Ta-Chau Chang

ABSTRACT. Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.

The mechanism of inhibition of telomerase by drugs is a key factor in an understanding of guanine-quadruplex complex stabilization during human cancer. This study describes a simulated annealing docking and molecular dynamics simulation to investigate a synthesized potent inhibitor, 3,6-bis(1-methyl-4-vinylpyridinium iodine) carbazole (BMVC), which stabilizes the quadruplex structure of the human telomeric DNA sequence d[AG(3)(T(2)AG(3))(3)] and inhibits telomerase activity. The compound was predicted to selectively interact with the quadruplex structure. During our simulation, the binding affinities were calculated and used to predict the best drug-binding sites as well as enhanced selectivity compared with other compounds. Our studies suggest that the simulation results quite coincide with the experimental results. In addition, molecular modeling shows that a 2:1 binding model involving the external binding of BMVC to both ends of the G-quartet of d[AG(3)(T(2)AG(3))(3)] is the most stable binding mode and this agrees with the absorbance titration results that show two binding sites. Of particular interest is that one pyridinium ring and carbazole moiety of the BMVC can stack well at the end of G-quartet. This implies that BMVC is a good human quadruplex stabilizer and also a good telomerase inhibitor.

The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R 2 > 0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

We reported that non-targeting siRNA (NT-siRNA) stress induces non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) expression to cooperate with exoribonuclease XRN2 for releasing the stress [Wei,P.C., Lo,W.T., Su,M.I., Shew,J.Y. and Lee, W. H. (2011) Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress. Nucleic Acids Res., 40, 323-332]. However, how NT-siRNA stress inducing NPGPx expression remains elusive. In this communication, we showed that the proximal promoter of NPGPx contained a mixed G-quadruplex (G4) structure, and disrupting the structure diminished NT-siRNA induced NPGPx promoter activity. We also demonstrated that nucleolin (NCL) specifically bonded to the G4-containing sequences to replace the originally bound Sp1 at the NPGPx promoter on NT-siRNA stress. Consistently, overexpression of NCL further increased NPGPx promoter activity, whereas depletion of NCL desensitized NPGPx promoter to NT-siRNA stress. These results suggest that the cis-element with mixed G4 structure at the NPGPx promoter plays an essential role for its transactivation mediated by NCL to release cells from NT-siRNA stress.

Because various non-parallel G-quadruplexes of human telomeric sequences in K+ solution can be converted to a parallel G-quadruplex by adding polyethylene glycol (PEG) as a co-solvent, we have taken advantage of this property of PEG to study the covalent attachment of a PEG unit to a G-quadruplex ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC). The hybrid ligand with the PEG unit, BMVC-8C3O or BMVC-6C2O by substituting either the tetraethylene glycol or the triethylene glycol terminated with a methyl-piperidinium cation in N-9 position of BMVC, not only induces structural change from different non-parallel G-quadruplexes to a parallel G-quadruplex but also increases the melting temperature of human telomeres in K+ solution by more than 45 degrees C. In addition, our ligand work provides further confidence that the local water structure plays the key to induce conformational change of human telomere.

Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. We have introduced a G-quadruplex (G4) ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy. The GROs that form parallel G4 structures, such as PU22, T40214 and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form non-parallel G4 structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the fluorescence resonant energy transfer studies of fluorophore-labeled GROs show that the parallel G4 structures can be retained in CL1-0 cells, whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the non-parallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs.

ABSTRACT. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8  ns and that of interaction with the duplex structure of a calf thymus is ∼1.2  ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

A novel method based on emulsion/filtration is introduced for G-quadruplex DNA structural separation. We first synthesized a lipophilic analogue of BMVC, 3,6-Bis(1-methyl-4-vinylpyridinium)-9-(12'-bromododecyl) carbazole diiodide (BMVC-12C-Br), which can form an oil-in-water (o/w) phase emulsion. Due to the binding preferences of BMVC-12C-Br emulsion to some specific DNA structures, the large emulsion ( approximately 2 microm) bound DNA was separated from the small free DNA in the filtrate by a 0.22 microm pore size MCE membrane. This method is able to isolate the non-parallel G-quadruplexes from the parallel G-quadruplexes and the linear duplexes from both G-quadruplexes. In addition, this method allows us not only to determine the absence of the parallel G-quadruplexes of d(T(2)AG(3))(4) and the presence of the parallel G-quadruplexes of d(T(2)AG(3))(2) in K(+) solution, but also to verify structural conversion from antiparallel to parallel G-quadruplexes of d[AG(3)(T(2)AG(3))(3)] in K(+) solution under molecular PEG condition. Moreover, this emulsion can separate the non-parallel G-quadruplexes of d(G(3)CGCG(3)AGGAAG(5)CG(3)) monomer from the parallel G-quadruplexes of its dimer in K(+) solution. Together with NMR spectra, one can simplify the spectra for both the free DNA and the bound DNA to establish a spectrum-structure correlation for further structural analysis.

