Enhancement of thermal conductive pathway of boron nitride coated polymethylsilsesquioxane composite.

We report here in the fabrication of enhanced thermal conductive pathway nanocomposites of boron nitride (BN)-coated polymethylsilsesquioxane (PMSQ) composite beads using isopropyl alcohol (IPA) as a mixing medium. Exfoliated and size-reduced boron nitride particles were successfully coated on the PMSQ beads and explained by surface charge differences. A homogeneous dispersion and coating of BN on the PMSQ beads using IPA medium was confirmed by SEM. Each condition of the composite powder was carried into the stainless still mould and then hot pressed in an electrically heated hot press machine. Three-dimensional percolation networks and conductive pathways created by exfoliated BN were precisely formed in the nanocomposites. The thermal conductivity of nanocomposites was measured by multiplying specific gravity, specific heat, and thermal diffusivity, based upon the laser flash method. Densification of the composite resulted in better thermal properties. For an epoxy reinforced composite with 30 vol% BN and PMSQ, a thermal conductivity of nine times higher than that of pristine PMSQ was observed.

Superparamagnetic particle dynamics and mixing in a rotating capillary tube with a stationary magnetic field.

The dynamics of superparamagnetic particles subject to competing magnetic and viscous drag forces have been examined with a uniform, stationary, external magnetic field. In this approach, competing drag and magnetic forces were created in a fluid suspension of superparamagnetic particles that was confined in a capillary tube; competing viscous drag and magnetic forces were established by rotating the tube. A critical Mason number was determined for conditions under which the rotation of the capillary prevents the formation of chains from individual particles. The statistics of chain length were investigated by image analysis while varying parameters such as the rotation speed and the viscosity of the liquid. The measurements showed that the rate of particle chain formation was decreased with increased viscosity and rotation speed ; the particle dynamics could be quantified by the same dimensionless Mason number that has been demonstrated for rotating magnetic fields. The potential for enhancement of mixing in a bioassay was assessed using a fast chemical reaction that was diffusion-limited. Reducing the Mason below the critical value, so that chains were formed in the fluid, gave rise to a modest improvement in the time to completion of the reaction.

Accelerated immunoassays based on magnetic particle dynamics in a rotating capillary tube with stationary magnetic field.

A rapid and simple magnetic particle-based immunoassay has been demonstrated in a capillary mixing system. Antibody-coated micrometer size superparamagnetic polystyrene (SPP) particles were used in an assay for rabbit IgG in a sandwich (noncompetitive) format. The kinetics of the assay was compared between a plate-based system and a single capillary tube. The interaction between the antigen (R-IgG) and the antibody (anti-R-IgG) that was carried by the SPP particles in a rotating capillary was tested under a stationary magnetic field. Competing magnetic and viscous drag forces helped to enhance the interaction between the analyte and the capture antibodies on the particles. The dimensionless Mason number (Mn) was employed to characterize the magnetic particle dynamics; a previously determined critical Mason number (Mn(c)) was employed as a guide to the appropriate experimental conditions of magnetic field strength and rotational speed of the capillary. The advantage of the rotating capillary system included a short assay time and a reduced reactive volume (20 µL). The results show that the immunoassay kinetics were improved by the formation of chains of the SPP particles for the conditions that corresponded to the critical Mason number.

Effect of cerium oxide nanoparticles on inflammation in vascular endothelial cells.

Because vascular endothelial cell inflammation is critical in the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs) to ultrafine particles induces an inflammatory response. To test the hypothesis, we incubated HAECs for 4 h with different concentrations (0.001-50 microg/ml) of CeO(2) nanoparticles and subsequently measured mRNA levels of the three inflammatory markers intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)-8, and monocyte chemotactic protein (MCP-1) using real-time polymerase chain reaction (PCR). Ceria nanoparticles caused very little inflammatory response in HAECs, even at the highest dose. This material is apparently rather benign in comparison with Y(2)O(3) and ZnO nanoparticles that we have studied previously. These results suggest that inflammation in HAECs following acute exposure to metal oxide nanoparticles depends strongly on particle composition.

Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector.

BACKGROUND: The use of retroviral vectors has shown an actual clinical benefit in a few inherited diseases. However, the occurrence of cases of leukemia after the X-SCID gene therapy trial raised concerns about the safety of insertional mutagenesis inherent to the biology of the retrovirus. Although the retrovirus has long been known to integrate into the host chromosome, and thus have the potential to activate the nearby gene, there has been no convenient method of studying or assaying such a cis-activation phenomenon. METHODS: In the present study, we report an in vitro assay system in which the effect of retroviral integration on the expression of the neighbouring gene can be studied. In this system, a retroviral vector and the neighbouring reporter gene were constructed in a single plasmid as if it had integrated into the chromosome. RESULTS: Using this assay, we found that the full-length long terminal repeat (LTR) could indeed activate the neighbouring gene expression from a distance and the magnitude of its activation was highly increased when this LTR was placed in the vicinity of the transcription start site of the gene, whereas the truncated LTR exerted little influence. CONCLUSIONS: This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.

Control of splicing efficiency by the mouse histone H2a element in a murine leukemia virus-based retroviral vector.

While using various human complementary DNA (cDNA) sequences in the context of the murine leukemia virus (MLV)-based retroviral vector, it was found that a retroviral vector containing some human cDNA sequences produces unusually low viral titer. One of those sequences is that for the human IL-1 receptor antagonist protein (IL1RN). The RNA analysis showed that a cryptic splice acceptor sequence is present in the middle of its coding region, resulting in the deletion of the packaging signal sequence and the removal of some coding sequences that lead to low viral titer and a low level of the transgene product. We tested whether the mouse Hist2h2aa1 element (mH2aE), previously shown to suppress the splicing function, could inhibit the cryptic splicing in the context of MLV-based retroviral vectors. It was found that the mH2aE could efficiently suppress such unwanted splicing event, thus increasing the amount of unspliced transcript, which eventually led to the increase in the level of IL1RN expression and viral titer. The mH2aE could also be used to control unusually high splicing activity. Our data suggested that the mH2aE could be used for the fine-tuning of the splicing process, thus improving the level of gene expression and viral titer in the context of retroviral vectors.

Construction of a retroviral vector production system with the minimum possibility of a homologous recombination.

A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia virus (MLV)-based packaging constructs contained significantly long 5' and/or 3' untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed.

MicroRNA maturation: stepwise processing and subcellular localization.

MicroRNAs (miRNAs) constitute a novel, phylogenetically extensive family of small RNAs ( approximately 22 nucleotides) with potential roles in gene regulation. Apart from the finding that miRNAs are produced by Dicer from the precursors of approximately 70 nucleotides (pre-miRNAs), little is known about miRNA biogenesis. Some miRNA genes have been found in close conjunction, suggesting that they are expressed as single transcriptional units. Here, we present in vivo and in vitro evidence that these clustered miRNAs are expressed polycistronically and are processed through at least two sequential steps: (i) generation of the approximately 70 nucleotide pre-miRNAs from the longer transcripts (termed pri-miRNAs); and (ii) processing of pre-miRNAs into mature miRNAs. Subcellular localization studies showed that the first and second steps are compartmentalized into the nucleus and cytoplasm, respectively, and that the pre-miRNA serves as the substrate for nuclear export. Our study suggests that the regulation of miRNA expression may occur at multiple levels, including the two processing steps and the nuclear export step. These data will provide a framework for further studies on miRNA biogenesis.