Biomedical subjects

Heng-Phon Too

Publications and source records attributed to Heng-Phon Too.

14 recordsLinked to original sources

PubMed2006

Establishing efficient siRNA knockdown in mouse embryonic stem cells.

FAM-labeled oligo dT (FAMdT) was utilized as a means to gauge efficient transfection of small nucleic acids into mouse embryonic stem cell (mESC) colonies. Using colonies grown overnight, transfection was restricted largely to the periphery of the colonies with only a 40% decrease in Oct-3/4 RNA transcript levels following cognate Oct-3/4, small interfering RNA (siRNA) delivery. However, transfection of mESC 4 h after seeding gave greater than 90% cells being successfully transfected based on quantitative real-time PCR detection of approximately 90% Oct-3/4 RNA transcript knockdown. This method provides an economical and efficient means by which to determine effective transfection conditions, and establish efficient siRNA knockdown of reportedly difficult to transfect cell lines such as mESC.

Animals↗

PubMed2006

Absolute quantification of gene expression in biomaterials research using real-time PCR.

One major measurement of tissue-engineered constructs efficacy and performance is determining expression levels of genes of interest at the molecular level. This measurement is commonly carried out with reverse transcription-polymerase chain reaction (RT-PCR). In this study, we described a novel method in achieving absolute quantification of gene expression using real-time PCR (aqPCR). This novel method did not require molecular cloning steps to prepare the standards for quantification comparison. Standards were linear double-stranded DNA molecules instead of the typical gene-in-plasmid format. aqPCR could also be used to give relative quantification comparisons between samples simply by dividing the copy numbers readings of the gene of interest with that of the normalization gene. RNA was extracted from monolayer and from polycaprolactone scaffold cultures and assayed for beta-actin and osteocalcin genes. We compared our aqPCR method with end-point PCR since end-point PCR is still a common means of measuring gene expression in the biomaterials field. This study showed that aqPCR was a better method to quantify gene expression than end-point PCR. With our described linear DNA standards method, we were able to obtain not only relative quantification of osteocalcin and beta-actin expression level but also actual copy numbers of osteocalcin and beta-actin for the monolayer culture and to be 1.34 x 10(4) and 1.45 x 10(7) copies, respectively and for the scaffold cultures to be 772 and 2.83 x 10(5) copies, respectively per starting total RNA mass of 10 ng. The standards curves made from these linear DNA standards showed good linearity (R(2)=0.9964 and 0.9902 for osteocalcin and beta-actin standards graphs), ranged from 10 to 10(9) copies and of comparable accuracy to current absolute quantification real-time PCR methods (which used plasmid standards obtained through molecular cloning methods). Our method might be a viable and more user-friendly alternative to current absolute quantification real-time PCR protocols.

Biocompatible Materials↗

PubMed2006

A bis(p-sulfonatophenyl)phenylphosphine-based synthesis of hollow Pt nanospheres.

We report herewith the synthesis of hollow Pt nanospheres by using bis(p-sulfonatophenyl)phenylphosphine to selectively remove the Ag cores of Ag-Pt core-shell nanoparticles. Core-shell Ag-Pt nanoparticles were first obtained by the successive reduction method with a discontinuous Pt shell to allow the BSPP passage. Transmission electron microscopy imaging of the core-shell Ag-Pt nanoparticles before and after BSPP dissolution showed little changes in the particle size, indicating that the removal of the Ag cores had occurred isomorphously. The hollow Pt nanospheres, together with the predecessor Ag-Pt core-shell particles of the same size, were transferred from water to toluene and surface modified by dodecylamine in toluene. This allows the catalytic activities of solid and hollow Pt particles in room temperature methanol oxidation reaction to be compared under conditions of identical particle size and the same surface environment. The measured higher specific activity of the Pt hollow nanospheres could then be attributed unambiguously to the larger specific surface area prevalent in the porous hollow structure.

Nanostructures↗

PubMed2006

Glial cell-line derived neurotrophic factor and neurturin regulate the expressions of distinct miRNA precursors through the activation of GFRalpha2.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related neurotrophic factors that have both been shown to prevent the degeneration of dopaminergic neurons in vitro and in vivo. NTN and GDNF are thought to bind with different affinities to the GDNF family receptor alpha-2 (GFRalpha2), and can activate the same multi-component receptor system consisting of GFRalpha2, receptor tyrosine kinase Ret (RET) and NCAM. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that regulate gene expression through translational repression or RNA degradation. miRNAs have diverse functions, including regulating differentiation, proliferation and apoptosis in several organisms. It is currently unknown whether GDNF and NTN regulate the expression of miRNAs through activation of the same multi-component receptor system. Using quantitative real-time PCR, we measured the expression of some miRNA precursors in human BE(2)-C cells that express GFRalpha2 but not GFRalpha1. GDNF and NTN differentially regulate the expression of distinct miRNA precursors through the activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2). This study showed that the expression of distinct miRNA precursors is differentially regulated by specific ligands through the activation of GFRalpha2.

