Total Osteopontin and Its Isoform OPN4 Are Differently Expressed in Respiratory Samples during Influenza A(H1N1)pdm09 Infection and Progression.

Influenza A virus (IAV) infection affects the human respiratory tract, causing an acute and highly contagious disease. Individuals with comorbidities and in the extremes of age are classified as risk groups for serious clinical outcomes. However, part of the severe infections and fatalities are observed among young healthy individuals. Noteworthy, influenza infections lack specific prognostic biomarkers that would predict the disease severity. Osteopontin (OPN) has been proposed as a biomarker in a few human malignancies and its differential modulation has been observed during viral infections. However, OPN expression levels in the primary site of IAV infection have not been previously investigated. Therefore, we evaluated the transcriptional expression patterns of total OPN (tOPN) and its splicing isoforms (OPNa, OPNb, OPNc, OPN4, and OPN5) in 176 respiratory secretion samples collected from human influenza A(H1N1)pdm09 cases and a group of 65 IAV-negative controls. IAV samples were differentially classified according to their disease severity. tOPN was more frequently detected in IAV samples (34.1%) when compared with the negative controls (18.5%) (p < 0.05), as well as in fatal (59.1%) versus non-fatal IAV samples (30.5%) (p < 0.01). OPN4 splice variant transcript was more prevalent in IAV cases (78.4%) than in the negative controls (66.1%) (p = 0.05) and in severe cases (85.7%) in relation to the non-severe ones (69.2%) (p < 0.01). OPN4 detection was also associated with severity symptoms such as dyspnea (p < 0.05), respiratory failure (p < 0.05), and oxygen saturation < 95% (p < 0.05). In addition, the OPN4 expression level was increased in the fatal cases of respiratory samples. Our data indicated that tOPN and OPN4 had a more pronounced expression pattern in IAV respiratory samples, pointing to the potential use of these molecules as biomarkers to evaluate disease outcomes.

Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling.

Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood-brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Low prevalence of influenza A strains with resistance markers in Brazil during 2017-2019 seasons.

The influenza A virus (IAV) is of a major public health concern as it causes annual epidemics and has the potential to cause pandemics. At present, the neuraminidase inhibitors (NAIs) are the most widely used anti-influenza drugs, but, more recently, the drug baloxavir marboxil (BXM), a polymerase inhibitor, has also been licensed in some countries. Mutations in the viral genes that encode the antiviral targets can lead to treatment resistance. Worldwide, a low prevalence of antiviral resistant strains has been reported. Despite that, this situation can change rapidly, and resistant strain surveillance is a priority. Thus, the aim of this was to evaluate Brazilian IAVs antiviral resistance from 2017 to 2019 through the identification of viral mutations associated with reduced inhibition of the drugs and by testing the susceptibility of IAV isolates to oseltamivir (OST), the most widely used NAI drug in the country. Initially, we analyzed 282 influenza A(H1N1)pdm09 and 455 A(H3N2) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S247N was detected in one A(H1N1)pdm09 strain. We also identified NA:I222V (n = 6) and NA:N329K (n = 1) in A(H3N2) strains. In addition, we performed a molecular screening for NA:H275Y in 437 A(H1N1)pdm09 samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC50 values for 222 A(H1N1)pdm09 and 83 A(H3N2) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H3N2) virus presenting with PA:E119D AAS. Moreover, the majority of the IAV sequences had the M2:S31N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis.

Identification of Hypericin as a Candidate Repurposed Therapeutic Agent for COVID-19 and Its Potential Anti-SARS-CoV-2 Activity.

The COVID-19 pandemic has had an unprecedented impact on the global economy and public health. Its etiologic agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible, pathogenic and has a rapid global spread. Currently, the increase in the number of new confirmed cases has been slowed down due to the increase of vaccination in some regions of the world. Still, the rise of new variants has influenced the detection of additional waves of rising cases that some countries have experienced. Since the virus replication cycle is composed of many distinct stages, some viral proteins related to them, as the main-protease (Mpro) and RNA dependent RNA polymerase (RdRp), constitute individual potential antiviral targets. In this study, we challenged the mentioned enzymes against compounds pre-approved by health regulatory agencies in a virtual screening and later in Molecular Mechanics/Poisson-Bolzmann Surface Area (MM/PBSA) analysis. Our results showed that, among the identified potential drugs with anti-SARS-CoV-2 properties, Hypericin, an important component of the Hypericum perforatum that presents antiviral and antitumoral properties, binds with high affinity to viral Mpro and RdRp. Furthermore, we evaluated the activity of Hypericin anti-SARS-CoV-2 replication in an in vitro model of Vero-E6 infected cells. Therefore, we show that Hypericin inhibited viral replication in a dose dependent manner. Moreover, the cytotoxicity of the compound, in cultured cells, was evaluated, but no significant activity was found. Thus, the results observed in this study indicate that Hypericin is an excellent candidate for repurposing for the treatment of COVID-19, with possible inhibition of two important phases of virus maturation.

Susceptibility of lung cancer patients to COVID-19: A review of the pandemic data from multiple nationalities.

