Copper regulation disturbance linked to oxidative stress and cell death during Zika virus infection in human astrocytes.

The Zika virus (ZIKV) caused neurological abnormalities in more than 3500 Brazilian newborns between 2015 and 2020. Data have pointed to oxidative stress in astrocytes as well as to dysregulations in neural cell proliferation and cell cycle as important events accounting for the cell death and neurological complications observed in Congenital Zika Syndrome. Copper imbalance has been shown to induce similar alterations in other pathologies, and disturbances in copper homeostasis have already been described in viral infections. Here, we investigated copper homeostasis imbalance as a factor that could contribute to the cytotoxic effects of ZIKV infection in astrocytes. Human induced pluripotent stem cell-derived astrocytes were infected with ZIKV; changes in the gene expression of copper homeostasis proteins were analyzed. The effect of the administration of CuCl2 or a copper chelator on oxidative stress, cell viability and percentage of infection were also studied. ZIKV infection leads to a downregulation of one of the transporters mediating copper release, ATP7B protein. We also observed the activation of mechanisms that counteract high copper levels, including the synthesis of copper chaperones and the reduction of the copper importer protein CTR1. Finally, we show that chelator-mediated copper sequestration in ZIKV-infected astrocytes reduces the levels of reactive oxygen species and improves cell viability, but does not change the overall percentage of infected cells. In summary, our results show that copper homeostasis imbalance plays a role in the pathology of ZIKV in astrocytes, indicating that it may also be a factor accounting for the developmental abnormalities in the central nervous system following viral infection. Evaluating micronutrient levels and the use of copper chelators in pregnant women susceptible to ZIKV infection may be promising strategies to manage novel cases of congenital ZIKV syndrome.

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

Bovine Lactoferrin Induces Cell Death in Human Prostate Cancer Cells.

Bovine lactoferrin (bLf) is a multifunctional protein widely associated with anticancer activity. Prostate cancer is the second most frequent type of cancer worldwide. This study was aimed at evaluating the influence of bLf on cell viability, cell cycle progression, reactive oxygen species (ROS) production, and rate of apoptosis in the human prostate cancer cell line (DU-145). MTT assay and trypan blue exclusion were used to analyze cell viability. Morphological changes were analyzed through optical microscopy after 24 h and 48 h of bLf treatment. FITC-bLf internalization and cellular damage were observed within 24 h by confocal fluorescence microscopy. Cell cycle analyses were performed by flow cytometry and propidium iodide. For caspases 3/7 activation and reactive oxygen species production evaluation, cells were live-imaged using the high-throughput system Operetta. The cell viability assays demonstrated that bLf induces cell death and morphological changes after 24 h and 48 h of treatment compared to control on DU-145 cells. The bLf internalization was detected in DU-145 cells, G1-phase arrest of the cell cycle, caspase 3/7 activation, and increased oxidative stress on bLf-treated cells. Our data support that bLf has an important anticancer activity, thus offering new perspectives in preventing and treating prostate cancer.

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres.

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.

Microbiome associated with the tetrodotoxin-bearing anuran Brachycephalus pitanga.

The genus Brachycephalus includes small species of aposematic anurans known as microendemic, occurring in the mountains of the Atlantic Forest. Brachycephalus ephippium, B. nodoterga and B. pernix have been reported to contain the neurotoxin tetrodotoxin in skin and viscera. The biological conservation of several Brachycephalus species is currently threatened by climate change, deforestation, and the pandemic caused by the fungus Batrachochytrium dendrobatidis (Bd). Despite the well-known importance of amphibians' associated bacteria in the defensive role against pathogens, there is still a poor understanding of amphibian microbiome composition. The present study investigated the composition of B. pitanga microbial community and the presence of TTX in the host and in cultures of bacterial isolates, using a combination of metagenomics, bacterial culture isolation, mass spectrometry and metabolomic analyses. Results of culture-dependent and -independent analyses characterized the microbial communities associated with the skin and viscera of B. pitanga. Mass spectrometry analysis indicated the presence of TTX in host tissues, while bacterial production of TTX was not observed under the experimental conditions used in this investigation. This is the first report confirming the occurrence of TTX in B. pitanga.

Non-permissive SARS-CoV-2 infection in human neurospheres.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that lead to damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins six, eight, 18 and tumor necrosis factor-alpha (TNF-α) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

Non-permissive SARS-CoV-2 infection in human neurospheres.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

Inhibition of SARS-CoV-2 infection in human iPSC-derived cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoarchitecture and beating.

SARS-CoV-2 infects cardiac cells and causes heart dysfunction. Conditions such as myocarditis and arrhythmia have been reported in COVID-19 patients. The Sigma-1 receptor (S1R) is a ubiquitously expressed chaperone that plays a central role in cardiomyocyte function. S1R has been proposed as a therapeutic target because it may affect SARS-CoV-2 replication; however, the impact of the inhibition of S1R in human cardiomyocytes remains to be described. In this study, we investigated the consequences of S1R inhibition in iPSC-derived human cardiomyocytes (hiPSC-CM). SARS-CoV-2 infection in hiPSC-CM was productive and reduced cell survival. S1R inhibition decreased both the number of infected cells and viral particles after 48 hours. S1R inhibition also prevented the release of pro-inflammatory cytokines and cell death. Although the S1R antagonist NE-100 triggered those protective effects, it compromised cytoskeleton integrity by downregulating the expression of structural-related genes and reducing beating frequency. Our findings suggest that the detrimental effects of S1R inhibition in human cardiomyocytes' integrity may abrogate its therapeutic potential against COVID and should be carefully considered.

The cyanobacterial saxitoxin exacerbates neural cell death and brain malformations induced by Zika virus.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.