A Stereological Approach to Quantify Immunohistochemical Staining in Zebrafish Larvae.

Immunohistochemistry (IHC) is a powerful research tool to localize specific antigens in whole-mount or tissue sections of embryos with labeled antibodies based on antigen-antibody interactions. Stereological methods are nowadays an essential tool to quantify cells or other types of structures in an unbiased and reproducible manner. In this chapter, a general protocol for a stereological estimation of the relative volume density of each structural component (Vv), which can be applied to any organ/structural component, will be described.

Anti-Inflammatory, Anti-Oxidative and Anti-Apoptotic Effects of Thymol and 24-Epibrassinolide in Zebrafish Larvae.

Thymol (THY) and 24-epibrassinolide (24-EPI) are two examples of plant-based products with promising therapeutic effects. In this study, we investigated the anti-inflammatory, antioxidant and anti-apoptotic effects of the THY and 24-EPI. We used zebrafish (Danio rerio) larvae transgenic line (Tg(mpxGFP)i114) to evaluate the recruitment of neutrophils as an inflammatory marker to the site of injury after tail fin amputation. In another experiment, wild-type AB larvae were exposed to a well known pro-inflammatory substance, copper (CuSO4), and then exposed for 4 h to THY, 24-EPI or diclofenac (DIC), a known anti-inflammatory drug. In this model, the antioxidant (levels of reactive oxygen species-ROS) and anti-apoptotic (cell death) effects were evaluated in vivo, as well as biochemical parameters such as the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), the biotransformation activity of glutathione-S-transferase, the levels of glutathione reduced and oxidated, lipid peroxidation, acetylcholinesterase activity, lactate dehydrogenase activity, and levels of nitric acid (NO). Both compounds decreased the recruitment of neutrophils in Tg(mpxGFP)i114, as well as showed in vivo antioxidant effects by reducing ROS production and anti-apoptotic effects in addition to a decrease in NO compared to CuSO4. The observed data substantiate the potential of the natural compounds THY and 24-EPI as anti-inflammatory and antioxidant agents in this species. These results support the need for further research to understand the molecular pathways involved, particularly their effect on NO.

Protective effects of 24-epibrassinolide against the 6-OHDA zebrafish model of Parkinson's disease.

The molecular processes behind Parkinson's disease (PD) remain under debate although mitochondrial oxidative stress generation has been proposed as a fundamental contributor. In this context, different brassinosteroids have shown neuroprotective action hampering oxidative stress. This study determined the effects of 24-Epibrassinolide (24-EPI) against 6-hydroxydopamine- (6-OHDA-) induced toxicity using the zebrafish embryonic model. Embryos were exposed to 250 µM 6-OHDA or co-exposed to 24-EPI (0.01, 0.1, and 1 µM) for 3 days, starting at 48 h post-fertilization (hpf). During the experimental period, developmental parameters were assessed. At 120 hpf, larvae were tested for behavioural phenotypes with different biochemical biomarkers and tyrosine hydroxylase- (TH-) reactive neurons being also assessed. Exposure to 6-OHDA induced a decrease in body length while no other morphological phenotypes were noticed. A significant decrease in TH-neurons immunofluorescence, a decreased locomotion (speed and distance moved), and an increased absolute angle were found in 6-OHDA-exposed embryos. These outcomes were rescuable by the co-exposure with 24-EPI. Surprisingly, the direct effects of 6-OHDA on reactive oxygen species (ROS) were not observed in the present study supporting the involvement of other molecular pathways in the 6-OHDA-induced effects during embryonic development. Overall, the results obtained confirm PD-like symptoms induced by 6-OHDA during embryonic development which were reverted by 24-EPI. Although antioxidative signalling pathways deserve further scrutiny, the findings support the further investigation of 24-EPI neuroprotective effects.

Mitigation of nicotine-induced developmental effects by 24-epibrassinolide in zebrafish.

