Browsing by Author "Huang, Sheng-Cih"

Now showing 1 - 14 of 14

Characterization and epitope mapping of Dengue virus type 1 specific monoclonal antibodies
(2017-10-02)
Chen, Wen-Hung
;
Chou, Feng-Pai
;
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Cheng, Chuan-Hung
Background: Dengue virus (DV) infection causes a spectrum of clinical diseases ranging from dengue fever to a life-threatening dengue hemorrhagic fever. Four distinct serotypes (DV1-4), which have similar genome sequences and envelope protein (E protein) antigenic properties, were divided. Among these 4 serotypes, DV1 usually causes predominant infections and fast diagnosis and effective treatments are urgently required to prevent further hospitalization and casualties. Methods: To develop antibodies specifically targeting and neutralizing DV1, we immunized mice with UV-inactivated DV1 viral particles and recombinant DV1 E protein from residue 1 to 395 (E395), and then generated 12 anti-E monoclonal antibodies (mAbs) as the candidates for a series of characterized assays such as ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Results: Among the mAbs, 10 out of 12 showed cross-reactivity to four DV serotypes as well as Japanese encephalitis virus (JEV) in different cross-reactivity patterns. Two particular mAbs, DV1-E1 and DV1-E2, exhibited strong binding specificity and neutralizing activity against DV1 and showed no cross-reactivity to DV2, DV3, DV4 or JEV-infected cells as characterized by ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Using peptide coated indirect ELISA, we localized the neutralizing determinants of the strongly inhibitory mAbs to a sequence-unique epitope on the later-ridge of domain III of the DV1 E protein, centered near residues T346 and D360 ((346)TQNGRLITANPIVTD(360)). Interestingly, the amino acid sequence of the epitope region is highly conserved among different genotypes of DV1 but diverse from DV2, DV3, DV4 serotypes and other flaviviruses. Conclusions: Our results showed two selected mAbs DV1-E1 and DV1-E2 can specifically target and significantly neutralize DV1. With further research these two mAbs might be applied in the development of DV1 specific serologic diagnosis and used as a feasible treatment option for DV1 infection. The identification of DV1 mAbs epitope with key residues can also provide vital information for vaccine design.
Detection of chlorophylls in spores of seven ferns
(2017-03-01)
Tseng, Mei-Hwei
;
Lin, Kuei-Huei
;
Huang, Yi-Jia
;
Chang, Ya-Lan
;
Huang, Sheng-Cih
Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, "cryptochlorophyllous spores", is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.
Effective Construction of a High-Capacity Boronic Acid Layer on a Quartz Crystal Microbalance Chip for High-Density Antibody Immobilization
(2019-01-01)
Lin, Pei-Heng
;
Huang, Sheng-Cih
;
Chen, Kuang-Po
;
Li, Bor-Ran
;
Li, Yaw-Kuen
Boronic acids (BAs) provide strong potential in orientation immobilization of antibody and the modification method is crucial for efficiency optimization. A highly effective method has been developed for rapid antibody immobilization on gold electrodes through the electrodeposition of a BA-containing linker in this study. Aniline-based BA forms a condense layer while antibody could automatically immobilize on the surface of the electrode. Compare to traditional self-assembled monolayer method, the electrodeposition process dramatically reduces the modification time from days to seconds. It also enhances the immobilized efficiency from 95 to 408 (ng/cm(2)) with a strong preference being exhibited for shorter aniline-based linkers.
Enantioselective Synthesis of Hydantoin and Diketopiperazine-Fused Tetrahydroisoquinolines via Pictet-Spengler Reaction
(2019-04-01)
Liu, Shih-I
;
Haung, Jia-Yun
;
Barve, Indrajeet J.
;
Huang, Sheng-Cih
;
Sun, Chung-Ming
An enantioselective synthesis of iso-, isothio-, and isoselenohydantoin and diketopiperazine-fused tetrahydroisoquinolines from L-Dopa was reported. The route consists of an Pictet-Spengler reaction of (S)-2-amino-3-(3,4-dimethoxyphenyl)propanoates with various aldehydes to afford diastereomeric tetrahydroisoquinolines. Next step, the tetrahydroisoquinolines were further reacted with iso-, isothio-, or isoselenocyanates to construct hydantoin. Similarly, the diketopiperazine moiety was constructed by subjecting tetrahydroisoquinolines to a condensation reaction with chloroacetyl chloride followed by nucleophilic addition with various primary amines.
