Yen-Peng Ho

Ru(II) complexes of N4 and N2O2 macrocyclic Schiff base ligands: Their antibacterial and antifungal studies

Reactions of [RuCl2(DMSO)4] with some of the biologically active macrocyclic Schiff base ligands containing N4 and N2O2 donor group yielded a number of stable complexes, effecting complete displacement of DMSO groups from the complex. The interaction of tetradentate ligand with [RuCl2(DMSO)4] gave neutral complexes of the type [RuCl2(L)] [where L=tetradentate macrocyclic ligand]. These complexes were characterized by elemental, IR, 1H, 13C NMR, mass, electronic, thermal, molar conductance and magnetic susceptibility measurements. An octahedral geometry has been proposed for all complexes. All the macrocycles and macrocyclic Ru(II) complexes along with existing antibacterial drugs were screened for antibacterial activity against Gram +ve (Bacillus subtilis, Staphylococcus aureus) and Gram -ve (Escherichia coli, Klebsiella pneumonia) bacteria. All these compounds were found to be more active when compared to streptomycin and ampicillin. The representative macrocyclic Schiff bases and their complexes were also tested in vitro to evaluate their activity against fungi, namely, Aspergillus flavus and Fusarium species.

Mono and bis-6-arylbenzimidazo[1,2-c]quinazolines: a new class of antimicrobial agents.

With the aim of obtaining novel biologically active compounds, we have synthesized a series of mono, bis-2-o-arylideneaminophenylbenzimidazoles and a second series of corresponding mono, bis-6-arylbenzimidazo[1,2-c]quinazolines respectively. The target benzimidazo[1,2-c]quinazoline compounds were obtained by the condensation of 2-(o-aminophenyl)benzimidazole with mono and di carbonyl compounds, followed by oxidative cyclisation of the resulting mono and bis-2-o-arylideneaminophenylbenzimidazoles.All the products were characterized via IR, (1)H NMR, (13)C NMR, MS and elemental analysis. The antimicrobial activities of all quinazolines against various bacteria and fungi were evaluated. Among the compounds tested IVd, IVe exhibited good antibacterial and antifungal activities while IIIb, IIIc also showed notable antimicrobial activity with reference to standard drugs Ampicillin and Ketoconazole respectively.

Identification of Pathogens by Mass Spectrometry

Abstract Background: Mass spectrometry (MS) is a suitable technology for microorganism identification and characterization. Content: This review summarizes the MS-based methods currently used for the analyses of pathogens. Direct analysis of whole pathogenic microbial cells using MS without sample fractionation reveals specific biomarkers for taxonomy and provides rapid and high-throughput capabilities. MS coupled with various chromatography- and affinity-based techniques simplifies the complexity of the signals of the microbial biomarkers and provides more accurate results. Affinity-based methods, including those employing nanotechnology, can be used to concentrate traces of target microorganisms from sample solutions and, thereby, improve detection limits. Approaches combining amplification of nucleic acid targets from pathogens with MS-based detection are alternatives to biomarker analyses. Many data analysis methods, including multivariate analysis and bioinformatics approaches, have been developed for microbial identification. The review concludes with some current clinical applications of MS in the identification and typing of infectious microorganisms, as well as some perspectives. Summary: Advances in instrumentation (separation and mass analysis), ionization techniques, and biological methodologies will all enhance the capabilities of MS for the analysis of pathogens.

Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laeviss oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

Advances in mass spectrometry for the identification of pathogens.

Abstract Mass spectrometry (MS) has become an important technique to identify microbial biomarkers. The rapid and accurate MS identification of microorganisms without any extensive pretreatment of samples is now possible. This review summarizes MS methods that are currently utilized in microbial analyses. Affinity methods are effective to clean, enrich, and investigate microorganisms from complex matrices. Functionalized magnetic nanoparticles might concentrate traces of target microorganisms from sample solutions. Therefore, nanoparticle‐based techniques have a favorable detection limit. MS coupled with various chromatographic techniques, such as liquid chromatography and capillary electrophoresis, reduces the complexity of microbial biomarkers and yields reliable results. The direct analysis of whole pathogenic microbial cells with matrix‐assisted laser desorption/ionization MS without sample separation reveals specific biomarkers for taxonomy, and has the advantages of simplicity, rapidity, and high‐throughput measurements. The MS detection of polymerase chain reaction (PCR)‐amplified microbial nucleic acids provides an alternative to biomarker analysis. This review will conclude with some current applications of MS in the identification of pathogens.

