Identificação de negevírus em mosquitos colhidos no sul de Portugal e análise da sua replicação

O género Negevirus foi descrito pela primeira vez em 2013, tendo sido proposto como um novo grupo taxonómico de vírus específicos de insetos com uma ampla distribuição geográfica, e caracterizado por uma considerável diversidade de hospedeiros, entre os quais flebótomos do género Lutzomyia e mosquitos, maioritariamente do género Culex mas também Anopheles e Aedes. Em Portugal, a primeira identificação de um negevírus foi obtida de um lote de mosquitos da espécie Oc. caspius, após o seu coisolamento com um flavivírus específico de insetos OCFVPT. A génese dos efeitos citopáticos exuberantes e de desenvolvimento rápido decorrentes da replicação destes vírus em culturas de células de inseto, a sua origem e evolução desconhecidas, as semelhanças de algumas das proteínas que codificam com as de vírus de plantas, e o seu potencial impacto na fitness e/ou na eventual competência vetorial dos insetos em que são encontrados, são questões que têm contribuído para o crescente interesse sobre o estudo destes vírus. No decurso do trabalho que aqui se apresenta foi efetuada a deteção, isolamento e caracterização genética de novos negevírus de mosquitos colhidos no sul de Portugal, tentativamente definida a sua gama de hospedeiros e analisada a sua estratégia de replicação, usando células de inseto mantidas em cultura. Através da utilização de RT-PCR e de primers degenerados (na sua maioria) foi possível identificar novas estirpes de negevírus, presentes em quatro espécies de mosquitos diferentes capturados no sul de Portugal: Ochlerotatus caspius, Cx. pipiens, Cx. theileri e Cx. univittatus. Inferências filogenéticas baseadas no alinhamento de sequências codificantes da polimerase viral, amplificadas de negevírus isolados em culturas de células C6/36, demonstraram a ocorrência de um novo grupo monofilético, filogeneticamente próximo do grupo monofilético do qual constam negevírus de tipo Negev e pertencente ao grupo taxonómico proposto recentemente, Nelorpivirus. A análise da replicação de negevírus in vitro permitiu observar a presença de efeitos citopáticos extensos e de desenvolvimento rápido, embora sem indicação óbvia da entrada das células infetadas em apoptose. Estes vírus comprovaram replicar de forma muito rápida, tendo sido possível detetar genomas virais no sobrenadante de culturas celulares C6/36 infetadas, 4 horas após o início da infeção. Neste trabalho foi também construído um vetor plasmídico, designado pIC111-V, o qual deverá permitir a expressão transitória de proteínas em células de inseto como consequência da presença de um promotor de transcrição de baculovírus reconhecido constitutivamente nestas células. A sua utilização na transfeção de células de inseto revelou poder representar um passo importante na melhoria das metodologias de análise proteómica nestas células.The genus Negevirus, described for the first time in 2013, was proposed as a new taxon of insect specific viruses with wide geographic distribution and characterized by a considerable host diversity, including Lutzomyia sandflies and mosquitoes mostly from the Culex, Anopheles and Aedes genera. In Portugal, the first identification of a negevirus was obtained as a result of its co-isolation with an insect-specific flavivirus, OCFVPT. The genesis of exuberant cytopathic effects and the fast replication of these viruses in insect cell cultures, its source and evolution, the similarities of the proteins they encode with those encoded by plant viruses and the potential impact in the fitness and/or in the possible vector competence of the insects where these virus are found, are questions that fuel the growing interest in the study of these viruses. In this report, detection, isolation and genetic characterization of new negeviruses of mosquitoes collected in the south of Portugal have been made, tentatively defining its host range and analyzing its replication strategy using insect cells maintained in culture. By using RT-PCR and degenerate primers (mostly) it was possible to identify new negeviruses strains, present in four different species of mosquitoes trapped in the south of Portugal: Ochlerotatus caspius, Cx. pipiens, Cx. theileri e Cx. univittatus. Phylogenetic inferences based on alignments of coding sequences of the viral polymerase, amplified from isolated negeviruses in C6/36 cell cultures, showed the occurrence of a new monophyletic group, phylogenetically close to that which contains negeviruses Negev-like and that belongs to the proposed recently, Nelorpivirus taxon. The analysis of negeviruses replication in vitro allowed us to observe the presence of extensive cytopathic effects with fast development, although with no obvious indication that infected cells undergo extensive apoptosis. Viral replication was confirmed to occur quickly, and it was possible to detect viral genomes in the infected C6/36 cell culture supernatant, 4 hours after the onset of infection. In this report, it was also constructed a plasmid vector, designated pIC111-V, which should allow the transient expression of proteins in insect cells, as a consequent of the presence of a baculovirus transcriptional promoter. Its use in transfection of insect cells transfection may be an important step in the improving of the proteomics analysis methodologies in these cells

A Gene Expression Signature to Select Hepatocellular Carcinoma Patients for Liver Transplantation

Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.OBJECTIVE: To propose a new decision algorithm combining biomarkers measured in a tumor biopsy with clinical variables, to predict recurrence after liver transplantation (LT). SUMMARY BACKGROUND DATA: Liver cancer is one of the most frequent causes of cancer-related mortality. LT is the best treatment for hepatocellular carcinoma (HCC) patients but the scarcity of organs makes patient selection a critical step. Additionally, clinical criteria widely applied in patient eligibility decisions miss potentially curable patients while selecting patients that relapse after transplantation. METHODS: A literature systematic review singled out candidate biomarkers whose RNA levels were assessed by quantitative PCR in tumor tissue from 138 HCC patients submitted to LT (>5 y follow up, 32% beyond Milan criteria). The resulting four gene signature was combined with clinical variables to develop a decision algorithm using machine learning approaches. The method was named HepatoPredict. RESULTS: HepatoPredict identifies 99% disease-free patients (>5 y) from a retrospective cohort, including many outside clinical criteria (16%-24%), thus reducing the false negative rate. This increased sensitivity is accompanied by an increased positive predictive value (88,5%-94,4%) without any loss of long-term overall survival or recurrence rates for patients deemed eligible by HepatoPredict; those deemed ineligible display marked reduction of survival and increased recurrence in the short and long term. CONCLUSIONS: HepatoPredict outperforms conventional clinical-pathologic selection criteria, (Milan, UCSF) providing superior prognostic information. Accurately identifying which patients most likely benefit from LT enables an objective stratification of waiting lists and information-based allocation of optimal versus suboptimal organs.publishersversionepub_ahead_of_prin

Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis

The uploaded article version is the Epub Ahead of Print version of the article, posted online 8 May 2018. It has been submitted to peer-review.The deposited article version contains attached the supplementary materials within the pdf.Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.Fundação para a Ciência e a Tecnologia–Harvard Medical School Program Portugal grant: (HMSP-CT/SAU-ICT/0075/2009); Liga Portuguesa Contra o Cancro; European Molecular Biology Organization Installation; Sociedade Portuguesa de Gastroenterologia.N/

Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4 h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3 h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon