Migration of Styrene in Yogurt and Dairy Products Packaged in Polystyrene: Results from Market Samples.

The European Food Safety Authority is re-evaluating styrene for assessing the safety of food contact materials (FCM) such as polystyrene (PS) and started a systematic review of the data on migration levels in food. A restriction for styrene is expected in the near future. The main food contact application of PS is dairy packaging, mainly at refrigerated storage. In this study, seventeen dairy products packed in PS taken from the Italian and German markets were investigated. Styrene concentrations in the refrigerated dairy products (yogurt, cream) ranged from 5 to 30 µg/kg at the best before date, while in single serving portions of coffee creamer, which were stored at room temperature until the best before date of approx. 190 days, 401 µg/kg were measured. Among several parameters, the ratio between the surface contact area of the package and the quantity of the food packed, the time/temperature conditions of production/filling and storage of the products were identified as the main factors influencing styrene migration into food under realistic conditions. Yogurts fermented in the pots for approximately 8 h at 40-50 °C showed higher styrene levels than those fermented in an incubator and filled at 20 °C. The fat content might influence the styrene level but the effect, if any, was too small in relation to the variability of other parameters. Levels of styrene migrating into 50% ethanol food simulant under standardized condition (10 days/40 °C) were found to be much higher than levels in refrigerated foods. This raises the question as to whether compliance testing for PS plastics should be adapted taking into consideration the correlation between migration testing by laboratory simulations and migration into real food.

Brain-Derived Neurotrophic Factor/Tropomyosin Receptor Kinase B Signaling Controls Excitability and Long-Term Depression in Oval Nucleus of the BNST.

Dysregulation of proteins involved in synaptic plasticity is associated with pathologies in the CNS, including psychiatric disorders. The bed nucleus of the stria terminalis (BNST), a brain region of the extended amygdala circuit, has been identified as the critical hub responsible for fear responses related to stress coping and pathologic systems states. Here, we report that one particular nucleus, the oval nucleus of the BNST (ovBNST), is rich in brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) receptor. Whole-cell patch-clamp recordings of neurons from male mouse ovBNST in vitro showed that the BDNF/TrkB interaction causes a hyperpolarizing shift of the membrane potential from resting value, mediated by an inwardly rectifying potassium current, resulting in reduced neuronal excitability in all major types of ovBNST neurons. Furthermore, BDNF/TrkB signaling mediated long-term depression (LTD) at postsynaptic sites in ovBNST neurons. LTD of ovBNST neurons was prevented by a BDNF scavenger or in the presence of TrkB inhibitors, indicating the contribution to LTD induction. Our data identify BDNF/TrkB signaling as a critical regulator of synaptic activity in ovBNST, which acts at postsynaptic sites to dampen excitability at short and long time scales. Given the central role of ovBNST in mediating maladaptive behaviors associated with stress exposure, our findings suggest a synaptic entry point of the BDNF/TrkB system for adaptation to stressful environmental encounters.

On the objectivity, reliability, and validity of deep learning enabled bioimage analyses.

Bioimage analysis of fluorescent labels is widely used in the life sciences. Recent advances in deep learning (DL) allow automating time-consuming manual image analysis processes based on annotated training data. However, manual annotation of fluorescent features with a low signal-to-noise ratio is somewhat subjective. Training DL models on subjective annotations may be instable or yield biased models. In turn, these models may be unable to reliably detect biological effects. An analysis pipeline integrating data annotation, ground truth estimation, and model training can mitigate this risk. To evaluate this integrated process, we compared different DL-based analysis approaches. With data from two model organisms (mice, zebrafish) and five laboratories, we show that ground truth estimation from multiple human annotators helps to establish objectivity in fluorescent feature annotations. Furthermore, ensembles of multiple models trained on the estimated ground truth establish reliability and validity. Our research provides guidelines for reproducible DL-based bioimage analyses.