Cells, Cultured↗

PubMed2005

Inhibition of DNA hybridization by small metal nanoparticles.

The inhibition of DNA hybridization by small metal nanoparticles has been examined in detail. DNA melting point analysis showed that the oligonucleotides adsorb strongly and nonspecifically on small metal nanoparticles, inhibiting the hybridization of complementary DNA sequences in common buffered solutions. The nonspecific interaction is even strong enough to disrupt pre-existing hydrogen bonds in short double-stranded DNA. The nonspecific interaction could be weakened by increasing the particle size. As an example, a core-shell assisted method was used to successfully assemble Pt nanoparticles by DNA hybridization that could not be done otherwise.

DNA, Single-Stranded↗

PubMed2005

Core-shell Ag-Au nanoparticles from replacement reaction in organic medium.

The replacement reaction between hydrophobized Ag nanoparticles and hydrophobized AuCl4- in toluene has been examined in detail. The conclusions obtained under our experimental conditions are different from those reported in the literature in three aspects: (1) a detectable contraction of the Ag nanoparticle sacrificial templates during the course of the reaction is shown; (2) the deposition of Au on the shrunken Ag templates inhibits further Ag oxidation, resulting in the formation of core-shell Ag-Au nanoparticles instead of Au nanoshells; and (3) the significant red-shift in the surface plasmon resonance (SPR) band is more of a consequence of shape and chemical composition changes rather than as an indication of Au nanoshell formation. Solvent and temperature are influential environmental factors that determine the structure and composition of nanoparticles formed by the replacement reaction.

Journal Article↗

PubMed2005

Tissue expression of alternatively spliced GFRalpha1, NCAM and RET isoforms and the distinct functional consequence of ligand-induced activation of GFRalpha1 isoforms.

Glial-cell-line-derived neurotrophic factor (GDNF) exerts its effect through a multi-component receptor system consisting of GFRalpha1, RET and NCAM. Two highly homologous alternatively spliced GFRalpha1 isoforms (GFRalpha1a and GFRalpha1b) have previously been identified. In this study, isoform specific real-time PCR assays were used to quantify the expression levels of GFRalpha1, RET and NCAM isoforms in murine embryonic and adult tissues. The expression levels of GFRalpha1b were found to be comparable to that of GFRalpha1a in peripheral tissues. However, GFRalpha1a was the predominant isoform expressed in the whole brain. The co-expressions of GFRalpha1 and the co-receptors were developmentally regulated and differentially expressed in some tissues. Microarray analyses of GFRalpha1 isoforms transfected cells stimulated with NTN showed distinct and non-overlapping gene profiles. These observations are consistent with the emerging view that the combinatorial interactions of the spliced isoforms of GFRalpha, RET and NCAM may contribute to the pleiotropic biological responses.

Alternative Splicing↗

PubMed2005

A phase-transfer identification of core-shell structures in Ag-Pt nanoparticles.

Ag-Pt nanoparticles with a confirmed core-shell structure could only be formed by the successive reduction method using Ag nanoparticles as the seeds. The core-shell structure could be conveniently inferred from the transferability of the particles from water to toluene. Independent measurements by UV-vis spectroscopy, transmission electron microscopy, energy-dispersive X-ray analysis, and X-ray photoelectron spectroscopy were used to validate the experimental results. The reverse order of synthesis using Pt nanoparticles as the seeds did not result in any core-shell product. Instead a physical mixture of Ag nanoparticles and the original Pt seeds was obtained under the same experimental conditions.

Journal Article↗

PubMed2005

Phase transfer identification of core-shell structures in Ag-Au bimetallic nanoparticles.

The difference between the transfer of poly(vinylpyrrolidone)-protected Au and Ag nanoparticles from an aqueous solution to toluene was used to develop a simple experimental procedure that can positively identify the formation of bimetallic Ag-Au nanoparticles with the core-shell structure. The method was validated by UV-visible spectroscopy, EDX, and TEM measurements. Using this technique, we have found that core-shell nanoparticles of Ag-Au were formed by the seed-mediated growth method using Ag nanoparticles as the seeds. The reversed order of using Au nanoparticles as the seeds, on the contrary, could only produce a physical mixture of Ag-Au core-shell nanoparticles and isolated Ag nanoparticles.

Gold↗

PubMed2004

A highly efficient phase transfer method for preparing alkylamine-stabilized Ru, Pt, and Au nanoparticles.

