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Few studies have been published on occurrence and distribution on microplastics (MPs) in invertebrates from the Arctic. We still need to develop harmonised methods to enable good comparison between studies taking into account recovery rates, size ranges, shapes and polymer types. Here, we review studies on MPs in invertebrates from the Arctic and present suggestions on sampling protocols and potential indicator species. Since information on MPs in Arctic invertebrates is vastly lacking, we recommend to at least include suspension feeding bivalves like mussels in monitoring programmes to function as indicator species in the Arctic. Mussels have also been suggested as indicator species for MP monitoring in coastal regions further south. Although we recognise the challenge with particle selection and egestion in mussels as well as the relatively low concentrations of MPs in Arctic waters, uptake levels seem to represent recent exposures. More research is needed to understand these selection processes and how they affect the bioaccumulation processes. Future research should include studies on whether different functional groups of invertebrates have different exposures to MPs, e.g., if there are differences between sessile versus motile species or different feeding strategies. More knowledge on monitoring strategies for pelagic and benthic species is needed. Microplastics in arctic invertebrates- Status on occurrence and recommendations for future monitoring

Anthropogenic releases of plastics, persistent organic pollutants (POPs), and heavy metals can impact the environment, including aquatic ecosystems. Nanoplastics (NPs) have recently emerged as pervasive environmental pollutants that have the ability to adsorb POPs and can cause stress in organisms. Among POPs, DDT and its metabolites are ubiquitous environmental pollutants due to their long persistence. Despite the discontinued use of DDT in Europe, DDT and its metabolites (primarily p,p′-DDE) are still found at detectable levels in fish feed used in salmon aquaculture. Our study aimed to look at the individual and combined toxicity of NPs (50 mg/L polystyrene) and DDE (100 μg/L) using zebrafish larvae as a model. We found no significant morphological, cardiac, respiratory, or behavioural changes in zebrafish larvae exposed to NPs alone. Conversely, morphological, cardiac and respiratory alterations were observed in zebrafish larvae exposed to DDE and NPs + DDE. Interestingly, behavioural changes were only observed in zebrafish larvae exposed to NPs + DDE. These findings were supported by RNA-seq results, which showed that some cardiac, vascular, and immunogenic pathways were downregulated only in zebrafish larvae exposed to NPs + DDE. In summary, we found an enhanced toxicological impact of DDE when combined with NPs. publishedVersion

Microplastic particles are ubiquitous in the environment. However, little is known about their uptake and effects in humans or mammalian model organisms. Here, we studied the effects of pristine polyamide (15–20 µm) and polyethylene (40–48 µm) particles after oral ingestion in rats. The animals received feed containing microplastic particles (0.1% polyamide or polyethylene, or a mixture of both polymers) or a control diet without microplastic particles, for 5 weeks. The permeability of the duodenum was investigated in an Ussing chamber, whereas gene expression and concentration of tight junction proteins were measured in gut tissue and plasma. Microplastic particles were quantified by pyrolysis-gas chromatography/mass spectrometry in rats’ feces. Rats fed with microplastic particles had higher duodenal permeability. Expression of gene coding for the tight junction protein occludin (OCLN) was higher in PE treated animals compared to control or the PA group. No changes in the expression of the gene coding for zonula occludens protein 1 were detected. Occludin protein concentrations were below the limit of detection of the applied method in both gut and plasma. Zonula occludens protein 1 concentrations in the gut were significantly higher in groups exposed to PA and PE as compared to control, while zonula occludens protein 1 concentrations in plasma did not show significant changes. These results demonstrated that short-term exposure to a dose of 0.1% (w/w) microplastic particles in feed had limited effects on duodenal permeability, expression of pro-inflammatory protein genes and tight junction protein genes in the duodenum. publishedVersion

The Arctic Monitoring and Assessment Programme (AMAP) has published a plan and guidelines for the monitoring of litter and microplastics (MP) in the Arctic. Here we look beyond suggestions for immediate monitoring and discuss challenges, opportunities and future strategies in the long-term monitoring of litter and MP in the Arctic. Challenges are related to environmental conditions, lack of harmonization and standardization of measurements, and long-term coordinated and harmonized data storage. Furthermore, major knowledge gaps exist with regard to benchmark levels, transport, sources and effects, which should be considered in future monitoring strategies. Their development could build on the existing infrastructure and networks established in other monitoring initiatives in the Arctic, while taking into account specific requirements for litter and MP monitoring. Knowledge existing in northern and Indigenous communities, as well as their research priorities, should be integrated into collaborative approaches. The monitoring plan for litter and MP in the Arctic allows for an ecosystem-based approach, which will improve the understanding of linkages between environmental media of the Arctic, as well as links to the global problem of litter and MP pollution. Future monitoring of litter and microplastics in the Arctic – challenges, opportunities and strategies

