both seasons. Toluene and m/p-xylene were the most abundant organic contaminant observed in this study. Indoor to outdoor (I/O) ratios of BTEX compounds were generally greater than unity and ranged from 0.2 to 8.7 and 0.2 to 4.3 in winter and summer, respectively. Statistical analysis and I/O ratios showed that the dominant pollution sources mainly came from indoors. The observed mean concentrations of TVOC lie within the second group of the Molhave criteria of indoor air quality, indicating a multifactorial exposure range. The estimated lifetime cancer risk (LCR) due to benzene in this study exceeded the value of 1?×?10^?6 recommended by USEPA, and the hazard quotient (HQ) of non-cancer risk came under an acceptable range.     

Determination of organophosphorus fire retardants and plasticizers in wastewater samples using MAE-SPME with GC-ICPMS and GC-TOFMS detection

Determination of organophosphorus fire retardants and plasticizers at trace levels in wastewater is described. In this work, microwave assisted extraction (MAE) and solid-phase microextraction (SPME) are used for sample preparation to extract and preconcentrate the analytes, followed by analysis by gas chromatography coupled to inductively coupled plasma mass spectrometry (GC-ICP-MS) for phosphorus-specific detection. Gas chromatography coupled to time of flight mass spectrometry (GC-TOF-MS) was used to confirm the organphosphorus fire retardants in wastewater. The detection limits of organophosphorus fire retardants (OPFRs) were 29 ng L(-1) for tri-n-butyl phosphate (TnBP), 45 ng for L(-1) for tris(2-butoxyethyl)phosphate (TBEP), and 50 ng L(-1) for tris(2-ethylhexyl)phosphate (TEHP). Optimized extraction conditions were performed at 65 degrees C for 30 min and with 10% NaCl. Application of MAE during the sample preparation prior to the SPME allowed the detection of tris(2-ethylhexyl) phosphate, which has been difficult to determine in previous work. Application of the method to wastewater samples resulted in detecting 3.1 microg L(-1) P from TnBP, 5.0 microg L(-1) P from TBEP, and 4.0 microg L(-1) P from TEHP. The presence of these compounds were also confirmed by SPME-GC-TOF-MS.     

The effect of thiacloprid formulation on DNA/chromosome damage and changes in GST activity in bovine peripheral lymphocytes

The potential genotoxic effect of thiacloprid formulation on bovine peripheral lymphocytes was evaluated using the comet assay and the cytogenetic endpoints: chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MNi). Whole blood cultures were treated with the insecticide at concentrations of 30, 60, 120, 240 and 480 μg mL^?1 for 24, 48 h and/or 2 h of incubation. A statistically significant increase in the frequency of DNA damage, as well as in unstable chromosome aberrations (% breaks) were found after exposure to the insecticide at concentrations ranging from 120 to 480 μg mL^?1 (P < 0.05, P < 0.01, P < 0.001). For the detection of stable structural chromosome aberrations (e.g., translocations) and numerical aberrations by the FISH method, three whole chromosome painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) were used in our experiments. We observed numerical aberrations, but without any statistical significance. Regarding the sister chromatid exchanges, no significant elevation in the SCE frequencies was found after 24-h exposure to the insecticide. A dose-related response in the SCE induction was obtained in bovine cultures after the prolonged time of exposure (48 h) to thiacloprid formulation at concentrations ranging from 120 to 480 μg mL^?1 in each donor (P < 0.05, P < 0.01), which was associated with a reduction of the PI (P < 0.05, P < 0.01). The insecticide failed to produce MNi; however, a significant reduction of CBPI was observed. Using real-time PCR, a decrease in the expression of bovine glutathione S-transferase M3 (GSTM3) was detected at the lowest dose. The higher concentrations of thiacloprid formulation caused an increase in the mRNA expression.     

Relevant parameters for assessing the environmental impact of some pyridinium, ammonium and pyrrolidinium based ionic liquids

Several physico-chemical properties relevant to determine the environmental impact of ionic liquids - aqueous solubility, octanol/water partition coefficient, chromatographically derived lipophilicity and infinite dilution diffusion coefficients in water - were measured in ionic liquids based on pyridinium, ammonium and pyrrolidinium cations with bis(trifluoromethylsulfonyl)imide anions. The influence of the presence of hydroxyl or ester groups in the physico-chemical properties of these liquids was checked. It appeared that the presence of functional oxygenated moieties reduces the lipophilicity of ionic liquids and so decreases the risk of bioaccumulation in environment.     

