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This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

Nontarget mass spectrometry has great potential to reveal patterns of water contamination globally through community science, but few studies are conducted in low-income countries, nor with open-source workflows, and few datasets are FAIR (Findable, Accessible, Interoperable, Reusable). Water was collected from urban and rural rivers around Dhaka, Bangladesh, and analyzed by liquid chromatography high-resolution mass spectrometry in four ionization modes (electrospray ionization ±, atmospheric pressure chemical ionization ±) with data-independent MS2 acquisition. The acquisition strategy was complementary: 19,427 and 7365 features were unique to ESI and APCI, respectively. The complexity of water pollution was revealed by >26,000 unique molecular features resolved by MS-DIAL, among which >20,000 correlated with urban sources in Dhaka. A major wastewater treatment plant was not a dominant pollution source, consistent with major contributions from uncontrolled urban drainage, a result that encourages development of further wastewater infrastructures. Matching of deconvoluted MS2 spectra to public libraries resulted in 62 confident annotations (i.e., Level 1-2a) and allowed semiquantification of 42 analytes including pharmaceuticals, pesticides, and personal care products. In silicostructure prediction for the top 100 unknown molecular features associated with an urban source allowed 15 additional chemicals of anthropogenic origin to be annotated (i.e., Level 3). The authentic MS2 spectra were uploaded to MassBank Europe, mass spectral data were openly shared on the MassIVE repository, a tool (i.e., MASST) that could be used for community science environmental surveillance was demonstrated, and current limitations were discussed.

Air pollution and rapid chemical intensification are major threats to the environment and human health. Today, we have produced over 350,000 chemicals, and current testing strategies do not meet the demands. Therefore, it is important to develop new approach methodologies (NAMs) that can help fill current information gaps. Toxicology needs to evolve from hazard and risk assessments based on morphological endpoints in animal tests towards a mechanism-driven integrated approach that better includes computational modelling as well as molecular, human, and in vitrodata. The application of new science and technology such as different types of imaging and omics methods can allow faster collection of high-quality toxicological data for hazard identification and better prediction of toxicological potential using advanced in silicoapproaches including machine learning. A shift toward active prevention of pollution through a safe and sustainable-by-design approach based on cutting-edge science could significantly help safeguard the population and planetary health. Moreover, it is necessary to improve the understanding of how interactions among chemical mixtures, climate change, infectious agents, and other stressors that constitute the exposome, may affect biota and human health. Individual responses to current exposures and susceptibility to disease are influenced by factors such as genetics, epigenetics, physiology, and health status, which involve changes in biological pathways caused by own previous exposures or even ancestral exposures. It is therefore important to better consider individual exposomes and susceptibility in future risk assessments and precision medicine. This review describes the central role of mechanistic toxicology in chemical safety and in the cross-disciplinary efforts needed to characterize the exposome and its complex interactions in detail.

ABSTRACTBreastmilk has many documented beneficial effects on the developing human infant, but the components of breastmilk that influence these developmental pathways have not been fully elucidated. Increasing evidence suggests that non-coding RNAs encapsulated in extracellular vesicles (EVs) represent an important mechanism of communication between the mother and child. Long non-coding RNAs (lncRNAs) are of particular interest given their key role in gene expression and development. However, it is not known whether breastmilk EVs contain lncRNAs. We used qRT-PCR to determine whether EVs isolated from human breastmilk contain lncRNAs previously reported to be important for developmental processes. We detected 55 of the 87 screened lncRNAs in EVs from the 30 analyzed breastmilk samples, and CRNDE, DANCR, GAS5, SRA1 and ZFAS1 were detected in >90% of the samples. GAS5, SNHG8 and ZFAS1 levels were highly correlated (Spearman's rho > 0.9; P< 0.0001), which may indicate that the loading of these lncRNAs into breastmilk EVs is regulated by the same pathways. The detected lncRNAs are important epigenetic regulators involved in processes such as immune cell regulation and metabolism. They may target a repertoire of recipient cells in offspring and could be essential for child development and health. Further experimental and epidemiological studies are warranted to determine the impact of breastmilk EV-encapsulated lnRNAs in mother to child signaling.

Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

Some aluminium alloys with Mg-Si age-hardening are used in vehicle radiators. For cost reasons they are preferably delivered in a naturally aged temper. Estimated minimum time of natural ageing between brazing and when the radiator is taken into service is 14 days. At the service temperature of 95°C, the radiator material will continue to age harden. For accelerated durability testing it is vital to use a radiator with the strength and ageing response of a service radiator. We investigated whether the full 14 days of natural ageing were needed, or if the time could be shortened. Since a vehicle is not in constant use, the radiator temperature will vary over time. We therefore compared cyclic ageing between ambient temperature and 95°C to continuous ageing at 95°C. The Sapa Heat Transfer alloys FA7870 (for headers) and FA7850 (for tubes) were subjected to different ageing times at different temperatures. Tensile and hardness were performed to assess the ageing effect. It was found that natural ageing reduced hardening during the subsequent ageing at service temperature ageing effect, an effect that was most pronounced for the first four days. There was no difference between continuous and cyclic ageing.

In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen-18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations.

Many pathological processes are not directly correlated to dramatic alterations in protein levels. The changes in local concentrations of important proteins in a subset of cells or at specific loci are likely to play a significant role in disease etiologies, but the precise location might be unknown, or the concentration might be too small to be adequately sampled for traditional proteomic techniques. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a unique analytical method that combines analysis of multiple molecular species and of their distribution in a single platform. As reproducibility is essential for successful biomarker discovery, it is important to systematically assess data quality in biologically relevant MALDI IMS experiments. In the present study, we applied four simple tools to study the reproducibility for individual sections, within-group variation, and between-group variation of data acquired from brain sections of 21 animals divided into three treatment groups. We also characterized protein changes in distinct regions of the striatum from six-month-old rats treated neonatally (postnatal days 9–10) with the cyanobacterial toxin β-N-methylamino-l-alanine (BMAA), which has been implicated in neurodegenerative diseases. The results showed that optimized experimental settings can yield high-quality MALDI IMS data with relatively low variation (14% to 15% coefficient of variance) that allow the characterization of subtle changes in protein expression in various subregions of the brain. This was further exemplified by the dose-dependent reduction of myelin basic protein in the caudate putamen and the nucleus accumbens of adult rats neonatally treated with BMAA (150 and 460 mg/kg). The reduction in myelin basic protein was confirmed through immunohistochemistry and indicates that developmental exposure to BMAA may induce structural effects on axonal growth and/or directly on the proliferation of oligodendrocytes and myelination, which might be important for the previously shown BMAA-induced long-term cognitive impairments.

-N-methylamino- l-alanine (BMAA), a neurotoxic amino acid produced by cyanobacteria, has been suggested to be involved in the etiology of a neurodegenerative disease complex which includes Parkinson-dementia complex (PDC). In PDC, neuromelanin-containing neurons in substantia nigra are degenerated. Many PDC patients also have an uncommon pigmentary retinopathy. The aim of this study was to investigate the distribution of 3H-BMAA in mice and frogs, with emphasis on pigment-containing tissues. Using autoradiography, a distinct retention of 3H-BMAA was observed in melanin-containing tissues such as the eye and neuromelanin-containing neurons in frog brain. Analysis of the binding of 3H-BMAA to Sepia melanin in vitro demonstrated two apparent binding sites. In vitro-studies with synthetic melanin revealed a stronger interaction of 3H-BMAA with melanin during synthesis than the binding to preformed melanin. Long-term exposure to BMAA may lead to bioaccumulation in melanin- and neuromelanin-containing cells causing high intracellular levels, and potentially changed melanin characteristics via incorporation of BMAA into the melanin polymer. Interaction of BMAA with melanin may be a possible link between PDC and pigmentary retinopathy.