Nicotine is a highly addictive substance that can cause teratogenic impacts in the embryo through redox-dependent pathways. As antioxidants, naturally occurring chemicals can protect cells from redox imbalance. The purpose of this study was to evaluate the effectiveness of 24-epibrassinolide (24-EPI), a natural brassinosteroid with well-known antioxidant properties, in protecting zebrafish embryos against nicotine's teratogenic effects. For 96 h, embryos (2 h post-fertilization - hpf) were exposed to 100 µM nicotine, co-exposed with 24-EPI (0.01, 0.1, and 1 µM), and 24-EPI alone (1 µM). Lethal and sublethal developmental characteristics were evaluated during exposure. Biochemical tests were performed at the conclusion of the exposure, and distinct behavioural paradigms were analysed 24 h later. Nicotine exposure resulted in a higher proportion of larvae with deformities, which were decreased following co-exposure to 24-EPI. Nicotine exposure also caused an increase in oxidative stress as observed by the increased activity of superoxide dismutase and catalase accompanied by an increase in the malondialdehyde levels. Besides, metabolic changes were noticed as observed by the increased lactate dehydrogenase activity that were hypothesised to be associated to nicotine-induced hypoxia which may be responsible for the increased oxidative damage. In addition, locomotor deficits were observed as well as a decrease in the acetylcholinesterase activity denoting nicotine-induced cognitive dysfunction. However, co-exposure to 24-EPI alleviated behavioural deficits and improved nicotine-induced emotional states. Overall, and although further studies are required to clarify these effects, 24-EPI showed promising ameliorative properties against the teratogenic effects induced by nicotine.

Evaluation of the developmental effects of a glyphosate-based herbicide complexed with copper, zinc, and manganese metals in zebrafish.

The use of glyphosate-based herbicides (GBH) has increased dramatically, being currently the most used herbicides worldwide. Glyphosate acts as a chelating agent, capable of chelate metals. The synergistic effects of metals and agrochemicals may pose an environmental problem as they have been shown to induce neurological abnormalities and behavioural changes in aquatic species. However, as their ecotoxicity effects are poorly understood, evaluating the impacts of GBH complexed with metals is an ecological priority. The main objective of the study was to evaluate the potentially toxic effects caused by exposure to a GBH (1 µg a.i. mL-1), alone or complexed with metals (Copper, Manganese, and Zinc (100 µg L-1)), at environmentally relevant concentrations, during the early period of zebrafish (Danio rerio) embryo development (96 h post-fertilization), a promising model for in vivo developmental studies. To clarify the mechanisms of toxicity involved, lethal and sublethal development endpoints were assessed. At the end of the exposure, biochemical and cell death parameters were evaluated and, 24 h later, different behavioural responses were assessed. The results showed that metals induced higher levels of toxicity. Copper caused high mortality, low hatching, malformations, and changes in biochemical parameters, such as decreased Catalase (CAT) activity, increased Glutathione Peroxidase (GPx), Glutathione S-Transferase (GST), reduced Glutathione (GSH) and decreased Acetylcholinesterase (AChE) activity, also inducing apoptosis and changes in larval behaviour. Manganese increased the activity of SODs enzymes. Zinc increased mortality, reactive oxygen species (ROS) levels, superoxide dismutase activity (SODs) and caused a decrease in AChE activity. Embryos/larvae exposed to the combination of GBH/Metal also showed teratogenic effects during their development but in smaller proportions than the metal alone. Although more studies are needed, the results suggest that GBH may interfere with the mechanisms of metal toxicity at the biochemical, physiological, and behavioural levels of zebrafish.

Microplastics and copper induce apoptosis, alter neurocircuits, and cause behavioral changes in zebrafish (Danio rerio) brain.

The knowledge regarding the neurological and behavioral toxic effects associated with microplastics (MPs) and heavy metals exposure is still scarce. The present study aimed to evaluate the potential chronic (30 days) toxic effects of MPs (2 mg/L) and copper (Cu, 25 µg/L), alone or combined, in the zebrafish (Danio rerio) brain antioxidant system, cell proliferation/death, cholinergic-, serotonergic- and dopaminergic pathways and, consequently, in locomotor, anxiety, and social behaviors. Our findings showed that MPs and Cu exposure modulated the antioxidant system of zebrafish brain, with superoxide dismutase (SOD) and glutathione reductase (GR) having higher activity in the Cu25 +MPs group, but glutathione peroxidase (GPx) being inhibited in MPs, Cu25 and Cu25 +MPs. Moreover, an increase in acetylcholinesterase (AChE) activity was observed in all exposed groups. When considering neurogenesis genes, a downregulation of proliferating cell nuclear antigen (pcna) was noticed in zebrafish exposed to the mixture treatment, while for dopaminergic system-related genes (th and slc6a3) an upregulation was observed in MPs, Cu25 and Cu25 +MPs groups. An increase in apoptosis-related genes expression (casp8, casp9 and casp3) was observed in the MPs exposed group. Changes in zebrafish behavior, particularly in mean speed, total distance moved, inactivity in the aquaria, and social/shoaling behavior was also observed in the MPs and Cu exposed groups. Overall, our results highlight the multiplicity of toxic effects of MPs, alone or combined with Cu, in zebrafish brain, namely apoptosis and alterations in adult neurogenesis, neurocircuits and, consequently, behavior.

