In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 replication in living Buffalo green monkey kidney (BGMK) cells via Tat peptide delivery. A nuclease-resistant MB containing 2-Omethyl RNA bases with phosphorothioate internucleotide linkages was designed to specifically target an 18-bp 5 noncoding region of the viral genome. For intracellular delivery, a cell-penetrating Tat peptide was conjugated to the MB by using a thiol-maleimide linkage. Presence of the Tat peptide enabled nearly 100% intracellular delivery within 15 min. When the conjugate was introduced into BGMK cell monolayers infected with coxsackievirus B6, a discernible fluorescence was observed at 30 min after infection, and as few as 1 infectious viral particle could be detected within 2 h. The stability and the intracellular delivery properties of the modified MBs enabled real-time monitoring of the cell-to-cell spreading of viral infection. These results suggest that the Tatmodified, nuclease-resistant MBs may be powerful tools for improving our understanding of the dynamic behavior of viral replication and for therapeutic studies of antiviral treatments.detection ͉ infectious diseases ͉ real time
Here we describe a new hybrid fluorescent nanoprobe composed of a nuclease-resistant molecular beacon (MB) backbone, CdSe-ZnS core-shell quantum dots (QDs) as donors, and gold nanoparticles (Au NPs) as quenchers, for the real-time visualization of virus replication in living cells. By using a Au NP-MB to QD ratio of 6 : 1, a 7.3-fold increase in fluorescent signal was achieved upon target binding. For living cell experiments, a hexahistidine-appended Tat peptide was self-assembled onto the QD surface to provide nearly 100% non-invasive delivery of the QD-MB-Au NP probes within 2 h. By directly visualizing the fluorescent complexes formed with the newly synthesized viral RNA, this QD-MB-Au NP probe provided sensitive and real-time detection of infectious viruses as well as the real-time visualization of cell-to-cell virus spreading.
Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5 noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.Hepatitis A virus (HAV), a positive single-stranded RNA virus, was previously classified as enterovirus type 72 (14) and has now been classified in the Hepatovirus genus of the Picornaviridae family (15). These single-plus-stranded RNA viruses can directly translate plus-strand RNA genomes into protein using the host ribosomes. The plus strand is transported to the infected cell via specific membrane vesicles, where it is copied into full-length minus strands. These minus strands then serve as templates for the synthesis of plus-strand genomic RNA molecules (1, 2).HAV is known to cause acute liver infection with a discrete onset of symptoms (e.g., fever, malaise, and nausea), followed in several days by jaundice. Person-to-person transmission through the fecal-oral route is the primary means of HAV transmission. Outbreaks and periodic cases also occur from exposure to fecally contaminated food or water (Centers for Disease Control and Prevention). Most picornaviruses are lytic, causing distinctive cytopathic effects and replicating in an 8-h cycle under one-step growth conditions. The growth of HAV in cell culture systems, however, is much slower and nonlytic and does not produce a detectable cytopathic effect in infected cells (13). Cromeans et al. reported the isolation of a cytopathic HAV variant from the rapidly replicating isolate HM-175 that is lytic for fetal rhesus monkey kidney (FRhK-4) cells (4). However, complete lysis still did not occur until 5 to 6 days postinfection (p.i.).Conventional methods for detecting infectious HAV rely on viral propagation in cell culture, radioimmunoassay, immunofluorescence, or plaque assay; these methods are difficult to perform, and it may take weeks before the viruses reach sufficiently high amo...
Aims: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) or mesenchymal stem cells (MSCs) facilitate post-infarct recovery, but the potential benefit of combination therapy using MSCs and hESC-CMs has not been examined. Our objective was to define the gene expression changes in donor and host-derived cells that are induced in vivo after co-transplantation of cardiomyocytes with and without mesenchymal stem cells expressing the prosurvival gene heme oxygenase 1. Methods and results: Human MSCs were engineered to over-express heme oxygenase-1 (HO-1) following lentiviral vector-mediated transduction. Athymic nude rats were subjected to myocardial infarction and received hESC-CMs alone, hESC-CMs plus human MSCs, hESC-CMs plus MSCs overexpressing HO-1, or saline. Real time PCR identified gene expression changes. Cardiac function was assessed by angiography. Co-transplantation of unmodified MSCs plus hESC-CMs elevated CXCR4, HGF, and IGF expression over levels induced by injection of hESC-derived cardiomyocytes alone. In animals co-transplanted with MSC over-expressing HO-1, the expression of these genes was further elevated. Gene expression levels of VEGF, TGF-β, CCL2, SMAD7, STAT3 and cardiomyocyte transcription factors were highest in the HO-1 MSC plus hESC-CM group at 30 days. Human CD31+, CD34+, isl-1+, NXK2.5 and c-kit+ transcripts were elevated. Rodent genes encoding NKX2.5, troponin T and CD31 were elevated and cell cycle genes were induced. Ejection fraction improved by six to seven percent. Conclusions: Co-administration of HO-1 MSCs plus hESC-CMs increased expression of pro-survival and angiogenesis-promoting genes in human cells and transcripts of cardiac and endothelial cell markers in rodent cells, consistent with activation of tissue repair in both transplanted hESC-CMs and the host heart.
Thermal dissipation is an important issue for power devices. In this work, the impact of thermal effects on the performance of Cu electroplated GaN-based high-electron-mobility transistors (HEMTs) are considered. Electrical, thermometry and micro-Raman characterization techniques were used to correlate the effects of improved heat dissipation on device performance for GaN HEMTs with different thicknesses of Si substrate (50, 100, 150 μm), with and without an additional electroplated Cu layer. GaN HEMTs on electroplated Cu on Si (≤50 μm) demonstrate an enhanced on/off current ratio compared to bare Si substrate by a factor of ~400 (from 9.61 × 105 to 4.03 × 108). Of particular importance, surface temperature measurements reveal a much lower channel temperature for thinner HEMT devices with electroplated Cu samples compared to those without.
ABSTRACT:The mass transport of methanol mixed with ferric chloride hexahydrate (FeCl 3 ⅐ 6H 2 O) in poly(methyl methacrylate) and poly(methyl methacrylate)/iron carbonate particulate (p) nanocomposites is prepared by chemical vapor crystallization and the resulting materials, which are subjected to characterization to evaluate thermal and optical properties, have been investigated. Mass transport is an anomalous and endothermic process and satisfies the van't Hoff plot. We have prepared successfully poly(methyl methacrylate)(PMMA)/iron carbonate particulates nanocomposites using CO 2 gas slowly diffused into saturated solvent mixture-treated poly(methyl methacrylate) for 48 h. After SEM observation, approximately 80 nm iron carbonate particulates were precipitated and evenly distributed in the poly(methyl methacrylate) matrix. In comparison with solvent mixture-treated PMMA, the cut-off wavelength of transmittance in nanocomposites shifts to the shorter wavelength side (red shift). The presence of nanoscale iron carbonate particulates increased the glass transition temperature of the nanocomposites as determined by differential scanning calorimeter, and the glass transition temperature increased with increasing content of nanoscale iron carbonate particulates. The FTIR spectra of solvent mixture-treated poly(methyl methacrylate) and nanocomposites are also studied.
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