Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: Preliminary observations on maternal samples

Monoclonal antibody to fetal haemoglobin (a₂γy₂) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-γ with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-γ-positive cells sorted from four different pregnant women are described here, using fluorescence in situ hybridization (FISH) with chromosome-specific probes to identify fetal cells. Our data demonstrate that far fewer candidate fetal cells are sorted with this protocol than by current cell surface staining methods that employ the monoclonal antibody CD71. This results in increased fetal cell sorting purities. With this protocol, standard FISH techniques require modification due to the rigorous fixation with 4 per cent paraformaldehyde. Our initial data indicate the promise of this approach.     

Detection of fetal HLA-DQα sequences in maternal blood: A gender-independent technique of fetal cell identification

The objective of this study was to detect fetal HLA-DQα gene sequences in maternal blood. HLA-DQα genotypes of 70 pregnant women and their partners were determined for type A1. We specifically sought couples where the father, but not the mother, had genotype A1. In 12 women, maternal blood samples were flow-sorted. Candidate fetal cells were isolated and amplified by using PCR primers specific for a paternal HLA-DQα A1 allele. Fetal HLA-DQα A1 genotype was predicted from sorted cells; amniocytes or cheek swabs were used for confirmation. Six of twelve sorted samples had amplification products indicating the presence of the HLA-DQα A1 allele; 6/12 did not. Prediction of the fetal genotype was 100 per cent correct, as determined by subsequent amplification of amniocytes or cheek swabs. We conclude that paternally inherited uniquely fetal HLA-DQα gene sequences can be identified in maternal blood. This system permits the identification of fetal cells independent of fetal gender, and has the potential for non-invasive prenatal diagnosis of paternally inherited conditions.     

Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood

Fetal nucleated erythrocytes (NRBC) in maternal blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from maternal blood by flow sorting. Mononuclear blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in maternal blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from maternal blood, permitting genetic analysis by the polymerase chain reaction.     

Fetal renal anomalies and genetic syndromes

Renal abnormalities are some of the commonest and most easily detectable anomalies on ultrasound. Many are an isolated finding but the prognosis may be altered considerably by the detection of other anomalies which could indicate a genetic disorder or syndrome. It is often easier to detect presupposed anomalies and the purpose of this article is to introduce and discuss those syndromes that may present with a renal abnormality on ultrasound. Common renal findings are presented with the range of additional anomalies that should be sought and suggested diagnostic tests. It should be remembered that although for many genetic conditions specific mutation analysis is now available, this usually requires pre‒pregnancy investigations. Furthermore, in some cases the definitive diagnosis may not be suspected until post mortem. By this time it may be too late to establish a cell line to confirm the suspicion using laboratory methods. It is therefore important to take tissue samples antenatally where possible, or at delivery, as postnatal samples may have a high culture failure rate. Copyright © 2001 John Wiley & Sons, Ltd.     

美国和五大湖双边共同关注区域的白肚燕(双色树燕)雏鸟的有机污染状况

白肚燕,又名双色树燕,栖居于五大湖流域中27个双边共同关注区域(AOCs),从2010年到2014年对其进行了污染物暴露风险评估,以协助管理者和监管者评估五大湖AOCs的污染状况。本文比较了AOCs和附近非AOCs雏鸟体内污染物浓度的差异。AOCs白肚燕雏鸟尸体中多氯联苯(PCB)和多溴联苯醚的含量分别为30%和33%,低于非AOCs的平均浓度。AOCs白肚燕雏鸟胃含物中多环芳烃(PAH)浓度和血浆中全氟化合物浓度分别为67%和64%,也低于非AOCs的平均浓度。但是与已有生殖效应记载的高PCB污染区相比,有些AOCs雏鸟尸体内的PCBs浓度只是存在小幅升高。一些AOCs食物中PAHs的浓度足够高以致引起可测量的生理响应。在AOCs中,全氟化合物全氟辛烷磺酸在血浆中的最高浓度出现在Raisin河(密歇根州,美国;几何平均数330 ng/mL),但远低于预估毒性的参考值(1 700 ng/mL)。之前有研究报道雏鸟胃含物中PAH和PCB的浓度以及尸体中PCBs的含量与沉积物中污染物的含量显著相关,从而可加强白肚燕在评估沉积物污染生物有效性方面的应用。
精选自Thomas W. Custer, Christine M. Custer, Paul M. Dummer, Diana Goldberg, J. Christian Franson, Richard A. Erickson. Organic contamination in tree swallow (Tachycineta bicolor) nestlings at United States and binational Great Lakes areas of concern. Environmental Toxicology and Chemistry: Volume 36, Issue 3, pages 735–748, July 2017. DOI: 10.1002/etc.3598
详情请见http://onlinelibrary.wiley.com/wol1/doi/10.1002/etc.3598/full
     