Ligand-Functionalization of BPEI-Coated YVO4:Bi3+,Eu3+ Nanophosphors for Tumor-Cell-Targeted Imaging Applications
(2013-11-01)
Chen, Yi-Chin
;
Huang, Sheng-Cih
;
Wang, Yu-Kuo
;
Liu, Yuan-Ting
;
Wu, Tung-Kung
In this study, surface-functionalized, branched polyethylenimine (BPEI)-modified YVO4:Bi3+,Eu3+ nanocrystals (NCs) were successfully synthesized by a simple, rapid, solvent-free hydrothermal method. The BPEI-coated YVO4:Bi3+,Eu3+ NCs with high crystallinity show broad-band excitation in the =250 to 400nm near-ultraviolet (NUV) region and exhibit a sharp-line emission band centered at =619nm under excitation at =350nm. The surface amino groups contributed by the capping agent, BPEI, not only improve the dispersibility and water/buffer stability of the BPEI-coated YVO4:Bi3+,Eu3+ NCs, but also provide a capability for specifically targeted biomolecule conjugation. Folic acid (FA) and epidermal growth factor (EGF) were further attached to the BPEI-coated YVO4:Bi3+,Eu3+ NCs and exhibited effective positioning of fluorescent NCs toward the targeted folate receptor overexpressed in HeLa cells or EGFR overexpressed in A431 cells with low cytotoxicity. These results demonstrate that the ligand-functionalized, BPEI-coated YVO4:Bi3+, Eu3+ NCs show great potential as a new-generation biological luminescent bioprobe for bioimaging applications. Moreover, the unique luminescence properties of BPEI-coated YVO4:Bi3+,Eu3+ NCs show potential to combine with a UVA photosensitizing drug to produce both detective and therapeutic effects for human skin cancer therapy.
Multiple Pleomorphic Tetramers of Thermostable Direct Hemolysin from Grimontia hollisae in Exerting Hemolysis and Membrane Binding
(2019-07-08)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Chang, Chin-Yuan
;
Huang, Wan-Ting
;
Liao, Man-Jun
Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell-cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh-TDH), a pore-forming toxin. The toxin crystalized in the same space group of P2(1)2(1)2, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer-I, -II, and -III. A central pore with comparable depth of similar to 50 angstrom but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.
Potential antitumor therapeutic application of Grimontia hollisae thermostable direct hemolysin mutants
(2015-04-01)
Huang, Sheng-Cih
;
Wang, Yu-Kuo
;
Huang, Wan-Ting
;
Kuo, Tsam-Ming
;
Yip, Bak-Sau
We report on the preparation of a new type of immunotoxin by conjugation of an epidermal growth factor receptor (EGFR)-binding peptide and an R46E mutation of thermostable direct hemolysin from Grimontia hollisae, (Gh-TDHR46E/EB). The hybrid immunotoxin was purified to homogeneity and showed a single band with slight slower mobility than that of Gh-TDHR46E. Cytotoxicity assay of Gh-TDHR46E/EB on EGFR highly, moderately, low, and non-expressed cells, A431, MDA-MB-231, HeLa, and HEK293 cells, respectively, showed apparent cytotoxicity on A431 and MDA-MB-231 cells but not on HeLa or HEK293 cells. In contrast, no cytotoxicity was observed for these cells treated with either Gh-TDHR46E or EB alone, indicating enhanced cytotoxic efficacy of Gh-TDHR46E by the EGFR binding moiety. Further antitumor activity assay of Gh-TDHR46E/EB in a xenograft model of athymic nude mice showed obvious shrinkage of tumor size and degeneration, necrosis, and lesions of tumor tissues compared to the normal tissues. Therefore, the combination of Gh-TDHR46E with target affinity agents opens new possibilities for pharmacological treatment of cancers and potentiates the anticancer drug's effect.
Pregnenolonyl-alpha-glucoside exhibits marked anti-cancer and CYP17A1 enzymatic inhibitory activities
(2020-02-07)
Chou, Feng-Pai
;
Hsu, Wen-Chen
;
Huang, Sheng-Cih
;
Chang, Chin-Yuan
;
Chiou, Ya-Sheng
We report here that pregnenolonyl-alpha-glucoside (2), a steryl glycoside synthesized directly from pregnenolone and glucose via a consecutive multienzyme-catalyzed process, exhibits marked dose-dependent cytotoxic activity against HT29, AGS, and ES-2 cells with IC50 values of 23.5 to 50.9 mu M. An in vitro CYP17A1 binding pattern assay and protein-ligand docking model support that 2, like abiraterone, binds in the active site heme iron pocket of CYP17A1.