Research in biology generates many image datasets, mostly from microscopy. These images have to be analyzed, and much of this analysis relies on a human expert looking at the images and manually annotating features. Image datasets are often large, and human annotation can be subjective, so automating image analysis is highly desirable. This is where machine learning algorithms, such as deep learning, have proven to be useful. In order for deep learning algorithms to work first they have to be 'trained'. Deep learning algorithms are trained by being given a training dataset that has been annotated by human experts. The algorithms extract the relevant features to look out for from this training dataset and can then look for these features in other image data. However, it is also worth noting that because these models try to mimic the annotation behavior presented to them during training as well as possible, they can sometimes also mimic an expert's subjectivity when annotating data. Segebarth, Griebel et al. asked whether this was the case, whether it had an impact on the outcome of the image data analysis, and whether it was possible to avoid this problem when using deep learning for imaging dataset analysis. For this research, Segebarth, Griebel et al. used microscopy images of mouse brain sections, where a protein called cFOS had been labeled with a fluorescent tag. This protein typically controls the rate at which DNA information is copied into RNA, leading to the production of proteins. Its activity can be influenced experimentally by testing the behaviors of mice. Thus, this experimental manipulation can be used to evaluate the results of deep learning-based image analyses. First, the fluorescent images were interpreted manually by a group of human experts. Then, their results were used to train a large variety of deep learning models. Models were trained either on the results of an individual expert or on the results pooled from all experts to come up with a consensus model, a deep learning model that learned from the personal annotation preferences of all experts. This made it possible to test whether training a model on multiple experts reduces the risk of subjectivity. As the training of deep learning models is random, Segebarth, Griebel et al. also tested whether combining the predictions from multiple models in a so-called model ensemble improves the consistency of the analyses. For evaluation, the annotations of the deep learning models were compared to those of the human experts, to ensure that the results were not influenced by the subjective behavior of one person. The results of all bioimage annotations were finally compared to the experimental results from analyzing the mice's behaviors in order to check whether the models were able to find the behavioral effect on cFOS. Segebarth, Griebel et al. concluded that combining the expert knowledge of multiple experts reduces the subjectivity of bioimage annotation by deep learning algorithms. Combining such consensus information in a group of deep learning models improves the quality of bioimage analysis, so that the results are reliable, transparent and less subjective.

Evidence for impaired extinction learning in humans after distal stress exposure.

Stressful or traumatic events can be risk factors for anxiety or trauma- and stressor-related disorders. In this regard, it has been shown that stress affects aversive learning and memory processes. In rodents, stress exposure 10 days prior to fear acquisition impairs fear extinction. However, in humans the effect of distal stress on fear conditioning is sparse. Therefore, we examined the influence of distal stress on fear memory in humans in two studies. In Study 1, participants underwent either socially evaluated cold-pressor test (SECPT) or sham procedure 10 days or 40 min before a fear conditioning paradigm (four groups, N = 78). In Study 2, context effects were examined by conducting SECPT and sham procedures 10 days prior conditioning either in the later fear conditioning context or in another context (three groups, N = 69). During acquisition phase, one geometrical shape (conditioned stimulus, CS+) was paired with painful electric shocks (unconditioned stimulus, US), but never a second shape (CS-). Extinction phase was identical to acquisition, but without US delivery. Importantly, for Study 1 these phases were conducted on one day, while for Study 2 on two separated days. Successful fear acquisition was indicated by aversive ratings and startle potentiation to CS+ versus CS- in both studies. Interestingly, participants stressed 10 days earlier showed impaired extinction on the implicit level (startle potentiation to CS+ vs. CS-) in Study 1 and only in the acquisition context on the explicit level (aversive ratings for CS+ vs. CS-) in Study 2. In sum, distal stress may strengthen later acquired fear memories and thereby impair fear extinction. This finding could have clinical implications, showing that prior stress exposure sensitizes later aversive processing and impairs therapy.

Siloxane in baking moulds, emission to indoor air and migration to food during baking with an electric oven.

