RESUMO
Microscopic lasers represent a promising tool for the development of cutting-edge photonic devices thanks to their ability to enhance light-matter interaction at the microscale. In this work, we realize liquid microlasers with tunable emission by exploiting the self-formation of three-dimensional liquid droplets into a polymeric matrix driven by viscoelastic dewetting. We design a flexible device to be used as a smart photonic label which is detachable and reusable on various types of substrates such as paper or fabric. The innovative lasing emission mechanism proposed here is based on whispering gallery mode emission coupled to random lasing, the latter prompted by the inclusion of dielectric compounds into the active gain medium. The wide possibility of modulating the emission wavelength of the microlasers by acting on different parameters, such as the cavity size, type and volume fraction of the dielectrics, and gain medium, offers a multitude of spectroscopic encoding schemes for the realization of photonic barcodes and labels to be employed in anticounterfeiting applications and multiplexed bioassays.
RESUMO
Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.
Assuntos
Fibroblastos/metabolismo , Ouro/química , Raios Infravermelhos , Nanopartículas Metálicas/química , Análise de Célula Única , Síncrotrons , Ultrassonografia , Animais , Sobrevivência Celular , Camundongos , Micronúcleo Germinativo/metabolismo , Células NIH 3T3 , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Canonical Wnt signaling is critical for the regulation of bone formation, however, recent evidence suggests that the non-canonical Wnt agonist Wnt5a stimulates human osteoblastogenesis through its co-receptor Ror2. The effects of MM cells on non-canonical Wnt signaling and the effect of the activation of this pathway on MM-induced osteoblast exhaustion are not known and were investigated in this study. We found that the osteogenic differentiation of bone marrow hMSCs toward osteoprogenitor cells (PreOB) significantly increased Ror2 expression, and that MM cells inhibit Ror2 expression by PreOB in co-culture by inhibiting the non-canonical Wnt5a signaling. The activation of the non-canonical Wnt pathway in hMSCs by means of Wnt5a treatment and the overexpression of Wnt5 or Ror2 by lentiviral vectors increased the osteogenic differentiation of hMSCs and blunted the inhibitory effect of MM in co-culture. Consistently, Wnt5a inhibition by specific small interfering RNA reduced the hMSC expression of osteogenic markers. Our findings demonstrate that the Wnt5a/Ror2 pathway is involved in the pathophysiology of MM-induced bone disease and that the activation of the non-canonical Wnt5a/Ror2 pathway in hMSCs increases osteogenic differentiation and may counterbalance the inhibitory effect of MM cells.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Mieloma Múltiplo/patologia , Osteoblastos/citologia , Osteogênese , Proteínas Proto-Oncogênicas/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Técnicas de Cocultura , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células-Tronco/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
VP2 is the outermost Bluetongue virus (BTV) antigenic protein, forming triskelion motifs on the virion surface. Although VP2 has been expressed successfully through many systems, its paracrine expression as a soluble form by mammalian cells represents a difficult task. In the present paper two fragments of VP2 have been expressed successfully into the medium of transiently transfected mammalian cells through a fusion peptides strategy. The crude conditioned medium containing the secreted peptide could be employed for immunodiagnostic assay development or vaccine purposes.
Assuntos
Vírus Bluetongue/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Meios de Cultura/química , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BoHV-4 replication cycle is dependent on the S-phase of the cell-cycle at the stage of viral DNA synthesis. Because p21 is a rate-limiting regulator of the G1/S-phase transition and up-regulated by DNA-damaging agents, in this study p21 expression in BoHV-4 infected cells was investigated. The p21 promoter was found to be highly activated in a dose- and time-dependent manner following BoHV-4 infection only in cells which are permissive for BoHV-4 replication. Thus p21 expression reports on BoHV-4 replication and could represent a host cell defensive response to infection-associated cellular damage.
Assuntos
Doenças dos Bovinos/virologia , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/fisiologia , Infecções Tumorais por Vírus/veterinária , Replicação Viral , Animais , Biomarcadores , Bovinos , Doenças dos Bovinos/metabolismo , Linhagem Celular , Genes Reporter , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno , Camundongos , Regiões Promotoras Genéticas , Fatores de Tempo , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologiaRESUMO
A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.