Physicochemical and biological characterization of novel macrocycles derived from o-phthalaldehyde

A series of novel macrocyclic compounds were synthesized by the condensation of o-phthalaldehyde with aromatic amino alcohols followed by treatment with 1,2-dibromoethane or 1,3-dibromopropane in non-template method. The structural features of the isolated macrocycles have been determined from the microanalytical, IR, (1)H, (13)C NMR and mass spectral studies. Antimicrobial activities of these macrocyclic compounds were tested against the gram-positive (Bacillus subtilis, Staphylococcus aureus) and gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria and found to exhibit potential antibacterial activity. The macrocycles were also tested in vitro to evaluate their activity against fungi, namely, Aspergillus flavus (A. flavus) and Fusarium species.

Digestion Completeness of Microwave-Assisted and Conventional Trypsin-Catalyzed Reactions

Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60°C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern.

Investigating the effects of protein patterns on microorganism identification by high-performance liquid chromatography–mass spectrometry and protein database searches

High-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) has been employed for separation and detection of protein biomarkers from E. coli samples. LC-MS is suitable for microbial identification because it can couple on-line with sample clean-up devices and is readily amenable to automation. In this work, we have investigated the effects of sample preparation methods on the detection of bacterial proteins by LC-MS. Many factors effect the degree of variations in the protein patterns (i.e. number and masses of proteins). For example, changing the polarity as well as pH of the extraction solvent may control the number of detected proteins. It is also noted that the protein patterns can vary even when the total ion chromatography plots seem to be the same under the same sample preparation conditions. Further, we have tested experimentally the influence of LC-MS-analyzed protein patterns (molecular masses between 2000 and 60,000) on microbial identification by protein database searches. This is in contrast to the current database search approach, where only the masses of smaller proteins (< or = 20,000) from direct matrix-assisted laser/desorption ionization MS are used. In spite of the variations in protein patterns, all the database search results show that the best matches come from the correct microorganism.

Proteomic comparative analysis of pathogenic strain 232 and avirulent strain J of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is an important pathogen of pigs causing enzootic pneumonia of swine. The pathogen remains largely enigmatic as far as the host-pathogen interactions are concerned. In the present study, the protein profiles of two strains of M. hyopneumoniae were compared by two-dimensional gel electrophoresis and mass spectrometry. The results indicate that the major adhesin P97, the 50-kDa protein derived from P159 adhesin, and the 43-kDa cleavage product of P102 are expressed at much higher levels in the pathogenic strain 232. In contrast, the avirulent strain J switches its focus to metabolism and expresses more glyceraldehyde 3-phosphate dehydrogenase in gluconeogenesis and lactate dehydrogenase, pyruvate dehydrogenase, and phosphate acetyltransferase in the pyruvate metabolism pathway. We speculate that the avirulent strain may have developed better capabilities to cope with the rich environment during repeated inoculations. Simultaneously, the capability to infect host cells may become less important so that the adhesion-related protein genes are down-regulated.

Laser induced popcornlike conformational transition of nanodiamond as a nanoknife

Nanodiamond (ND) is surrounded by layers of graphite on its surface. This unique structure feature creates unusual fluorescence spectra, which can be used as an indicator to monitor its surface modification. Meanwhile, the impurity, nitroso (CNO) inside the ND can be photolyzed by two-photon absorption, releasing NO to facilitate the formation of a sp3 diamond structure in the core of ND and transforming it into a sp2 graphite structure. Such a conformational transition enlarges the size of ND from 8to90nm, resulting in a popcornlike structure. This transition reaction may be useful as nanoknives in biomedical application.