Linear and cyclic volatile methylsiloxanes (l-VMS and c-VMS) are man-made chemicals with no natural source. They have been widely used in cosmetics, personal care products, coatings and many other products. As a consequence of their wide use, VMS can be found in different environmental media, as well as in humans. We bought 14 new silicone baking moulds and 3 metallic moulds from the market and used them in different baking experiments. Four of the silicone baking moulds were produced in Germany, two in Italy, four in China, and for the other moulds were no information available. The metal forms were all produced in Germany. VMS were measured in the indoor air throughout the baking process and at the edge and in the center of the finished cakes using a GC/MS system. Additionally, the particle number concentration (PNC) and particle size distribution were measured in the indoor air. The highest median concentrations of VMS were observed immediately following baking: 301 µg/m3 of D7, 212 µg/m3 of D6, and 130 µg/m3 of D8. The silicone moulds containing the highest concentrations of c-VMS corresponded with distinctly higher concentrations of the compounds in indoor air. Using a mould for more than one baking cycle reduced the indoor air concentrations substantially. Samples collected from the edge of the cake had higher concentrations relative to samples from the center, with a mean initial concentration of 6.6 mg/kg of D15, 3.9 mg/kg of D9, 3.7 mg/kg of D12, and 4.8 mg/kg of D18. D3 to D5 were measured only at very low concentrations. Before starting the experiment, an average PNC of 7300 particles/cm3 was observed in the room's air, while a PNC of 140,000 particles/cm3 was observed around the electric stove while it was baking, but this PNC slowly decreased after the oven was switched off. Baking with 4 of the moulds exceeded the German indoor precaution guide value for c-VMS, but the health hazard guide value was not reached during every experiment. Compared to other exposure routes, c-VMS contamination of cake from silicone moulds seems to be low, as demonstrated by the low concentrations of D4 and D6 measured. For less volatile c-VMS > D6 the results of the study indicate that food might play a more important role for daily intake. As a general rule, silicone moulds should be used only after precleaning and while strictly following the temperature suggestions of the producers.

Oxytocin Signaling in the Lateral Septum Prevents Social Fear during Lactation.

Oxytocin (OXT)-mediated behavioral responses to social and stressful cues have extensively been studied in male rodents. Here, we investigated the capacity of brain OXT receptor (OXTR) signaling in the lateral septum (LS) to prevent social fear expression in female mice using the social-fear-conditioning paradigm. Utilizing the activated OXT system during lactation, we show that lactating mice did not express fear 24 hr after social fear conditioning. Supporting the role of OXTR signaling in the LS in attenuation of social fear, synthetic OXT infusion or overexpression of OXTR in the LS diminished social fear expression, whereas constitutive OXTR knockout severely impaired social fear extinction in virgin mice. Subsequently, both pharmacological blockade of local OXTRs in the LS and chemogenetic silencing of supraoptic nucleus OXTergic afferents to the LS increased social fear expression in lactating mice. Hence, LS-projecting OXT neurons suppress social fear in female mice.

Rapid identification of polystyrene foam wastes containing hexabromocyclododecane or its alternative polymeric brominated flame retardant by X-ray fluorescence spectroscopy.

The brominated flame retardant hexabromocyclododecane (HBCDD) was added to Annex A of the list of persistent organic pollutants (POPs) of the Stockholm Convention. Thus, production and use of HBCDD will be banned, and the recycling of HBCDD-containing foam waste will be restricted. In reaction a special polymeric brominated flame retardant (PolyFR) was developed to replace HBCDD in expanded and extruded polystyrene foams for building and construction applications. A decision has to be made at some future time whether expanded and extruded polystyrene foam waste is to be subjected to incineration (with HBCDD) or to recycling (without HBCDD). Therefore, an appropriate and rapid field method is required to distinguish between foams containing HBCDD and foams free from HBCDD. Here we present a screening method for identifying HBCDD containing expanded and extruded polystyrene foams. The test principle is based on the fact that PolyFR (a brominated polymeric macromolecule) is not extractable whereas HBCDD (a low molecular weight substance) is extractable. Following rapid extraction of HBCDD the brominated flame retardant is identified and quantified via bromine analysis using a handheld X-ray fluorescence instrument. The method was applied successfully to 27 expanded and extruded polystyrene foam samples (foams and extruded polystyrene foam raw materials), which were provided without any information about the applied flame retardant. The presence of HBCDD was confirmed for all HBCDD-positive samples in the test. A robustness test revealed a high degree of correctness and a high repeatability for the test system: samples containing HBCDD and HBCDD-free samples were identified correctly with relative standard deviations of quantitative results below 14%. Moreover, X-ray fluorescence spectroscopy test results agree well with HBCDD determinations performed in a laboratory with a gas chromatograph coupled to a flame ionisation detector.