A highly efficient phase-transfer method was developed to prepare alkylamine-stabilized nanoparticles of several noble metals. This method involved first mixing the metal hydrosols and an ethanol solution of dodecylamine and then extracting the dodecylamine-stabilized metal nanoparticles into toluene. The efficiency of this phase-transfer method was nearly 100%. Alkylamine-stabilized Ru, Pt, and Au nanoparticles 3.45, 4.33, and 7.89 nm in diameter, respectively, could be prepared this way. The self-assembly of dodecylamine-stabilized Pt and Au particles was also detected by transmission electron microscopy (TEM).

Amines↗

PubMed2004

Acetate stabilization of metal nanoparticles and its role in the preparation of metal nanoparticles in ethylene glycol.

Acetate-stabilized ruthenium nanoparticles were prepared by the NaBH4 reduction of the metal precursor salt at room temperature. Nanoparticles with a mean diameter of 2.20 nm and a standard deviation of 1.03 nm could be obtained under experimental conditions. The Ru nanoparticles so obtained could be easily extracted to a toluene solution of alkylamine, giving rise to alkylamine-stabilized Ru nanoparticles with a mean diameter of 2.96 nm and a standard deviation of 0.92 nm. The new found role of acetate stabilization was used to formulate a mechanism for the formation of metal (Pt, Ru) nanoparticles in ethylene glycol. In this mechanism metal nanoparticles are stabilized in ethylene glycol by adsorbed acetate ions, which are produced as a product of the OH- catalyzed reaction between the metal precursor salt and ethylene glycol.

Acetates↗

PubMed2004

Preparation and characterization of positively charged ruthenium nanoparticles.

Positively charged ruthenium nanoparticles were prepared by NaBH(4) reduction at room temperature and at pH values lower than 4.9. The ruthenium nanoparticles were characterized by zeta potential measurement, TEM, XPS, and XRD. Particles with a mean diameter of 1.8 nm and a standard deviation of 0.40 nm could be obtained under the experimental conditions. The surface charge on the particles is believed to originate from hydrated proton adsorption. The positively charged ruthenium nanoparticles could be used as the starting material for further functionalization by PVP, ethylenediamine, and dodecylamine.

Journal Article↗

PubMed2004

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery.

BACKGROUND: Gene delivery vectors that restrict the expression of a therapeutic gene to a particular type of cells are critical to gene therapy in a complex structure, such as the central nervous system. We constructed a nonviral vector for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA. METHODS AND RESULTS: The vector was a synthetic chimeric peptide composed of a targeting moiety derived from NGF loop 4 and a DNA-binding moiety of 10 lysine residues. The peptide activated signal transduction pathways of the NGF receptor TrkA in PC12 cells and supported the survival of the cells after serum deprivation. After forming complexes with plasmid DNA, the peptide dose-dependently increased reporter gene expression in PC12 cells, which could be inhibited by excess NGF. The peptide-mediated gene expression was not affected in PC12 cells by co-incubation with a blocking antibody against the low-affinity NGF receptor p75 and was significantly enhanced in NIH3T3 cells stably transfected with TrkA cDNA, suggesting the involvement of the high-affinity NGF receptor TrkA without the participation of p75. Moreover, the peptide did not assist gene transfer in TrkA-poor, but TrkB- and/or TrkC-positive primary cerebellar granule neurons and primary cortical glial cells. CONCLUSIONS: The chimeric peptide reported will be useful in gene delivery to and gene therapy of the nervous system and other tissues/organs with cells expressing TrkA.

Animals↗

PubMed2003

Real time PCR quantification of GFRalpha-2 alternatively spliced isoforms in murine brain and peripheral tissues.

The neurotrophic factor neurturin (NTN) is structurally related to the glial-derived neurotrophic factor (GDNF) and has been shown to prevent the degeneration of dopaminergic neurons both in vitro and in vivo. The preferred receptor for NTN is the GDNF family receptor alpha 2 (GFRalpha-2). To date, three protein-coding alternatively spliced GFRalpha-2 isoforms (GFRalpha-2a, GFRalpha-2b, GFRalpha-2c) have been identified in mammalian tissues. An accurate quantification of the expression levels is necessary when determining the contributions of these isoforms to NTN signaling in tissues. In this report, sequence independent real time RT-PCR is used to determine the expression levels of GFRalpha-2 isoforms at different developmental stages of the murine embryos, and in various adult tissues. In the adult murine brain, GFRalpha-2a was found to be the most abundant, GFRalpha-2c was slightly less and GFRalpha-2b was 10-fold lower. The testis did not appear to express significant levels of GFRalpha-2a, 2b or 2c, compared to the brain. A novel finding in this study is that in some tissues, including the adult brain, the expression levels of GFRalpha-2, as quantified by the amplification of the 3' sequences encoding the putative glycosyl-phosphatidylinositol anchor signal sequence, were significantly higher than the combined levels of GFRalpha-2a, GFRalpha-2b and GFRalpha-2c. This indicates the existence of yet to be identified forms of GFRalpha-2 in some tissues that may be of physiological significance.

Aging↗