Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity.

Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Molecular evidence for gender differences in the migratory behaviour of a small seabird.

Molecular sexing revealed an unexpectedly strong female bias in the sex ratio of pre-breeding European Storm Petrels (Hydrobates pelagicus), attracted to playback of conspecific calls during their northwards migration past SW Europe. This bias was consistent across seven years, ranging from 80.8% to 89.7% female (mean annual sex ratio ± SD = 85.5% female ±4.1%). The sex ratio did not differ significantly from unity (i.e., 50% female) among (i) Storm Petrel chicks at a breeding colony in NW France, (ii) adults found dead on beaches in Southern Portugal, (iii) breeding birds attending nest burrows in the UK, captured by hand, and (iv) adults captured near a breeding colony in the UK using copies of the same sound recordings as used in Southern Europe, indicating that females are not inherently more strongly attracted to playback calls than males. A morphological discriminant function analysis failed to provide a good separation of the sexes, showing the importance of molecular sexing for this species. We found no sex difference in the seasonal or nocturnal timing of migration past Southern Europe, but there was a significant tendency for birds to be caught in sex-specific aggregations. The preponderance of females captured in Southern Europe suggests that the sexes may differ in migration route or in their colony-prospecting behaviour during migration, at sites far away from their natal colonies. Such differences in migration behaviour between males and females are poorly understood but have implications for the vulnerability of seabirds to pollution and environmental change at sea during the non-breeding season.

Assessing Amphotericin B polymeric nanoparticles' toxicity through Zebrafish FET test (OECD Test Guideline 236) / Avaliação da toxicidade de nanopartículas poliméricas seguindo as diretrizes do OECD TG 236 - teste de toxicidade aguda em embrião de peixe

As nanopartículas poliméricas (NPPs) têm atraído a atenção da indústria farmacêutica devido a características como a construção de modelos de liberação controlada, melhoria da biodisponibilidade e redução de efeitos adversos. A utilização do zebrafish (Danio rerio) como modelo animal para estudos toxicológicos tem aumentado nos últimos anos, e o teste de toxicidade aguda em embriões de peixes (FET), tem sido amplamente difundido, devido à rapidez na obtenção de dados e à possibilidade de estimar a DL50 para futuros testes em mamíferos. Objetivo: Investigar a toxicidade de NPPs de policaprolactona (PCL) e poli (ácido lático) (PLA). Metodologia: O teste FET foi realizado seguindo as diretrizes estabelecidas no OECD TG 236. As suspensões de NPPs foram diluídas nas concentrações desejadas com solução de meio embriônico E3. Foram utilizados 20 embriões fertilizados para cada concentração testada e também para o controle negativo. As concentrações testadas foram 4 µg/mL; 8 µg/mL; 16 µg/ml; 32 µg/ml; e 64 µg/ml. Ao final de 120 horas, os animais foram contidos em uma gota de carboximetilcelulose (CMC) a 6%, em lâmina para microscopia, com a finalidade de serem fotografados e filmados por 10 segundos. Foi construída uma escala de pontuação de 0 a 4, onde zero significa nenhuma alteração morfológica aparente; 1 representa uma alteração morfológica não letal; 2 simboliza duas alterações morfológicas não letais; 3 corresponde a mais de duas alterações morfológicas não letais; e 4 significa morte ou presença de alterações incompatíveis com a vida determinadas no guia 236. Adicionalmente, também foram realizadas medidas de tamanho corporal, diâmetro dos olhos e batimentos cardíacos por minuto (bpm). Resultados: Os grupos expostos às NPPs apresentaram óbito e/ou malformações como ausência de somitos, edema pericárdico, atraso no desenvolvimento e ausência de insuflação da bexiga natatória, porém, sem diferir estatisticamente do controle negativo. Nas análises de bpm, tamanho corporal e diâmetro ocular, as larvas também não apresentaram diferenças estatísticas em relação ao controle negativo. Conclusão: Em relação à segurança das NPPs testadas, pode-se concluir que eles se mostraram seguros, pois não apresentaram diferenças estatísticas em relação ao grupo controle em nenhuma das concentrações testadas e em nenhum dos parâmetros avaliados.