We describe the isolation and characterization of an insect-specific flavivirus (ISF) from Ochlerotatus caspius (Pallas, 1771) mosquitoes collected in southern Portugal. The RNA genome of this virus, tentatively designated OCFVPT, for O. caspius flavivirus from Portugal, encodes a polyprotein showing all the features expected for a flavivirus. As frequently observed for ISF, the viral genomes seems to encode a putative Fairly Interesting Flavivirus ORF (FIFO)-like product, the synthesis of which would occur as a result of a -1 translation frameshift event. OCFVPT was isolated in the C6/36 Stegomyia albopicta (= Aedes albopictus) cell line where it replicates rapidly, but failed to replicate in Vero cells in common with other ISFs. Unlike some of the latter, however, the OCFVPT genome does not seem to be integrated in the mosquito cells we tested. Phylogenetic analyses based on partial ISF NS5 nucleotide sequences placed OCFVPT among recently published viral strains documented from mosquitoes collected in the Iberian Peninsula, while analyses of ORF/E/NS3/or NS5 amino acid sequences cluster OCFVPT with HANKV (Hanko virus), an ISF recently isolated from O. caspius mosquitoes collected in Finland. Taking into account the genetic relatedness with this virus, OCFVPT is not expected to be overtly cytopathic to C6/36 cells. The cytopathic effects associated with its presence in culture supernatants are postulated to be the result of the replication of a co-isolated putative new Negev-like virus.
Cancer remains a complex medical challenge and one of the leading causes of death worldwide. Nanomedicines have been proposed as innovative platforms to tackle these complex diseases, where the combination of several treatment strategies might enhance therapy success. Among these nanomedicines, nanoparticle mediated delivery of nucleic acids has been put forward as key instrument to modulate gene expression, be it targeted gene silencing, interference RNA mechanisms and/or gene edition. These novel delivery systems have strongly relied on nanoparticles and, in particular, gold nanoparticles (AuNPs) have paved the way for efficient delivery systems due to the possibility to fine-tune their size, shape and surface properties, coupled to the ease of functionalization with different biomolecules. Herein, we shall address the different molecular tools for modulation of expression of oncogenes and tumor suppressor genes and discuss the state-of-the-art of AuNP functionalization for nucleic acid delivery both in vitro and in vivo models. Furthermore, we shall highlight the clinical applications of these spherical AuNP based conjugates for gene delivery, current challenges, and future perspectives in nanomedicine.
Advances in nanotechnology and medical science have spurred the development of engineered nanomaterials and nanoparticles with particular focus on their applications in biomedicine. In particular, gold nanoparticles (AuNPs) have been the focus of great interest, due to their exquisite intrinsic properties, such as ease of synthesis and surface functionalization, tunable size and shape, lack of acute toxicity and favorable optical, electronic, and physicochemical features, which possess great value for application in biodetection and diagnostics purposes, including molecular sensing, photoimaging, and application under the form of portable and simple biosensors (e.g., lateral flow immunoassays that have been extensively exploited during the current COVID-19 pandemic). We shall discuss the main properties of AuNPs, their synthesis and conjugation to biorecognition moieties, and the current trends in sensing and detection in biomedicine and diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging K E Y W O R D S biodetection, bioimaging, diagnostics, gold nanoparticles, nanomedicine 1 | INTRODUCTION 1.1 | Nanoparticles for diagnosticsNanoparticle-based diagnosis has relied strongly on the continuous progresses in nanoscience and nanotechnology, particularly on the discovery and characterization of new nanomaterials and their applications in biomedicine. Several of these developments have stemmed from the fundamental research on the properties and functions of nanomaterials, where metal nanoparticles have a prominent position, particularly those of gold, iron, silver, platinum, copper, and cobalt (Doria et al., 2012;Habibullah et al., 2021).Beatriz B. Oliveira and Daniela Ferreira contributed equally to this study.
Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs.Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC.Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC.Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.
The genus Flavivirus (Flaviviridae) includes over seven dozen known enveloped viruses with ssRNA genomes, the evolution, geographic dispersion and epidemiology of which seems to have been shaped both by environmental constraints (involving ecological factors and human activities), and by their replication in vertebrate and invertebrate hosts. Most bona-fide flaviviruses have been classified as mosquito or tick-borne viruses, although for some no known invertebrate vector has yet been found. Over time, a divergent cluster including viral agents that seem to be specific of insect (Insect-Specific Flaviviruses-ISF) has been associated with the genus. The classification of ISF as flaviviruses is supported by their genomic organization, protein hydropathy plots, conserved polyprotein cleavage sites and enzyme domains, which are similar to those of classical flaviviruses. However, they are antigenically distinct, and share approximately the same level of nucleotide sequence identity with other members of the genus, as when compared to the members of the two other genera in the Flaviviridae family.This work reports the initial characterization, including near full-length sequence analysis, of an ISF which seems to form a divergent lineage within the ISF radiation (Figure 1), as indicated by phylogenetic analysis based on ac multiple sequence alignment of the NS5 flavivirus coding sequence. This virus, tentatively designated OCFV (Ochlerotatus caspius flavivirus) was isolated from clarified macerates of a pool of Aedes (Ochlerotatus) caspius (Pallas, 1771) adult mosquitoes (lab code number 174) using the Ae. albopictus-derived C6/36 cell line. The mosquitoes from which OCFV was isolated were collected using CDC-traps baited with CO2, and are amongst those found in high densities in the coastal, and estuarine, districts of Setúbal and Faro. Detection of viral genomes by RT-PCR using RNA directly extracted from supernatants of C6/36 infected cultures, collected 24h post-infection, indicated that the virus replicates rapidly in these cells. Nevertheless, but as expected for an ISF, it does not replicate in Vero cells. Unexpectedly, unlike most ISF OCFV seems to cause overt cytopathic effect in C6/36 cells, which rapidly detach from a solid support and round-up soon (24-48h) after infection. Electron-microscope analysis of thin-sections of C6/36 cells at 48h post-infection with OCFV revealed nuclear hyperplasy, and an evident enlargement of the intracisternal space of the nuclear envelope, which is filled by multiple sized vesicles. In some cells, a complex network of apparent membrane trabeculae was also clearly evident (Figure 2A). Viral particles seem to assemble at the endoplasmatic reticulum, from which viruses reach the cell surface by vesicular transport (Figure 2B). Unexpectedly for a flavivirus, but as previously observed, viral particles also seem to gemulate directly from the cytoplasmic membrane (Figure 2C).
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