Several studies have highlighted that cancer patients tend to be more susceptible to develop severe infection and to die from COVID-19. Certain medical conditions such as immunosuppression, presence of comorbidities, and underlying pulmonary damage are possible determinants of disease severity, especially in lung cancer patients. While recent studies have shown that lung cancer is one of the most prevalent tumor types among COVID-19 cancer patients, we still have an incomplete view of how data from several countries work as a whole. The aim of this review was to investigate COVID-19 prevalence in lung cancer patient cohorts and their probability to develop severe illness and death when compared to nonlung cancer patients from multiple nationalities, including countries that have been the epicenters of the pandemic. We also focus on some intrinsic lung cancer features that might influence COVID-19 outcomes. An integrative view of the susceptibility of lung cancer patients might be especially relevant to assist physicians in evaluating the risks of COVID-19 in these patients, and to foster better decisions on treatment delay.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells.

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Wastewater-Based Epidemiology (WBE) and Viral Detection in Polluted Surface Water: A Valuable Tool for COVID-19 Surveillance: A Brief Review

SARS-COV-2 is the causative agent of the current COVID-19 pandemic. Disease clinical manifestations range from asymptomatic to severe multiple organ damage. SARS-CoV-2 uses ACE2 as a cellular receptor, which is abundantly expressed in the small intestine, allowing viral replication in the gastrointestinal tract. Viral RNA has been detected in the stool of COVID-19 patients and viable viruses had been isolated in some of these samples. Thus, a putative role of SARS-CoV-2 fecal-oral transmission has been argued. SARS-CoV-2 is shed in human excreta and further disposed in the sewerage or in the environment, in poor basic sanitation settings. Wastewater-based epidemiology (WBE) is a valuable population level approach for monitoring viral pathogens and has been successfully used in di_erent contexts. This review summarizes the current global experience on SARS-CoV-2 WBE in distinct continents and viral detection in polluted surface water. The advantages and concerns of this strategy for SARS-CoV-2 surveillance are discussed. Outcomes suggest that WBE is a valuable early warning alert and a helpful complementary surveillance tool to subside public health response, to tailor containment and mitigation measures and to determine target populations for testing. In poor sanitation settings, contaminated rivers could be alternatively used as a source for environmental surveillance.

Identification of SARS-CoV-2 and additional respiratory pathogens cases under the investigation of COVID-19 initial phase in a Brazilian reference laboratory.

Coronavirus disease 2019 (COVID-19) surveillance, in Brazil, initiated shortly after its description, in China. Our aim was to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional pathogens in samples from the initial phase of the outbreak in Brazil, from late February to late March. From 707 samples analysed, 29 (4.1%) were SARS-CoV-2 positive. Fever and cough were their most prevalent symptoms. Co-detection of rhinovirus was observed in 2 (6.9%) cases. Additional pathogens were identified in 66.1% of the SARS-CoV-2 negative cases, mainly rhinovirus and influenza A(H1N1)pdm09. Thus, we emphasise the importance of differential diagnosis in COVID-19 suspected cases.

Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV in vitro and in vivo. However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.

The long non-coding RNA PCA3: an update of its functions and clinical applications as a biomarker in prostate cancer.

Prostate cancer antigen 3 (PCA3) is an overexpressed prostate long non-coding RNA (lncRNA), transcribed from an intronic region at the long arm of human chromosome 9q21-22. It has been described that PCA3 modulates prostate cancer (PCa) cell survival through modulating androgen receptor (AR) signaling, besides controlling the expression of several androgen responsive and cancer-related genes, including epithelial-mesenchymal transition (EMT) markers and those regulating gene expression and cell signaling. Also, PCA3 urine levels have been successfully used as a PCa diagnostic biomarker. In this review, we have highlighted recent findings regarding PCA3, addressing its gene structure, putative applications as a biomarker, a proposed origin of this lncRNA, roles in PCa biology and expression patterns. We also updated data regarding PCA3 interactions with cancer-related miRNAs and expression in other tissues and diseases beyond the prostate. Altogether, literature data indicate aberrant expression and dysregulated activity of PCA3, suggesting PCA3 as a promising relevant target that should be even further evaluated on its applicability for PCa detection and management.

Caracterização da expressão e função da trombospondina 2 no câncer de próstata / [Characterization of the expression and function of thrombospondin 2 in cancer of prostate]

A trombospondina 2 (TSP2) é uma glicoproteína secretada na matriz extracelular envolvida com inibição de crescimento e angiogênese tumorais. Sua expressão em tumores é bastante controversa, dependendo do tipo tumoral. A caracterização do nível de expressão e da função de TSP2 ainda não foi realizada no câncer de próstata (CaP). Os objetivos deste trabalho são caracterizar o perfil de expressão de TSP2 no CaP e sua potencial aplicação no diagnóstico diferencial entre CaP e hiperplasia prostática benigna (HPB), além de investigar sua função no CaP. Para tanto, o nível de expressão do transcrito TSP2 foi analisado por PCR quantitativo em tempo real, em amostras teciduais de CaP e HPB e em linhagens prostáticas tumorais e não tumorais. A expressão da proteína TSP2 foi avaliada por imunohistoquímica em microarranjo de tecidos contendo amostras de CaP e HPB e utilizando um sistema semi-automático de análise de imagem celular. Linhagens celulares de CaP (PC3 e DU145) foram tratadas com a proteína recombinante de TSP2 (recTSP2) (50 µg/mL), sendo realizadas análises de viabilidade e de ciclo celular, além de análise fosfo-proteômica na linhagem PC3 tratada com recTSP2, através de um arranjo contendo anticorpos para 46 proteínas fosforiladas. Observamos que o transcrito TSP2 apresentou menores níveis de expressão em amostras teciduais e linhagens de CaP, em comparação com amostras teciduais de HPB e linhagem não tumoral prostática (p<0,01). Em análise de curva ROC (Receiving Operating Curve), o transcrito TSP2 mostrou-se mais eficiente na distinção entre CaP e HPB (p<0,01) do que os níveis séricos de PSA (p=0,299). A expressão da proteína TSP2 foi observada no citoplasma das células dos compartimentos estromais e epiteliais de CaP e HPB. Notadamente, o escore da marcação estromal de TSP2 nos tecidos de CaP apresentou-se significativamente menor do que em HPB (p<0,01), enquanto o padrão de marcação epitelial mostrou- se similar entre as duas doenças. O tratamento das linhagens PC3 e DU145 com recTSP2 diminuiu a viabilidade destas células, além de aumentar o percentual de células na fase sub-G1 do ciclo celular. A análise fosfo-proteômica das células PC3 tratadas mostrou que resíduos das proteínas ERK1/2 e JNK apresentam menor nível de fosforilação, enquanto que resíduos das proteínas Akt, p70S6, p53, paxilina, RSK1/2/3 e p27 possuíam maior nível de fosforilação. Em conjunto, estes dados demonstram que TSP2 apresenta menor expressão no CaP, especialmente no compartimento estromal. Os níveis alterados de TSP2 apresentam potencial aplicação no diagnóstico diferencial de CaP e HPB. Adicionalmente, os resultados gerados por este estudo fazem a primeira descrição da potencial função de TSP2 no CaP, reforçando a possibilidade de TSP2 ser um inibidor tumoral em células de CaP, o que já foi demonstrado para outros tipos tumorais (AU)

Thrombospondin 2 (TSP2) is a glycoprotein that is secreted in the extracellular matrix and is related to tumor growth and angiogenesis inhibition. However, its expression in tumors is quite controversial, depending on the tumor type. Despite the characterization of TSP2 expression level and function has been shown for some tumors types, a detailed characterization in prostate cancer (PCa) has not been performed. This study aims to characterize TSP2 expression profile in PCa and its potential application in the differential diagnosis between PCa and benign prostatic hyperplasia (BPH), and to characterize TSP2 possible functions in PCa. TSP2 transcript expression levels were analyzed by real-time quantitative PCR in PCa and BPH tissue samples and tumor and non tumor prostate cell lines. Protein expression was evaluated in tissue microarray assay containing PCa and BPH tissue samples, by immunohistochemistry with an anti-TSP2 antibody, and using a semi- automated image analysis system. To investigate the functional role of TSP2 in PCa, PCa cell lines (DU145 and PC3) were treated with TSP2 recombinant protein (recTSP2) (50 µg/mL). Cell viability and cell cycle analysis were performed. There was also performed phospho-proteomic analysis in PC3 cells treated with recTSP2 through an array containing antibodies to 46 phosphorylated proteins. Our results showed that TSP2 transcript expression was down regulated in PCa tissue samples and cell lines, as compared to BPH tissue samples and non-tumor prostate cell line (p <0.01). ROC (Receiving Operating Curve) analyses showed that TSP2 transcript expression level was more efficient in distinguishing between PCa and BPH groups (p <0.01) than serum PSA (p = 0.299). TSP2 protein expression was observed in the cytoplasm of cells of epithelial and stromal compartments of PCa and BPH. Notably, TSP2 stromal staining score was significantly lower in PCa than in BPH (p <0.01), whereas the epithelial staining pattern was found to be similar for both diseases. PC3 and DU145 cell lines treatment with recTSP2 significantly reduced viability of these cells, and increase the percentage of cells in sub-G1 phase of the cell cycle. The phospho- proteomic analysis of treated PC3 cells showed that the proteins ERK1/2 and JNK presented lower phosphorylation level. However, Akt, p70S6, p53, p27, paxillin and RSK1/2/3 had a higher level of phosphorylation after treatment with recTSP2. Together, these data demonstrate that TSP2 is down regulated in PCa, especially in the stromal compartment. TSP2 diminished levels in PCa have potential application in the differential diagnosis of PCa and BPH. Additionally, the results generated in this study are the first description of the potential role of TSP2 in PCa, reinforcing the possibility that TSP2 is an inhibitor of PCa, which has been demonstrated for other tumor types. (AU)