Histological alterations and oxidative stress in adult zebrafish muscle after aluminium exposure.

Aluminium (Al) is among the most abundant metals in nature, and its presence in the environment is further increasing by anthropogenic activities. In water bodies, the Al concentrations ranged between 0.001 and 50 mg/L, raising concerns about the health of aquatic organisms. For this reason, zebrafish was chosen as the model, since it is well suited for ecotoxicological studies. Adult specimens were exposed to 11 mg/L of Al for 10, 15 and 20 days to assess both the morphology and the oxidative state of muscle tissue. Considering the involvement of ROS, the activity of the main antioxidant enzymes, metallothioneins contents, but also oxidative damage and enzymes involved in energy consumption and neuromuscular transmission were assessed. Collected data showed an increase in the thickness of the endomysium and resorbed myofibrils in the organisms exposed to Al for 10 days, and an increase of myotomes' size in the organisms exposed to Al for 15 days. Moreover, the organisms exposed for less time to Al, it was evident an activation of anaerobic metabolism and the increased activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and glutathione S-transferases. However, these effects stabilized with increasing exposure time. In addition, only after 20 days of treatment did the oxidative damage to the proteins and the activity of acetylcholinesterase increase while the levels of metallothioneins and the lipid peroxidation were lower for all treated animals when compared to the control group. Overall, the biochemical and histological changes induced by aluminium exposure in the muscular tissue represent a relevant contribution to understanding the environmental risk due to the diffusion of this metal within the aquatic compartment.

Anesthesia Overdose Versus Rapid Cooling for Euthanasia of Adult Zebrafish.

The rapid increase in zebrafish use needs to be accompanied by research into the refinement of procedures. The European (EU) Directive lists three possible euthanasia methods for fish: anesthetic overdose, electrical stunning, and concussion. However, for small fish such as zebrafish, concussion and electrical stunning are difficult to perform, leaving anesthetic overdose as the most used method. Our aim was to test the efficacy and side effects of anesthesia overdose using different anesthetics and the rapid cooling method to euthanize adult zebrafish. Adult mixed-sex AB zebrafish were randomly assigned to: 250 mg/L MS222; 20 mg/L propofol +100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; and rapid cooling (water at 2°C-4°C). Two minutes after opercular movement ceased, animals were transferred into clean water for 20 min and recovery assessed, or decapitated and used for biochemical analysis of the gills, muscle, liver, and brain; for the histological analysis of the gills and muscle; or for the assessment of cortisol levels. No animal recovered; rapid cooling was the quickest and etomidate overdose was the slowest method to cease the opercular movements. There were no major differences between euthanasia methods regarding the biochemical or histological data. Cortisol levels were higher in the rapid cooling group, but only when compared with the propofol/lidocaine group. The use of a physical method of euthanasia, such as rapid cooling, is essential when chemicals, such as anesthetics, may interfere with postmortem analyses. Although anesthetic overdose can be used without major effects on the analyses conducted in this work, rapid cooling can be another option with the advantage of being simple to administer, easily available, affordable, and very quick; this decreases the potential duration of suffering, being more humane. Therefore, a change in EU legislation should be considered to include additional humane options for euthanasia, such as rapid cooling, for zebrafish and other small tropical fish.

Microplastics- and copper-induced changes in neurogenesis and DNA methyltransferases in the early life stages of zebrafish.