Seafood plays a central role in global food and nutrition security. However, there is a lack of data on the concentration of nutrients and contaminants in fish and other seafood, especially in low- and middle-income countries. In order to assess the potential risks and benefits associated with seafood intake, reliable and up-to-date food composition data is crucial. The quality of food composition data is affected by several factors, such as sampling protocols and the suitability and quality of the methods applied for sample preparation and analysis. In this paper, we describe the sampling methodology and protocols related to the sampling of fish and other seafood and the corresponding analytical methods used to analyse the nutrient and contaminant content of such species. For nutrients, the determination of protein, fat, ash, energy, fatty acids, cholesterol, and amino acids is described, in addition to analyses for determination of the vitamin and mineral content in fish and other seafood. For contaminants, analyses for the determination of organic pollutants and microplastics are described. The methodology described in this paper is used for sampling data through scientific surveys in low- and middle-income countries with research vessel

Marine fish from the North East Atlantic Ocean (NEAO) are nutrient rich and considered a valuable economic resource. However, marine fish are also a major dietary source of several contaminants, including persistent organic pollutants (POPs) and heavy metals. Using one of the world’s largest seafood datasets (n > 25,000 individuals), comprising 12 commercially important fish species collected during 2006–2019 in the NEAO, we assessed the co-occurrence of elements and POPs, and evaluated potential risks to human consumers. Several positive correlations between concentrations of mercury (Hg), dioxins, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were observed. Concentrations of Hg, dioxins, PCBs and PBDEs increased from North to South and associations between marine sediment contamination, sea temperature, and fish Hg and POPs concentrations were identified using multi-linear regression (MLR) models. In general, Hg concentrations in fillet and liver of fish were positively associated with increases in both sediment contamination and sea temperature. POPs concentrations in both fillet and liver were positively associated with increases in sediment contamination, and only POPs concentrations in the liver of benthopelagic and demersal species were found to be positively correlated with sea temperature. Using a probabilistic approach to estimate human contaminant exposure from seafood, we showed that intake of pelagic species posed the highest risk of dioxins and dioxin-like PCBs (DL-PCBs) exposure, while intake of benthopelagic and demersal species posed the highest risk of Hg exposure. This study can serve as a model to further understand the distribution, co-occurrence, and trends of contaminants in seafood harvested from the NEAO and their potential risks to human consumers. publishedVersion

This review chapter is an overview of the published articles, thesis and report on pollution by microplastics in the Svalbard area. This work was supported by the Norwegian Polar Institute, Tromsø and Svalbard Integrated Arctic Earth Observing System - Knowledge Centre (SIOS-KC). The findings of microplastics in sea ice, snow, water, sediment, and biota samples in Svalbard show that the archipelago is not isolated from pollution generated in other parts of the world and plastic pollution in the region must be monitored

Seafood plays a central role in global food and nutrition security. However, there is a lack of data on the concentration of nutrients and contaminants in fish and other seafood, especially in low- and middle-income countries. In order to assess the potential risks and benefits associated with seafood intake, reliable and up-to-date food composition data is crucial. The quality of food composition data is affected by several factors, such as sampling protocols and the suitability and quality of the methods applied for sample preparation and analysis. In this paper, we describe the sampling methodology and protocols related to the sampling of fish and other seafood and the corresponding analytical methods used to analyse the nutrient and contaminant content of such species. For nutrients, the determination of protein, fat, ash, energy, fatty acids, cholesterol, and amino acids is described, in addition to analyses for determination of the vitamin and mineral content in fish and other seafood. For contaminants, analyses for the determination of organic pollutants and microplastics are described. The methodology described in this paper is used for sampling data through scientific surveys in low- and middle-income countries with research vessel Dr. Fridtjof Nansen under the EAF-Nansen Programme. The Programme aims to improve knowledge on the nutritional composition of fish and ensure the fish is safe to consume.•In this paper, we describe the sampling protocols used for sampling fish and other seafood during scientific surveys under the EAF-Nansen Programme.•This paper describes the methodology and quality control for analysing nutrients and contaminants in fish and other seafood., Graphical abstract Image, graphical abstract

