RESUMO
Ranavirus is a large nucleocytoplasmic DNA virus. Chinese giant salamander iridovirus (CGSIV) belongs to the ranavirus genus, and its replication involves a series of essential viral genes. Viral PCNA is a gene closely associated with viral replication. CGSIV-025L also encodes PCNA-like genes. We have described the function of CGSIV-025L in virus replication. The promoter of CGSIV-025L is activated during viral infection, and it is an early (E) gene that can be effectively transcribed after viral infection. CGSIV-025L overexpression promoted viral replication and viral DNA replication. siRNA interfered with CGSIV-025L expression and attenuated viral replication and viral DNA replication. The Δ025L-CGSIV strain with the deletion of CGSIV-025L could not replicate normally and could be rescued by the replenishment of 025L. CGSIV-025L was proven to be an essential gene for CGSIV by overexpression, interference, and deletion mutation experiments. CGSIV-025L was found to interact with CGSIV-062L by yeast two-hybrid, CoIP, and GST pulldown. Thus, the current study demonstrated that CGSIV-025L is an essential gene of CGSIV, which may be involved in viral infection by participating in viral DNA replication and interacting with replication-related proteins.
Assuntos
Infecções por Vírus de DNA , Iridovirus , Ranavirus , Animais , Iridovirus/genética , Genes Essenciais , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , DNA Viral/genética , Infecções por Vírus de DNA/veterinária , Replicação Viral , Ranavirus/genética , Genes Virais , Urodelos/genéticaRESUMO
A polynucleotide probe, call a polymeric sequence probe (PSP), was used to detect influenza A (Influenza A/WSN/33) NA (Neuraminidase) viral RNA in Madin-Darby canine kidney (MDCK) cells. The PSP is a single-stranded DNA molecule with ~2,000 tandem repeat fluorescence binding sites and target binding sites that can bind with multiple fluorescence complementary oligos and target viral RNA using a fluorescence in situ hybridization (FISH) process. A single viral RNA labeled by PSP can be directly observed in MDCK cells. The simple FISH protocol enables the observation and quantitative analysis of the infectious process and drug effects with ultrahigh sensitivity and spatial resolution.
Assuntos
Vírus da Influenza A/química , Vírus da Influenza A/genética , Polinucleotídeos/genética , Sondas RNA/química , Sondas RNA/genética , RNA Viral/análise , RNA Viral/genética , Animais , Cães , Hibridização in Situ Fluorescente , Células Madin Darby de Rim CaninoRESUMO
A polynucleotide probe, polymeric sequence probe (PSP), was developed for single molecular detections. PSP is a single-stranded DNA molecule with ~2000 tandem repeat target-binding sequences and label-binding sequences. A single PSP can bind to multiple fluorescent complementary oligos to generate a strong fluorescence signal. Single target molecules bound to PSPs can be clearly visualized by a conventional fluorescence microscope. An ultrasensitive PSP-based assay for Mycobacterium tuberculosis was demonstrated.
Assuntos
Sondas de DNA/química , DNA de Cadeia Simples/análise , Polinucleotídeos/química , Células Cultivadas , DNA de Cadeia Simples/genética , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/genéticaRESUMO
The effective and sustained delivery of DNA and siRNAs locally would increase the applicability of gene therapy in tissue regeneration and cancer therapy. One promising approach is to use hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the disease sites. However, this approach is currently limited by the inability to load concentrated and active gene delivery nanoparticles into the hydrogels due to the severe nanoparticle aggregation during the loading process. Here, we present a process to load concentrated and un-aggregated non-viral gene delivery nanoparticles, using DNA/polyethylene imine (PEI) polyplexes as an example, into neutral polyethylene glycol (PEG), negatively charged hyaluronic acid (HA) and protein fibrin hydrogels crosslinked through various chemistries. The encapsulated polyplexes are highly active both in vitro and in vivo. We believe this process will significantly advance the applications of hydrogel scaffold mediated non-viral gene delivery in tissue regeneration and cancer therapy.
Assuntos
DNA/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Polietilenoimina/química , Alicerces Teciduais/química , Animais , Cátions , Bovinos , Galinhas , Liofilização , Técnicas de Transferência de Genes , Humanos , Microscopia Confocal , Nanopartículas/química , Neovascularização Fisiológica , Polietilenoglicóis/químicaRESUMO
Proteins assembled on an Au nanowire are manipulated by an electrical potential applied on the nanowire, which leads to the modulation of molecular fluorescence. The molecular modality can be unequivocally correlated with the modulated fluorescence, which enables the specific fluorescence from a single target protein to be unambiguously distinguished from background noise and nonspecific fluorescence. As demonstrated through a thrombin assay, this simple method can significantly improve the sensitivity and specificity of the protein detection down to the single molecule level.
Assuntos
Ouro/química , Nanofios , Proteínas/química , Sequência de Bases , Fluorescência , Sensibilidade e EspecificidadeRESUMO
Nanoarrays of distinct DNA and protein biomolecules were fabricated by electrochemically controlling their assembly/release from Au nanoelectrodes on a chip. The surface density, ratio, and activity of the biomolecules assembled on each nanoelectrode in the array can be configured quantitatively and temporally by adjusting the electrochemical potential applied on the nanoelectrode. The dynamically configurable biomolecular nanoarray can potentially activate combinatorial interactions with microbiosystems under the control of an electronic circuit for biological and medical applications.