In this study, zebrafish embryos were exposed to microplastics (MPs, 2 mg/L) and copper (Cu, 60 and 125 µg/L), alone or combined, for 14 days, and the development of motor neurons was assessed through gene expression and immunohistochemistry. DNA methyltransferases (DNMTs) genes expression was also evaluated. The results showed a downregulation of neuronal proliferation (sox2, pcna), neurogenesis (neuroD, olig2), and motor neurons development (islet) related genes, implying potential deficits in the neurogenesis of the exposed zebrafish early life stages. Downregulation of the maintenance and de novo DNMTs expression was also found, indicating that the DNA methylation patterns could be modulated by MPs and Cu. A high relative volume of proliferating cell nuclear antigen (PCNA)-positive cells was found in the fish retina from the MPs exposed group, suggesting that MPs increased the rate of cellular division. In contrast, a significant decrease of PCNA-positive cells, and therefore a lower cell proliferation, was found in the retina and brain of zebrafish exposed to Cu and Cu + MPs, which could lead to cognitive and behavioral functions impairment. No alterations were found in the relative volume of ISL1&2-positive cells. This study contributes to the knowledge of the mechanisms by which MPs and Cu cause neurotoxicity, fundamental for a comprehensive and realistic ecological risk assessment in aquatic populations.

Oxidative stress, apoptosis and serotonergic system changes in zebrafish (Danio rerio) gills after long-term exposure to microplastics and copper.

Fish gills are in direct contact with the surrounding pollutants, and thus, potentially more vulnerable to microplastics (MPs) and heavy metals. The present study aimed to evaluate the long-term exposure effects of MPs and copper (Cu) in the gills of adult zebrafish (Danio rerio). To this end, zebrafish were exposed to MPs (2 mg/L), Cu (Cu25, 25 µg/L) and their mixture (Cu25 + MPs) for 30 days, and then oxidative stress, detoxification, antioxidant, metabolic and neurotoxicity enzymes/genes, as well serotonergic system and apoptosis genes, were evaluated in gills. In the mixture group, ROS levels were increased, while CAT and GPx activities were inhibited, indicating the induction of oxidative stress in zebrafish gills. This was followed by an increase of LPO levels and potential oxidative damage in zebrafish gills. The tryptophan hydroxylase 1a (tph1a) and caspase-3 (casp3) genes were significantly upregulated in Cu25 + MPs group, indicating a potential dysregulation of serotonin synthesis and apoptosis pathways, respectively. Overall, the present study contributes to improving the knowledge about the response of aquatic organisms to MPs and the potential ecological risk that these particles represent to the ecosystems.

Toxicity of microplastics and copper, alone or combined, in blackspot seabream (Pagellus bogaraveo) larvae.

Plastics pose serious risks for fish productivity and a potential constraint for food security. Newly hatched blackspot seabream larvae were exposed to microplastics (MPs), copper (Cu, 10-810 µg/L) and their mixtures (Cu+MPs), during 3 and 9 days. Biochemical biomarkers and the expression of antioxidant and neurotoxicity-related genes were evaluated. In the 3-day exposure, catalase and glutathione-S-transferase activities decreased in MPs, Cu and Cu+MPs groups, followed by an increase of lipid peroxidation in the Cu270 and Cu270 +MPs exposed larvae. In the 9-day exposure, ROS levels increased in MPs and Cu30 groups, but no significant oxidative damage was observed, suggesting that the antioxidant system overcome the induced oxidative stress. However, the acetylcholinesterase transcript was downregulated in MPs, Cu and Cu10+MPs groups, indicating that MPs effects in cholinergic neurotransmission may arise after longer exposures. Overall, MPs and Cu can reduce survival, induce oxidative stress, lipid peroxidation, neurotoxicity, and impact negatively fish larvae fitness.

24-Epibrassinolide modulates the neurodevelopmental outcomes of high caffeine exposure in zebrafish (Danio rerio) embryos.

Previous embryonic fish data have shown caffeine to induce potential teratogenic and long-term neurodevelopmental outcomes through oxidative stress-mediated apoptosis. In this context, antioxidants may have the potential to counteract the caffeine-induced effects. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proven antioxidant properties, against caffeine-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 0.5 mM caffeine, co-exposed to 24-EPI (0.01, 0.1 and 1 µM) and to 24-EPI alone (1 µM) for 96 h. During exposure, lethal and sublethal developmental parameters were evaluated. At the end of the exposure, biochemical evaluations were made, and 24 h after, different behavioural paradigms were assessed. An increased number of animals showing oedema and malformations were observed after caffeine exposure, while these were reduced after co-exposure to 24-EPI concentration, namely the tail curvature. The results showed oxidative stress and related parameters similar among treatments. Yet, caffeine exposure resulted in locomotor deficits (decreased speed and distance) and disrupted anxiety-like and avoidance responses. The co-exposure to caffeine and to the highest 24-EPI concentrations resulted in less pronounced behavioural deficits. Overall, there was an absence of effects in the embryo/larvae exposed solely to 24-EPI, while caffeine caused developmental and neurotoxic effects. Although further studies are needed, the results showed promising protective effects of the highest 24-EPI concentration tested against the toxicity induced by caffeine in zebrafish.

Apoptosis, oxidative stress and genotoxicity in developing zebrafish after aluminium exposure.

Aluminium is a non-essential metal and potentially toxic to organisms whose environmental concentration increases due to pollution. In our previous studies, the behavioral changes induced by aluminium were already shown on zebrafish, a model organism widely used for ecotoxicology screening. To examine in depth the knowledge about the toxicity mechanism induced by this metal, zebrafish embryos, at 6 hpf, have been exposed to 50, 100 and 200 µM of AlCl3 for 72 h. Phenotypic alterations, apoptosis and oxidative stress responses have been assessed by evaluations of antioxidant defence and changes in metabolism at the end of treatment. The mRNA expression level of c-fos, appa and appb as marker genes of neural development and function were analyzed by qPCR for the highest used concentration. The data showed that aluminium significantly affected the development of zebrafish inducing morphological alterations and cell death. The oxidative state of larvae was altered, although the formation of reactive oxygen species and the levels of metallothioneins, and the activity of some antioxidant enzymes, decreased at the maximum concentration tested. In addition, at this concentration, the expression of the evaluated genes increased. The comprehensive information obtained gives a realistic snapshot of the aluminium toxicity and provides new information on the mechanism of action of this metal.

Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure.

Microplastics (MPs, <5 mm) have been frequently detected in aquatic ecosystems, representing both health and ecological concerns. However data about the combined effects of MPs and other contaminants is still limited. This study aimed to evaluate the impact of MPs and the heavy metal copper (Cu) on zebrafish (Danio rerio) larvae development and behavior. Zebrafish embryos were subchronically exposed to MPs (2 mg/L), two sub-lethal concentrations of Cu (60 and 125 µg/L) and binary mixtures of MPs and Cu using the same concentrations, from 2-h post fertilization until 14 days post fertilization. Lethal and sub-lethal responses (mortality, hatching, body length) were evaluated during the embryogenesis period, and locomotor, avoidance, anxiety and shoaling behaviors, and acetylcholinesterase (AChE) activity were measured at 14 dpf. The results showed that survival of larvae was reduced in groups exposed to MPs, Cu and Cu+MPs. Regarding the behavioral patterns, the higher Cu concentration and mixtures decreased significantly the mean speed, the total distance traveled and the absolute turn angle, demonstrating an adverse effect on swimming competence of zebrafish larvae. Exposure to MPs and Cu, alone or combined, also affected avoidance behavior of zebrafish, with larvae not reacting to the aversive stimulus. There was a significant inhibition of AChE activity in larvae exposed to all experimental groups, compared to the control group. Moreover, a higher inhibition of AChE was noticed in larvae exposed to MPs and both Cu+MPs groups, comparatively to the Cu alone groups. Our findings demonstrate the adverse effects of MPs, alone or co-exposed with Cu, on fish early life stages behavior. This study highlights that MPs and heavy metals may have significant impacts on fish population fitness by disrupting locomotor and avoidance behaviors.

Development of antibodies against the notch ligand Delta-Like-1 by phage display with activity against breast cancer cells.

Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.

Zebrafish male differentiation: Do all testes go through a "juvenile ovary" stage?

Zebrafish (Danio rerio) studies describe before the onset of mature gonads differentiation all individuals go through a "juvenile ovary" stage. However, the sequential events of the early zebrafish gonad differentiation are still not described in full detail and recent works indicate that some individuals never form a "juvenile ovary" structure. Therefore, the present study aimed to confirm the existence of two processes of zebrafish male differentiation. For this purpose, every two days between 20 and 30 days post-fertilization (dpf) zebrafish were collected for a stereological analysis of the differentiating gonads. The histological evaluation showed that prior to 22 dpf, zebrafish gonads were still undifferentiated. At 24 dpf, some individuals started to present a "juvenile ovary" and from 26 to 30 dpf, it was possible to discern two processes of gonad development. The majority of the individuals (80 %) developed a "juvenile ovary", while in the remaining (20 %) it was not possible to detect this structure. The results of the present study show the existence of two distinct processes of zebrafish male gonad development, indicating that not all individuals go through the "juvenile ovary" stage.