Plastic pollution has become a major environmental concern due to its omnipresence and degradation to smaller particles. The potential toxicological effects of micro- and nanoplastic on biota have been investigated in a growing number of exposure studies. We have performed a comprehensive review of the main determining factors for plastic particle toxicity in the relevant exposure systems, from publications until including the year 2018. For a focused scope, effects of additives or other pollutants accumulated by the plastic particles are not included. In summary, current literature suggests that plastic particle toxicity depends on concentration, particle size, exposure time, particle condition, shape and polymer type. Furthermore, contaminant background, food availability, species, developmental stage and sex have major influence on the outcome of plastic particles exposures. Frequently reported effects were on body and population growth, energy metabolism, feeding, movement activity, physiological stress, oxidative stress, inflammation, the immune system, hormonal regulation, aberrant development, cell death, general toxicity and altered lipid metabolism. Several times reported were increased growth and food consumption, neuro-, liver- or kidney pathology and intestinal damage. Photosynthesis disruption was reported in studies investigating effects on phytoplankton. For the currently unquantified plastic particles below 10 μm, more toxic effects were reported in all aquatic life, as compared to plastic particles of larger size.

Iodine is a trace element required for the production of thyroid hormones, essential for metabolism, growth and brain development, particularly in the first trimester of pregnancy. Milk and lean fish are the main dietary sources of iodine in the Norwegian diet. Thus, the aim of the present study was to provide updated analysed values of iodine concentration in six fish species, 27 selected Norwegian iodine-rich dairy foods and Norwegian hen’s eggs. The iodine concentrations in the wild fish species varied between 18 μg/100 g (Atlantic halibut) and 1,210 μg/100 g (pollack). The iodine concentration of cow milk varied between 12 and 19 μg/100 g and the iodine concentration of the eggs varied between 23 and 43 μg/100 g. The results in this study deviate somewhat from the current iodine concentrations in the Norwegian Food Composition Table. This deviation may have a large impact on the assessment of the iodine intake. Hence, updated knowledge about the variation in iodine level of fish, milk, dairy products and hen’s egg are of great importance when estimating the iodine intake in the population. These data will contribute substantially to future estimations of dietary iodine intake and will be made available for the public Norwegian Food Composition Table.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT.

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT.

Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

The motor protein myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems.

Exocrine, endocrine, and neuroendocrine cells store hormones and neuropeptides in secretory granules (SGs), which undergo regulated exocytosis in response to an appropriate stimulus. These cargo proteins are sorted at the trans-Golgi network into forming immature secretory granules (ISGs). ISGs undergo maturation while they are transported to and within the F-actin-rich cortex. This process includes homotypic fusion of ISGs, acidification of their lumen, processing, and aggregation of cargo proteins as well as removal of excess membrane and missorted cargo. The resulting mature secretory granules (MSGs) are stored in the F-actin-rich cell cortex, perhaps as segregated pools exhibiting specific responses to stimuli for regulated exocytosis. During the last decade our understanding of the maturation of ISGs advanced substantially. The use of biochemical approaches led to the identification of membrane molecules mechanistically involved in this process. Furthermore, live cell imaging in combination with fluorescently tagged marker proteins of SGs provided insights into the dynamics of maturing ISGs, and the functional implications of cytoskeletal elements and motor proteins.

Large dense core vesicles (LDCVs) are storage organelles for regulated secretory proteins such as neuropeptides and hormones. During their formation at the trans-Golgi network regulated secretory proteins are selectively packaged into these organelles by aggregation and the involvement of a variety of sorting signals. The resulting immature LDCVs are transported along microtubules to the cell cortex, in which they move in an F-actin-dependent manner. During this dynamic phase, LDCVs undergo maturation, which includes acidification of the lumen, processing and aggregation of cargo proteins, and removal of proteins not distined for mature LDCVs. Finally, LDCVs undergo exocytosis to release their cargo into the extracellular matrix upon appropriate stimuli.

It emerges that myosin Va plays multiple roles in the trafficking of SGs (secretory granules). In addition to a function in the capture and transport of newly formed SGs in the F-actin-rich cortex, myosin Va is implicated in late transport events of these organelles, which precede their exocytosis. Consistent with these roles, interactions of myosin Va with an array of well-known proteins involved in regulated protein secretion have been documented.

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks. publishedVersion

The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CELLSEGM, the software presented in this work, is a MATLAB based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CELLSEGM has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CELLSEGM. The command-line interface of CELLSEGM facilitates scripting of the separate tools, all implemented in MATLAB, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CELLSEGM enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. publishedVersion