Environmental and socioeconomic assessment of impacts by mining activities—a case study in the Certej River catchment,Western Carpathians,Romania

Background, aim and scope

In the region of the Apuseni Mountains, part of the Western Carpathians in Romania, metal mining activities have a long-standing tradition. These mining industries created a clearly beneficial economic development in the region. But their activities also caused impairments to the environment, such as acid mine drainage (AMD) resulting in long-lasting heavy metal pollution of waters and sediments. The study, established in the context of the ESTROM programme, investigated the impact of metal mining activities both from environmental and socioeconomic perspectives and tried to incorporate the results of the two approaches into an integrated proposition for mitigation of mining-related issues.

Study site

The small Certej catchment, situated in the Southern Apuseni Mountains, covers an area of 78 km². About 4,500 inhabitants are living in the basin, in which metal mining was the main economic sector. An open pit and several abandoned underground mines are producing heavy metal-loaded acidic water that is discharged untreated into the main river. The solid wastes of mineral processing plants were deposited in several dumps and tailings impoundment embodying the acidic water-producing mineral pyrite.

Methods

The natural science team collected samples from surface waters, drinking water from dug wells and from groundwater. Filtered and total heavy metals, both after enrichment, and major cations were analysed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Major anions in waters, measured by ion chromatography, alkalinity and acidity were determined by titration. Solid samples were taken from river sediments and from the largest tailings dam. The latter were characterised by X-ray fluorescence and X-ray diffraction. Heavy metals in sediments were analysed after digestion. Simultaneously, the socioeconomic team performed a household survey to evaluate the perception of people related to the river and drinking water pollution by way of a logistic regression analysis.

Results and discussion

The inputs of acid mine waters drastically increased filtered heavy metal concentrations in the Certej River, e.g. Zn up to 130 mg L⁻¹, Fe 100 mg L⁻¹, Cu 2.9 mg L⁻¹, Cd 1.4 mgL⁻¹ as well as those of SO₄ up to 2.2 g L⁻¹. In addition, river water became acidic with pH values of pH 3. Concentrations of pollutant decreased slightly downstream due to dilution by waters from tributaries. Metal concentrations measured at headwater stations reflect background values. They fell in the range of the environmental quality standards proposed in the EU Water Framework Directive for dissolved heavy metals. The outflow of the large tailing impoundment and the groundwater downstream from two tailings dams exhibited the first sign of AMD, but they still had alkalinity.

Most dug wells analysed delivered a drinking water that exhibited no sign of AMD pollution, although these wells were a distance of 7 to 25 m from the contaminated river. It seems that the Certej River does not infiltrate significantly into the groundwater.

Pyrite was identified as the main sulphide mineral in the tailings dam that produces acidity and with calcite representing the AMD-neutralising mineral. The acid–base accounting proved that the potential acid-neutralising capacity in the solid phases would not be sufficient to prevent the production of acidic water in the future. Therefore, the open pits and mine waste deposits have to be seen as the sources for AMD at the present time, with a high long-term potential to produce even more AMD in the future.

The socioeconomic study showed that mining provided the major source of income. Over 45% of the households were partly or completely reliant on financial compensations as a result of mine closure. Unemployment was considered by the majority of the interviewed persons as the main cause of social problems in the area. The estimation of the explanatory factors by the logistic regression analysis revealed that education, household income, pollution conditions during the last years and familiarity with environmental problems were the main predictors influencing peoples’ opinion concerning whether the main river is strongly polluted. This model enabled one to predict correctly 77% of the observations reported. For the drinking water quality model, three predictors were relevant and they explained 66% of the observations.