A CE-resonance Raman spectroscopy (CE-RRS) method based on MEKC and sweeping-MEKC modes is described. A nonfluorescent compound, malachite green (MG), and a doubled Nd:YAG laser (532 nm, 300 mW) were selected as model compound and light source, respectively. In order to carry out a quantitative analysis of MG, a monochromator (effective bandwidth, 0.4 nm) was used to collect the specific Raman line at 1616 cm(-1) (N-phi and C-C stretch, corresponding to 582 nm when the wavelength of the exciting source was 532 nm). As a result, the LOD for MG was 10 ppm, based on the MEKC/RRS mode. This could be improved to 5 ppb when the sweeping-MEKC/RRS mode was applied. Furthermore, with the addition of nano-size silver colloids to the CE buffer the detection limits can be further improved, but the data obtained with surface-enhanced resonance Raman spectroscopy (SERRS) are less useful for quantitative purposes.

We have previously illustrated that the electron donor of carbazole moiety and the electron acceptor of methyl pyridinium cation in 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) molecule could form an intramolecular charge-transfer state. The intramolecular twist of the vinyl group in bridging the donor and acceptor plays an important role in the BMVC fluorescence. Here, we have synthesized three 9-aryl-substituted BMVC derivatives with different electronic properties for the design of the second generation of fluorescence probes. The steady-state solvatochromic studies show no appreciable change to the charge transfer of BMVC by substituting an anisole electron-donating group at 9-position of BMVC. However, substituting a 9-nitrobenzyl electron-withdrawing group in BMVC could restrict the charge transfer in the excited state. Moreover, the increase of the fluorescence yields of 9-anisole BMVC and 9-phenyl BMVC upon interaction with DNA is even higher than that in glycerol, while the fluorescence yield of 9-nitrobenzyl BMVC upon interaction with DNA is much lower than that in glycerol. Although 9-nitrobenzyl BMVC is a good G-quadruplex stabilizer, substituting an electron-withdrawing group at 9-position of BMVC is not recommended for the design of fluorescence probes. On the other hand, colocalization between 9-phenyl BMVC and MitoTracker Red in the merged image of cells indicates that the 9-phenyl BMVC is a potential fluorescent mitochondrial probe. (C) 2007 Elsevier B.V. All rights reserved.

G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na(+) or K(+)). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10 mM Na(+) solution at 37 °C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68 ± 0.01 and 1.63 ± 0.03 (s(-1)), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu(2+)-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions.

The gel assay, circular dichroism, and differential scanning calorimetry results all demonstrate that a major monomer component of bcl2mid exists at low [K(+)] and an additional dimer component appears at high [K(+)]. This implies that bcl2mid is a good candidate for elucidating the mechanisms of structural conversion between different G-quadruplexes. We further discovered that the conversion between the monomer and dimer forms of bcl2mid does not occur at room temperature but is detected when heated above the melting point. In addition, the use of the lithium cation to keep the same ionic strength in a K(+) solution favors the formation of the bcl2mid dimer. We also found that the bcl2mid dimer is more stable than the monomer. However, after the bcl2mid monomer is formed in a K(+) solution, there is no appreciable structural conversion from the monomer to the dimer detected with addition of Li(+) at room temperature. Furthermore, the spectral changes of bcl2mid when transitioning from sodium form to potassium form take place upon K(+) titration. The absence of the dimer form for bcl2mid after the direct addition of 150 mM [K(+)] at room temperature suggests that the spectral changes are not due to rapid unfolding and refolding. In addition, this work reveals the conditions that would be useful for NMR studies of G-quadruplexes.

A handheld device based on fluorescence of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) staining was established for the rapid, point-of-care screening of cancer cells (see Chang and co-workers, Analyst, 2007, 132, 745). Offering instant screening of cancer at low cost, here we apply this simple assay in clinical tests on fine needle aspirates of neck masses from 114 outpatients (115 specimens). The diagnostic accuracy of this simple method alone is ca. 80% (80/99). The combination of the BMVC test and the fine needle aspiration (FNA) cytology reduced the non-diagnosis from 17 cases in FNA cytology to 6 cases in the combined method. Moreover, an algorithm is proposed to improve the diagnostic accuracy of malignant neck lumps up to nearly 100%.

Earlier work from our laboratory has indicated that a hemerythrin-like protein was over-produced together with the particulate methane monooxygenase (pMMO) when Methylococcus capsulatus (Bath) was grown under high copper concentrations. A homologue of hemerythrin had not previously been found in any prokaryote. To confirm its identity as a hemerythrin, we have isolated and purified this protein by ion-exchange, gel-filtration and hydrophobic interaction chromatography, and characterized it by mass spectrometry, UV-visible, CD, EPR and resonance Raman spectroscopy. On the basis of biophysical and multiple sequence alignment analysis, the protein isolated from M. capsulatus (Bath) is in accord with hemerythrins previously reported from higher organisms. Determination of the Fe content in conjunction with molecular-weight estimation and mass analysis indicates that the native hemerythrin in M. capsulatus (Bath) is a monomer with molecular mass 14.8 kDa, in contrast to hemerythrins from other eukaryotic organisms, where they typically exist as a tetramer or higher oligomers. (c) 2008 Elsevier Inc. All rights reserved.

A simple handheld device based on the fluorescence analysis of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide ( BMVC) stained cells was established for routine screening and potentially for early detection of cancer cells at extremely low cost. Flow cytometry assay further supported the utility of this simple device, where a preliminary study of tissue biopsy showed highly encouraging results.

Different cellular accumulations with distinct fluorescence properties of BMVC in cancer cells from normal cells allow us to establish a simple and economic method for the diagnosis of cancer cells. With using a light emitting diode to excite the BMVC molecule, microarray fluorescence analysis of a cell-based glass chip provides an easy method towards the detection of a limited number of cancer cells.