Preliminary structure determination and functional residues characterization of thermostable direct hemolysin from Grimontia hollisae
(2009-08-16)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Wu, Tung-Kung
;
生物科技學系
;
Department of Biological Science and Technology
Purification, crystallization and preliminary X-ray analysis of a thermostable direct haemolysin from Grimontia hollisae
(2011-02-01)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Wu, Yi-Fang
;
Chen, Yu-Ching
;
Chen, Wen-Hung
Vibrio hollisae, a halophilic species recently reclassified as Grimontia hollisae, is a causative agent of gastroenteritis and septicaemia. One important pathogenic Vibrio factor, thermostable direct haemolysin (TDH), has been purified and crystallized in two crystal forms using the vapour-diffusion method. The crystals belonged to an orthorhombic space group, with unit-cell parameters a = 104.8, b = 112.4, c = 61.3 angstrom and a = 122.9, b = 123.3, c = 89.8 angstrom. The crystals contained either four or eight molecules per asymmetric unit, with predicted solvent contents of 49.4 and 46.3% and Matthews coefficients (V(M)) of 2.4 and 2.3 angstrom(3) Da(-1), respectively. These crystals were suitable for structure determination, which would yield structural details related to the cytotoxicity and oligomeric structure of this pore-forming toxin.
Purification, crystallization and preliminary X-ray crystallographic analysis of xylose reductase from Candida tropicalis
(2009-04-01)
Chen, Li-Chun
;
Huang, Sheng-Cih
;
Chuankhayan, Phimonphan
;
Chen, Chung-Der
;
Huang, Yen-Chieh
Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of D-xylose to xylitol, which is the first step in the metabolism of D-xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 angstrom resolution, the unit cell belongs to space group P3(1) or P3(2). Preliminary analysis indicated the presence of four XR molecules in the asymmetric unit, with 68.0% solvent content.
Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties
(2011)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Wu, Yi-Fang
;
Chen, Yu-Ching
;
Lin, Yen-Ling
Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 degrees C but is reactivated by additional heating above 80 degrees C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a beta-sheet to alpha-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.
Synthesis of side-chain regioregular and main-chain alternating poly(bichalcogenophene)s and an ABC-type periodic poly(terchalcogenophene)
(2020-04-21)
Liu, Huai-Hsuan
;
Liang, Wei-Wei
;
Lai, Yu-Ying
;
Su, Yen-Chen
;
Yang, Hau-Ren
Three unsymmetrical diiodobichalcogenophenes SSeI2, STeI2, and SeTeI2 and a diiodoterchalcogenophene SSeTeI2 were prepared. Grignard metathesis of SSeI2, STeI2, SeTeI2, and SSeTeI2 occurred regioselectively at the lighter chalcogenophene site because of its relatively lower electron density and less steric bulk. Nickel-catalyzed Kumada catalyst-transfer polycondensation of these Mg species provided a new class of side-chain regioregular and main-chain AB-type alternating poly(bichalcogenophene)s-PSSe, PSTe, and PSeTe-through a chain-growth mechanism. The ring-walking of the Ni catalyst from the lighter to the heavier chalcogenophene facilitated subsequent oxidative addition, thereby suppressing the possibility of chain-transfer or chain-termination. More significantly, the Ni catalyst could walk over the distance of three rings (ca. 1 nm)-from a thiophene unit via a selenophene unit to a tellurophene unit-to form PSSeTe, the first ABC-type regioregular and periodic poly(terchalcogenophene) comprising three different types of 3-hexylchalcogenophenes.
The Thermostable Direct Hemolysin from Grimontia hollisae Causes Acute Hepatotoxicity In Vitro and In Vivo
(2013-02-21)
Lin, Yan-Ren
;
Chen, Yao-Li
;
Wang, Keh-Bin
;
Wu, Yi-Fang
;
Wang, Yu-Kuo
Background: G. hollisae thermostable direct hemolysin (Gh-TDH) is produced by most strains of G. hollisae. This toxin has been reported to be absorbed in the intestines in humans. Secondary liver injury might be caused by venous return of the toxin through the portal system. We aimed to firstly analyze the in vitro and in vivo hepatotoxicity of Gh-TDH. Methods: Liver cells (primary human non-cancer cell and FL83B mouse cells) were treated and mice (BALB/c) were fed with this toxin to investigate its hepatotoxicity. Morphological examination and cytotoxicity assays using liver cells were also performed. Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of this protein in liver cells. Mice were subjected to liver function measurements and liver biopsies following toxin treatment and wild-type bacterial infection. PET (positron emission tomography)/CT (computed tomography) images were taken to assess liver metabolism during acute injury and recovery. Results: The effect of hepatotoxicity was dose and time dependent. Cellular localization showed that the toxin was initially located around the cellular margins and subsequently entered the nucleus. Liver function measurements and liver biopsies of the mice following treatment with toxin or infection with wild-type Grimontia hollisae showed elevated levels of transaminases and damage to the periportal area, respectively. The PET/CT images revealed that the reconstruction of the liver continued for at least one week after exposure to a single dose of the toxin or bacterial infection. Conclusions: The hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in the periportal area of the liver, and the liver became functionally insufficient.