Perfluoroalkyl and polyfluoroalkyl substances in consumer products.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are used in a wide range of products of all day life. Due to their toxicological potential, an emerging focus is directed towards their exposure to humans. This study investigated the PFAS load of consumer products in a broad perspective. Perfluoroalkyl sulfonic acids (C4, C6-C8, C10-PFSA), carboxylic acids (C4-C14-PFCA) and fluorotelomer alcohols (4:2, 6:2; 8:2 and 10:2 FTOH) were analysed in 115 random samples of consumer products including textiles (outdoor materials), carpets, cleaning and impregnating agents, leather samples, baking and sandwich papers, paper baking forms and ski waxes. PFCA and PFSA were analysed by HPLC-MS/MS, whereas FTOH were detected by GC/CI-MS. Consumer products such as cleaning agents or some baking and sandwich papers show low or negligible PFSA and PFCA contents. On the other hand, high PFAS levels were identified in ski waxes (up to about 2000 µg/kg PFOA), leather samples (up to about 200 µg/kg PFBA and 120 µg/kg PFBS), outdoor textiles (up to 19 µg/m(2) PFOA) and some other baking papers (up to 15 µg/m(2) PFOA). Moreover, some test samples like carpet and leather samples and outdoor materials exceeded the EU regulatory threshold value for PFOS (1 µg/m(2)). A diverse mixture of PFASs can be found in consumer products for all fields of daily use in varying concentrations. This study proves the importance of screening and monitoring of consumer products for PFAS loads and the necessity for an action to regulate the use of PFASs, especially PFOA, in consumer products.

Impact of industrial production and packaging processes on the concentration of per- and polyfluorinated compounds in milk and dairy products.

Perfluorinated alkylated compounds (PFAA) have been identified in milk and dairy products at sub ppb levels, however, knowledge on the impact of industrial milk processing on PFAA levels is rare. This study examined industrial milk processing first by analytical screening of products of a cooperating dairy, which varied in kind and number of processing steps. Second, amounts of PFAA in raw milk, cream, skim milk, butter milk, and butter were mass balanced in industrial production. For migration testing, unpacked butter was sampled from the production and exposed to original packaging at 5 °C for 45 days. Screening identified dairy products with high fat contents to bear higher loads of PFAA. The mass balance of butter production revealed a significant impact of phase separation processes on concentrations in fat rich and aqueous phases. Storage of butter in packaging coated with a fluorinated polymer increased butter levels of both PFAA and FTOH.

Detection of fluorotelomer alcohols in indoor environments and their relevance for human exposure.

Fluorotelomer alcohols (FTOH) are important precursors of perfluorinated carboxylic acids (PFCA). These neutral and volatile compounds are frequently found in indoor air and may contribute to the overall human exposure to per- and polyfluorinated alkyl substances (PFAS). In this study air samples of ten workplace environments and a car interior were analysed. In addition, extracts and emissions from selected outdoor textiles were analysed in order to establish their potential contribution to the indoor levels of the above-mentioned compounds. Concentrations of FTOHs measured in air ranged from 0.15 to 46.8, 0.25 to 286, and 0.11 to 57.5ng/m(3) for 6:2, 8:2 and 10:2 FTOHs, respectively. The highest concentrations in air were identified in shops selling outdoor clothing, indicating outdoor textiles to be a relevant source of FTOH in indoor workplace environments. Total amounts of FTOH in materials of outdoor textiles accounted for <0.8-7.6, 12.1-180.9 and 4.65-105.7µg/dm(2) for 6:2, 8:2 and 10:2 FTOHs, respectively. Emission from selected textiles revealed emission rates of up to 494ng/h. The measured data show that a) FTOHs are present in indoor textiles (e.g. carpets), b) they are released at ambient temperatures and c) indoor air of shops selling outdoor textiles contains the highest levels of FTOH. Exposure of humans to perfluorooctanoic acid (PFOA) through absorption of FTOH and subsequent degradation is discussed on the basis of indoor air levels. Calculation of indoor air-related exposure using the median of the measured air levels revealed that exposure is on the same order of magnitude as the recently reported dietary intakes for a background-exposed population. On the basis of the 95th percentile, indoor air exposure to PFOA was estimated to exceed dietary exposure. However, indoor air-related intakes of FTOH are far below the tolerable daily intake (TDI) of PFOA, indicating that there is no risk to health, even when assuming an unrealistic complete degradation of FTOH into PFOA.