Conclusions

Coupling the findings from the natural science and socioeconomic approaches, we may conclude that the impact of mining on the Certej River water is high, while drinking water in wells is not significantly affected. The perceptions of the respondents to pollution were to a large extent consistent with the measured results.

Recommendations and perspectives

The results of the study can be used by various stakeholders, mainly the mining company and local municipalities, in order to integrate them in their post-mining measures, thereby making them aware of the potential long-term impact of mining on the environment and on human health as well as on the local economy.

     

Long-term effect of temperature on bioaccumulation of dietary metals and metallothionein induction in Sparus aurata

Previous studies have demonstrated that the commercial feed of aquacultured fish contains trace amounts of toxic and essential metals which can accumulate in tissues and finally be ingested by consumers. Recently rising temperatures, associated to the global warming phenomenon, have been reported as a factor to be taken into consideration in ecotoxicology, since temperature-dependent alterations in bioavailability, toxicokinetics and biotransformation rates can be expected. Sparus aurata were kept at 22 °C, 27 °C and 30 °C for 3 months in order to determine the temperature effect on metallothionein induction and metal bioaccumulation from a non-experimentally contaminated commercial feed. A significant temperature-dependent accumulation of cadmium (Cd), copper (Cu), mercury (Hg), zinc (Zn), lead (Pb) and iron (Fe) was found in liver, together with that of manganese (Mn), Fe and Zn in muscle. Hg presented the highest bioaccumulation factor, and essential metal homeostasis was disturbed in both tissues at warm temperatures. An enhancement of hepatic metallothionein induction was found in fish exposed to the highest temperature.     

Effect of sequencing batch reactor operation on presence and concentration of tetracycline-resistant organisms.

The effect of sequencing batch reactor operation on presence and concentration of tetracycline-resistant organisms was studied as a function of organic loading rate (OLR) and solids retention time (SRT), with and without supplemented influent tetracycline. These effects were evaluated using bacterial counts, bacterial production, system growth rate, and percent resistance. These evaluation parameters were applied to both intermediate resistant and resistant heterotrophs, enterics, and lactose fermenters. Tetracycline intermediate resistant and resistant bacteria are defined as the survival of colonies on agar with 5 and 20 mg/L tetracycline, respectively. Based on these studies, increases in influent tetracycline concentration and OLR resulted in amplification of tetracycline resistance. Decreases in SRT also resulted in amplification of tetracycline resistance.     

Inferring extinction of mammals from sighting records, threats, and biological traits

For species with five or more sightings, quantitative techniques exist to test whether a species is extinct on the basis of distribution of sightings. However, 70% of purportedly extinct mammals are known from fewer than five sightings, and such models do not include some important indicators of the likelihood of extinction such as threats, biological traits, search effort, and demography. Previously, we developed a quantitative method that we based on species' traits in which we used Cox proportional hazards regression to calculate the probability of rediscovery of species regarded as extinct. Here, we used two versions of the Cox regression model to determine the probability of extinction in purportedly extinct mammals and compared the results of these two models with those of stationary Poisson, nonparametric, and Weibull sighting-distribution models. For mammals with five or more sightings, the stationary Poisson model categorized all but two critically endangered (flagged as possibly extinct) species in our data set as extinct, and results with this model were consistent with current categories of the International Union for the Conservation of Nature. The scores of probability of rediscovery for individual species in one version of our Cox regression model were correlated with scores assigned by the stationary Poisson model. Thus, we used this Cox regression model to determine the probability of extinction of mammals with sparse records. On the basis of the Cox regression model, the most likely mammals to be rediscovered were the Montane monkey-faced bat (Pteralopex pulchra), Armenian myotis (Myotis hajastanicus), Alcorn's pocket gopher (Pappogeomys alcorni), and Wimmer's shrew (Crocidura wimmeri). The Cox model categorized two species that have recently disappeared as extinct: the baiji (Lipotes vexillifer) and the Christmas Island pipistrelle (Pipistrellus murrayi). Our new method can be used to test whether species with few records or recent last-sighting dates are likely to be extinct.