Exposure to aluminium causes behavioural alterations and oxidative stress in the brain of adult zebrafish.

Aluminium (Al) water pollution is an increasing environmental problem. Accordingly, this study aimed to find out more about its toxic effects on aquatic organisms. Adult zebrafish were exposed to 11 mg/L of Al and the behavioural responses and its correlation with brain oxidative stress, antioxidant-defences, changes in metabolism and neurotransmission were assessed at 10, 15 and 20 days of exposure. The behavioural and locomotory responses, suggest an increase in the anxiety state, especially observed in animals exposed to Al for 15 days. The reactive oxygen species increased in a time-dependent trend, while the oxidative damage varied over exposure time. The activity of antioxidant enzymes, as superoxide dismutase, glutathione peroxidase and glutathione S-transferases, and the metallothioneins levels increased after short-term exposures and tended to decrease or stabilize at longer times. The results contribute to understand the toxic mechanisms activated by Al highlighting correlations like behavioural disorders and oxidative state.

Single and combined acute and subchronic toxic effects of microplastics and copper in zebrafish (Danio rerio) early life stages.

The evaluation of the interaction between microplastics (MPs) and heavy metals is of special importance for risk assessment. In this study, zebrafish (Danio rerio) were exposed to MPs (2 mg/L), two sub-lethal concentrations of copper (Cu, 60 and 125 µg/L) and their mixtures (Cu60 + MPs, Cu125 + MPs), from 2-h post-fertilization (hpf) until 14-days post-fertilization (dpf). Lethal and sublethal endpoints were evaluated, along with a set of biochemical and genetic biomarkers between 2 and 14 dpf. Exposure to MPs and Cu, single or combined, induced high mortality and oxidative stress in zebrafish larvae, with data showing that the antioxidant enzymes were inhibited at 6 dpf, increasing thereafter until 14 dpf, due to the accumulation of reactive oxygen species. MPs and Cu, single or combined, caused neurotoxicity in larvae by inhibiting acetylcholinesterase activity. There was an increased and significant effect of Cu + MPs groups on the evaluated biomarkers, concerning the corresponding Cu groups, suggesting that MPs may have a synergistic effect in relation to Cu. The Integrated Biomarker Response (IBR) evidenced that a higher degree of stress occurred at the larval period. Our findings highlight that MPs can act as a vector for heavy metals, therefore, influencing their bioavailability and toxicity in the organisms.

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

Toxicological effects induced on early life stages of zebrafish (Danio rerio) after an acute exposure to microplastics alone or co-exposed with copper.

Data about the toxicological interactions of MPs and heavy metals in biota is limited, particularly in fish early life stages. This study aimed to evaluate the toxicological effects of MPs and copper (Cu), alone or combined, in zebrafish early life stages. Embryos were exposed from 2 until 96-h post-fertilization (hpf) to MPs (2 mg/L), three sub-lethal concentrations of Cu (15, 60 and 125 µg/L) and binary mixtures containing Cu and MPs (Cu15+MPs, Cu60+MPs, Cu125+MPs). Lethal and sub-lethal parameters, histopathological changes, biochemical biomarkers, gene expression and behavior were assessed. Our findings showed that Cu and Cu + MPs decreased embryos survival and hatching rate. Increased ROS levels were observed in larvae exposed to the two lowest Cu and Cu + MPs groups, suggesting an induction of oxidative stress. An increased CAT and GPx activities were observed in Cu and Cu + MPs, implying a response of the antioxidant defense system to overcome the metal and MPs stress. The sod1 expression was downregulated in all Cu groups and in the two highest Cu + MPs exposed groups. AChE was significantly inhibited in Cu and Cu + MPs groups, indicating neurotoxicity. A disruption of avoidance and social behaviors were also noticed in the Cu125 and Cu125+MPs exposed larvae. Evidences of Cu-toxicity modulation by MPs were observed in some endpoints. Overall, the findings of this study highlight that Cu alone or co-exposed with MPs lead to oxidative stress, neurotoxicity and ultimately behavioral alterations in early life stages of zebrafish, while MPs alone do not produce significant effects on zebrafish larvae.