Mercury in wild mushrooms and underlying soil substrate from Koszalin, North-central Poland

Concentrations of total mercury were determined by cold-vapour atomic absorption spectroscopy (CV-AAS) in 221 caps and 221 stalks of 15 species of wild growing higher fungi/mushrooms and 221 samples of corresponding soil substrate collected in 1997-98 in Manowo County, near the city of Koszalin in North-central Poland. Mean mercury concentrations in caps and stalks of the mushroom species examined and soils varied between 30+/-31 and 920+/-280, 17+/-11 and 560+/-220, and 10+/-9 and 170+/-110 ng/g dry matter, respectively. Cap to stalk mercury concentration quotients were from 1.0+/-0.4 in poison pax (Paxillus involutus) to 2.8+/-0.7 in slippery jack (Suillus luteus). Brown cort (Cortinarius malicorius), fly agaric (Amanita muscaria), orange-brown ringless amanita (A. fulva), red-aspen bolete (Leccinum rufum) and mutagen milk cap (Lactarius necator) contained the highest concentrations of mercury both in caps and stalks, and mean concentrations varied between 600+/-750 and 920+/-280 and 370+/-470 and 560+/-220 ng/g dry matter, respectively. An estimate of daily intake of mercury from mushroom consumption indicated that the flesh of edible species of mushrooms may not pose hazards to human health even at a maximum consumption rate of 28 g/day. However, it should be noted that mercury intake from other foods will augment the daily intake rates. Species such as the sickener (Russula emetica), Geranium-scented russula (R. fellea) and poison pax (P. involutus) did not concentrate mercury as evidenced from the bioconcentration factors (BCFs: concentrations in mushroom/concentration in soil substrate), which were less than 1. Similarly, red-hot milk cap (L. rufus), rickstone funnel cap (Clitocybe geotropa) and European cow bolete (S. bovinus) were observed to be weak accumulators of mercury. Fly agaric (A. muscaria) accumulated great concentrations of mercury with BCFs reaching 73+/-42 and 38+/-22 in caps and stalks, respectively. Mercury BCFs of between 4.0+/-2.3 and 23+/-25 (caps) and 2.6+/-1.9 and 14+/-12 (stalks) were noted for the other mushroom species. Relatively great concentrations of mercury in fly agaric (A. muscaria) were due to preferential uptake of this element by this species.     

Land cover composition determinants in the Uckermark (NE Germany) over a 220-year period

There is broad consensus that land use/cover has changed in central Europe over millennia. However, few studies have addressed the roles of the anthropogenic (socio-economic) and natural (biophysical) factors driving these changes over shorter periods (e.g., 100 years). In this study, we analyse the determinants of land cover composition at three discrete time periods (c. 1780, 1890, and 2000). We hypothesise that different anthropogenic and natural factors are the determinants of the main land cover types (arable fields, grasslands, and forests) and that the effects of natural and anthropogenic factors on the main land cover types differ among landscapes with highly dynamic and more stable forest-open land distributions. The study was carried out in the Uckermark region located in northeastern Germany. We compiled data on natural and anthropogenic factors (e.g. forest cover and number of inhabitants) of 65 municipalities in four landscape sections of equal size (10 × 10 km). Landscape sections were selected to reflect different dynamics (high/low) in conversion from forest to arable fields or grasslands and vice versa from 1780 to 2000. Averaged linear mixed-effect models explained between 7.5 and 81.2 % of the variance. The unique effect of anthropogenic factors varied from 2.1 to 18.7 % and that of natural factors varied from 0.2 to 43.4 %. In four of six models that included both types of factors, the natural factors were more influential than the anthropogenic factors. Except in three cases, anthropogenic and natural factors showed opposite effects on land cover types in more dynamic and more stable windows. Though the Uckermark region has been influenced by human activity for thousands of years, natural factors were a major determinant of land cover composition during all time periods, whereas anthropogenic factors became more important only during the latest time period under investigation.     

Chemical characteristics of trace metals in PM10 and their concentrated weighted trajectory analysis at Central Delhi, India

Trace metals associated with PM₁₀ aerosols and their variation during day and nighttime as well as during different seasons have been studied for the year 2012. PCA analysis suggested 5 PCs, which accounted for 86.8% cumulative variance. PC1 accounted for 30% with a significant loading of metals of anthropogenic origin, while PC2 showed 28% variance with the loading of metals of crustal origin. These trace metals showed seasonal distinct day and night time characteristics. The concentrations of Cu, Pb, and Cd were found to be higher during nighttime in all the seasons. Only Fe was observed with significantly higher mean concentrations during daytime of all seasons except monsoon. The highest mean values of Cu, Cd, Zn, and Pb during post-monsoon might be attributed to winds advection over the regions of waste/biomass burning and industrial activities in Punjab and Haryana regions. Furthermore, concentration weighted trajectory analysis suggested that metals of crustal origin were contributed by long-range transport while metals of anthropogenic and industrial activities were contributed by regional/local source regions.