Kuo-Yuan Hwa

Synthesis of zinc oxide nanoparticles on graphene–carbon nanotube hybrid for glucose biosensor applications

Synthesis of zinc oxide nanoparticles incorporated graphene-carbon nanotubes hybrid (GR-CNT-ZnO) through a simple, one-pot method is demonstrated. The as-synthesized GR-CNT-ZnO composite is applied to fabricate an enzyme based glucose biosensor. The GOx immobilized on GR-CNT-ZnO composite exhibits well-defined redox peaks with a peak potential separation (ΔEp) of about 26 mV with enhanced peak currents, indicating a fast electron transfer at the modified electrode surface. The cyclic voltammetry measurements revealed that the modified film has high electrocatalytic ability towards glucose detection in the presence of oxygen. The proposed sensor has a wide linear detection range from 10 μM to 6.5 mM of glucose with a limit of detection (LOD) of 4.5 (±0.08) μM. In addition, the sensor possessed appreciable repeatability, reproducibility and remarkable stability for the sensitive determination of glucose. The practicality of this sensor has been demonstrated in human serum samples, with results being in good agreement with those determined using a standard photometric method.

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica

ABSTRACT A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis.

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV-vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of -0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10(-10) mol cm(-2) and 3.36 s(-1), respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM - 2 mM with LOD of 4.1 μM, (2) 2 mM - 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.

Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant

ABSTRACT Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

Glycosyl phosphatidylinositol-linked glycoconjugates: structure, biosynthesis and function.

The purpose of this review is to summarize the most recent advances on GPI research. Structural studies on GPI-linked glycoconjugates indicate that there are significant variations in different organisms, although there is a conserved core structure. Furthermore, structural studies suggest that in different cell types, there is an army of glycosyltransferases dedicated to the synthesis of GPI-linked glycoconjugates. Biochemical studies on the synthesis of these GPI-linked glycoconjugates suggest that not only many different enzymes are involved but also that enzymes from different cell types, involving in the conserved core structure can have different substrate specificity. Genetic cloning of the yeast genes involved in synthesizing the core structure suggests that many of these enzymes also have human homologues. However, paroxysmal nocturnal hemogobinuria (PNH) is the only known human disease associated with the synthesis of GPI-linked glycoconjugates. Functional studies suggest that GPI-anchor can act as a signal for protein sorting and localization. Furthermore, GPI-linked receptors play an important role in T-cell activation.

Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.

Both viral effect and immune‐mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome‐associated coronavirus (SARS‐CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type‐2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non‐SARS‐pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti‐epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross‐reactive epitopes on SARS‐CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti‐S2 Abs and IFN‐γ resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti‐S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS‐CoV infection may be implicated in SARS pathogenesis.

Structural analysis of the asparagine-linked glycans from the procyclic Trypanosoma brucei and its glycosylation mutants resistant to Concanavalin A killing

The variant surface glycoprotein of the bloodstream form of Trypanosoma brucei is known to be glycosylated with a range of structures including high mannose and complex types. In contrast, glycosylation in the procyclic form of the parasite appears to be restricted to a single Man(5)GlcNAc(2) structure, as found on its procyclin. To gain a better insight into the developmentally regulated glycosylation pattern, we have structurally defined the full range of N-linked glycans made by the procyclic trypanosomes, as well as two previously described glycosylation mutants generated under Con A selection. It was found that the wild type procyclic cells could synthesize a full range of high mannose type structures from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), with Man(5)GlcNAc(2) as the major component. In contrast, the two mutants mainly synthesized a truncated Man(4)GlcNAc(2) structure, Man alpha 1-3Man alpha 1-6(Man alpha1-3)Man be ta 1-4 GlcNAc beta 1-4GlcNAc, a significant portion of which was further extended by a single GlcNAc to form GlcNAc-Man(4)GlcNAc(2) and a single N-acetyllactosamine unit at the 3-arm position to form Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3(Man al pha 1- 3Man alpha 1-6)Man beta 1-4G lcNAc beta 1-4GlcNAc. The results suggest that the procyclic trypanosomes could be induced by Con A selection to synthesize limited hybrid type structures, but in general do not further process their N-linked glycans into multiantennary complex types as the blood stream forms do.

Molecular chaperones affect GTP cyclohydrolase I mutations in dopa-responsive dystonia.

Unstable GTP cyclohydrolase I (GCH) mutations in dopa‐responsive dystonia (DRD) can exert a dominant‐negative effect in the HeLa cell model, but in a batch of cells this effect could not be shown. Through differential display, we found a higher Hsc70 expression in the non–dominant‐negative cells. We further demonstrated that ectopic expression of Hsp40/Hsp70 stabilized the GCH mutant G201E. Moreover, Hsp90 inhibitor geldanamycin destroyed the wild‐type GCH level, and heat shock increased the synthesis of GCH protein. Therefore, the dominant‐negative effect produced by unstable proteins would be susceptible to the status of molecular chaperones, which could be the modifying genes and therapeutic targets for DRD and other genetic diseases. Ann Neurol 2004;55:875–878

Exchange of active site residues alters substrate specificity in extremely thermostable β-glycosidase from Thermococcus kodakarensis KOD1.

β-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with β-glucosidase, β-mannosidase, β-fucosidase and β-galactosidase activities. Sequence alignment with other β-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic β-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its β-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for β-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures.

A GHS-Based Intelligent Management Platform with IP-Camera Security System

We have described an e-platform, with an IP camera for managing toxic and hazardous chemicals within an organization. The system allows: (1) a worker to understand the globally harmonized system (GHS) of classification and labeling of chemicals and printout the GHS-based safety data sheets with 16 sections of listed information, according to the GHS, (2) a director of a chemical laboratory to inventory hazardous chemicals and to track the education training history of workers, and (3) an organization safety manager to review the inventory and apply for government licenses. And, the system has an emergency system connected to IP-cameras. It provides online image of storage environments of hazardous chemicals. With this intelligent information system with several automatic functions, a safer environment for storage and usage of hazardous chemicals can be implemented in an organization.